PURIFICATION AND CHARACTERIZATION OF
TRYPSIN FROM THE PYLORIC CECA OF
HOKI (Macruronus novaezealandiae)
BY
CHANGYING SHI
Department of Food Science & Agricultural Chemistry
Macdonald Campus, McGill University
Montreal, Canada
May, 2006
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science
© Changying Shi, 2006
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ABSTRACT
Fish viscera are produced in large quantities in the fishing industry and represent a waste
disposal and environmental pollution problem. However, this material is a rich source of
trypsins that may have sorne unique properties, such as high molecular activity at low
processing temperature, low thermostability, and high pH optimum/pH stability, for both
basic research and industrial applications. The main objectives of this project were to
extract, purify and characterize trypsin from the pyloric ceca of hoki (Macruronus
novaezealandiae), which is by far the most important commercial fish in New Zealand.
Trypsin was purified from the pyloric ceca of hoki by ammonium sulfate fractionation,
followed by acetone fractionation and affinity chromatography on SBTI-Sepharose 4B.
The purified extract was simultaneously desalted and concentrated by ultrafiltration, and
then characterized using N-a.-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate.
The affinity fraction migrated as a signal band in SDS-PAGE gels as weIl as in isoelectric
focusing gels. The molecular weight of the isolated trypsin was determined by SDSPAGE to be approximately 26,000 Da, whereas the MALDI-TOF MS method of analysis
indicated a molecular weight of23,791 Da. The isoelectric point was determined as 6.5.
The kinetic properties, temperature, pH and inhibition effects on the activity of the
purified trypsin were verified. On the basis of the kinetic properties, hoki trypsin showed
better amidase activity than bovine trypsin. The hoki trypsin.. hadalkaline'pR optimum
(pH 9.0) and was stable at a high pH. Roki trypsin had a higher optimum temperature (60
OC) and still had relative higher activity at lower temperature. On the other hand, hoki
trypsin was unstable at higher temperature. The enzyme was inhibited by weIl known
trypsin inhibitors (SB TI, aprotinin, benzamidine and PMSF). The N-terminal residues of
hoki trypsin, IVGGQECVPNSQPFMASLNY, displayed considerable homology with
other fish trypsins. Based on the above characteristics, it is suggested that the hoki
enzyme is authentic trypsin with potential for use in food industry and related
applications.
1
RÉSUMÉ
L'industrie de la pêche génère de grandes quantités de viscères, ce qui représente
d'importantes quantités de rejets de même qu'un problème environnemental au niveau de
la pollution. Or, ces résidus marins sont une source riche en trypsine ayant d'importantes
propriétés pour la recherche fondamentale et les applications industrielles: activité
moléculaire à faible température de mise en oeuvre, faible thermostabilité, pH optimum
élevé et conservation de la stabilité sur une large gamme de pH. Les objectifs de ce projet
sont: d'extraire, de purifier et de caractériser la trypsine des caecums pyloriques du hoki
(Macruronus novaezealandiae), le poisson le plus commercialisé en Nouvelle-Zélande.
La purification de la trypsine des caecums pyloriques du hoki se fait par
fractionnement à partir de sulfate d'ammonium, suivi par l'acétone et finalement, en
utilisant la chromatographie d'affinité avec phase SBTI-Sépharose 4B. L'ultrafiltration,
l'étape suivante, permet une séparation d'avec les sels et une concentration du produit,
qui peut alors être caractérisé en utilisant la N-a-benzoyl-DL-arginine-p-nitroanilide
(BAPNA) comme substrat. Les fractions chromatographiques lorsqu'analysées par SDSPAGE gels et isoelectrique focusing, migrent comme une bande. Par SDS-PAGE, la
masse molaire de la trypsine est évaluée à 26,000 Da, alors qu'elle est de 23,791 Da par
MALDI-TOF MS. Le point isoélectrique est de 6.5.
Les études de l'influence des propriétés cinétiques, de la température; du pH et
d'effets d'inhibition sur la trypsine purifiée ont aussi été effectuées. Sur la base des
propriétés cinétiques, la trypsine marine montre une meilleure activité amidase que celle
des bovins. Le pH optimal de la trypsine des hokis est de 9.0 et est aussi stable au-delà de
cette valeur. Avec sa température d'activité maximale à 60°C, elle montre aussi pour
toutes températures inférieures à ce point, une activité relative supérieure à sa vis-à-vis
bovine. Toutefois, elle est instable passé cette limite. L'enzyme est aussi inactivée par
les inhibiteurs conventionnels de la trypsine (SBTI, aprotinine, benzamidine et PMSF).
Le résidu N-terminal de la trypsine du hoki, IVGGQECVPNSQPFMASLNY, est très
similaire aux trypsines retrouvées chez les autres poissons. À partir de ces évidences, il
II
est probant que cette enzyme soit réellement de la trypsine, donc sujette à plusieurs
applications potentielles pour l'industrie alimentaire et connexe.
III
ACKNOWLEDGEMENTS
l would like to express my deep sense of gratitude to my supervisor Dr. Benjamin K.
Simpson for his guidance, advice and assistance in this research. His instructions were
always constructive and positive, helping me to finish my thesis with thorough
understanding of the problem.
Special gratitude goes to my thesis committee member Dr. Byong H. Lee for his helpful
advice and guidance. Aiso the help from Ms. Lise Stiebel and Ms. Barbara Laplaine are
gratefully acknowledged.
l would like to thank all ofmy fellow students and people in Dr Simpson's research group.
First, thanks a lot to Ms. Dan Li, who provided big help on my experiment guidance. l
would like to mention a few other colleagues for their support, encouragement and
friendship; they are Ms. Alberta Aryee and Mr. Rodrigo Santibanez. l also wish to
acknowledge the assistance of Dr. Sung Hun Yi and Dr. Martin Macouzet. Thanks to all
my friends, it was a pleasure studying and working with you.
l would like to express my deepest gratitude to my parents and my brother. Without their
support all this would be difficult. l owe them eternal gratitude.
At the end, the most important word of appreciation goes towards my loved husband
Juntao Luo. Without your love and understanding, l could have not archived this goal.
IV
TABLE OF CONTENTS
ABSTRACT ............................................................................................................... "...... .1
RÉSUMÉ ......................................................................................................................... .11
ACKNOWLEDGEMENT .............................................................................................. IV
TABLE OF CONTENTS ................................................................................................. V
LIST OF TABLES ........................................................................................................ VIII
LIST OF FIGURES ........................................................................................................ .1X
LIST OF ABBREVIATIONS .......................................................................................... XI
CHAPTER 1 GENERAL INTRODUCTION ................................................................. 1
CHAPTER II LITERATURE REVIEW ........................................................................ .4
2.1 Digestive proteases from marine fishes ....................................................................... .4
2.1.1 Mechanistic classification ..................................................................................... 4
2.1.1.1 Serine proteases ............................................................................................ 6
2.1.1.2 Cysteine proteases ......................................................................................... 9
2.1.1.3 Aspartic proteases ........................................................................................ 10
2.1.1.4 Metallo proteases ......................................................................................... 11
2.1.2 Some unique properties of digestive proteases from marine fish ....................... .12
2.1.3 Applications of digestive proteases from marine fish .......................................... 12
2.2 Trypsin of marine fishes .............................................................................................. 13
2.2.1 Active site and mechanism of catalysis ............................................................... 14
2.2.2 Substrates specificity ............................................................................................ 14
2.2.3 General properties of trypsin ................................................................................ 17
2.2.4 Purification of trypsin .......................................................................................... 21
2.2.5 Assay methods for trypsin activity.................................................................... .22
2.2.6 Applications oftrypsins from marine fishes .................................................... .23
2.2.7 Examples of previous research studies on trypsin from fish species .................. .25
CHAPTER III
PURIFICATION AND CHARACTERIZATION OF TRYPSIN
FROM THE PYLORIC CECA OF HOKI (Macruronus novaezealandiae) .............. 31
v
3.1 Abstract ........................................................................................................................ 32
3.2 Introduction ................................................................................................................. 32
3.3 Materials and methods ................................................................................................. 34
3.3.1 Biological specimen ............................................................................................ 34
3.3.2 Materials and instruments .................................................................................... 35
3.3.3 Extraction and purification oftrypsin ................................................................. 36
3.3.3.1 Extraction oftrypsin .................................................................................. 36
3.3.3.2 Purification oftrypsin ................................................................................ 36
3.3.4 Protein determination .......................................................................................... 38
3.3.5 Enzyme activity assay ......................................................................................... 38
3.3.5.1 Amidase activity ........................................................................................ 38
3.3.5.2 Esterase activity ....................................... ~ ....................... ,........................ 39
3.3.6 pH optimum and stability .................................................................................... 40
3.3.7 Temperature optimum and thermostability ........................................................ .40
3.3.8 Kinetics studies ................................................................................................... 41
3.3.9 Enzyme inhibition studies .................................................................................. .42
3.3.10 SDS polyacrylamide gel electrophoresis (SDS-PAGE) .................................... 43
3.3.10.1 Casting of gels ......................................................................................... 43
3.3.10.2 Sample preparation and SDS polyacrylamide gel electrophoresis ......... 44
3.3.10.3 Molecular weight determination ............................................................ .44
3.3.11 MALDI -TOF mass spectrometry ...................................................................... 45
3.3.12 Isoelectric focusing ............................................................................................ 45
3.3.12.1 Running conditions for IEF .................................................................... .46
3.3.12.2 Coomassie Blue staining ofPhastGel IEF media ................................... .47
3.3.12.3 Silver staining of PhastGel IEF media .................................................. 47
3.3.13 N-terminal amino acid sequence analysis ........................................................ .47
3.4 Results and discussions ............................................................................................... 48
3.4.1 Purification of hoki trypsin ................................................................................ .48
3.4.1.1 Recovery oftrypsin (affinity) fraction ..................................................... .48
3.4.1.2 Elution profile of the trypsin fraction on SBTI- Sepharose media .......... .48
3.4.1.3 Storage stability ofhoki trypsin fraction .................................................. .49
VI
3.4.2 pH optimum and stability ................................................................................... 50
3.4.3 Temperature optimum and thermostability ......................................................... 50
3.4.4 Kinetics studies ................................................................................................... 51
3.4.5 Enzyme inhibition studies ................................................................................... 52
3.4.6 Molecular weight determination ......................................................................... 52
3.4.6.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE) of hoki trypsin ... 52
3.4.6.2 MALDI-TOF mass spectrometry of hoki trypsin ..................................... 53
3.4.7 Isoelectric focusing ofhoki trypsin ..................................................................... 54
3.4.8 N-terminal amino acid sequence analysis ........................................................... 54
3.5 Conclusion .................................................................................................................... 55
CHAPTER IV GENERAL CONCLUSIONS AND RECOMMENDATIONS ........ 86
CHAPTER V REFERENCES ........................................................................................ 89
VII
LIST OF TABLES
Table 3.1
Formulations of SDS-PAGE resolving gel.. .............................................. 57
Table 3.2
Formulations of SDS-PAGE stacking gel ................................................. 58
Table 3.3
Solution for running e1ectrophoresis and staining gel .............................. 59
Table 3.4
Program for running IEF ........................................................................... 60
Table 3.5
Coomassie Brilliant Blue staining method for PhastGel IEF media ........ 61
Table 3.6
Coomassie Brilliant Blue staining solution for PhastGel IEF media ....... 62
Table 3.7
Silver staining method for PhastGel IEF media ........................................ 63
Table 3.8
Silver staining solutions for PhastGel IEF media ..................................... 64
Table 3.9
Purification of trypsin from the pyloric ceca of hoki .............................. 65
Table 3.10
Kinetic properties ofhoki trypsin in the hydrolysis
of BAPNA and TAME ............................................................................. 66
Table 3.11
Comparison of amidase and esterase activities
of hoki and bovine trypsins ..................................................................... 67
Table 3.12
The effect of inhibitors on the activity of hoki trypsin
using BAPNA as substrate at 25°C ......................................................... 68
Table 3.13
The N-terminal amino acid sequence ofhoki trypsin
compared to trypsins from other species ................................................. 69
VIII
LIST OF FIGURES
Figure 2.1
The hydrolysis reaction: enzymatic cleavage of a peptide bond ................. 5
Figure 2.2
A catalytic mechanism ofproteolysis: E (enzyme), S (substrate),
P and P' (resulting peptide), k (reaction velocity constant) .......................... 5
Figure 2.3
Schematic of the reaction mechanism in serine proteases.
Nucleophilic attack of serine on the peptide substrate
bound in the substrate binding cleft forms an acyl-enzyme
intermediate that is then hydrolyzed by solvent water ................................ 7
Figure 2.4
Substrates specificity of trypsin ................................................................. 15
Figure 3.1
Migration of each protein on SDS geL ..................................................... 70
Figure 3.2
PhastSystem (1) Separation compartment, (2) separation
and control unit, (3) development unit, (4) development chamber. .......... 71
Figure 3.3
Calibration curve for protein content determination using
bovine serum albumin (BSA) as standard. Aliquots of stock
BSA concentration of 1 mg/mL were used to prepare
the standard curve ...................................................................................... 72
Figure 3.4
Affinity chromatography on SBTI-Sepharose 4B.
Acetone fraction was applied to the affinity column equilibrated
in 0.05 M Tris-HCI containing 0.5 M NaCI and
0.02 M CaCb'2H2 0, pH 7.8. After washing with the same
equilibration buffer (fractions 10- 60), the adsorbed material
was eluted with 5 mM HCI (fractions 61-90). The effluent
was promptly neutralized with equilibration buffer (pH 7.8) .................... 73
Figure 3.5
Storage stability of trypsin fraction at 4°C ............................................... 74
Figure 3.6
Profile of change in hoki trypsin amidase activity with various
pH from 2 to 10 at 25°C. Buffer solutions are described in the
Materials and Methods section. Percentage of enzyme activity
was estimated based on the highest activity detected in this assay
as 100% ...................................................................................................... 75
IX
Figure 3.7
pH stability ofhoki trypsin after 30 min of incubation in various
buffers pH varying from 2 to Il at 25°C. Percentage of enzyme
activity was estimated based on the highest activity detected in
this assay as 100% ..................................................................................... 76
Figure 3.8
Effect oftemperature on the BAPNA activity ofhoki trypsin.
Activity was evaluated using 1 mM BAPNA as substrate at
pH 8.2 and changing temperature from 20 to 80°C. Percentage
of enzyme activity was estimated based on the highest activity
detected in this assay as 100% .................................................................. 77
Figure 3.9
Thermostability of hoki trypsin. Residual activity at pH 8
after incubation of enzyme extract with 1 mM BAPNA for
60 min at temperatures from 30to 70°C ...................................................... 78
Figure 3.10
Lineweave-Burk plots for trypsin kinetics ................................................ 79
Figure 3.11
SDS-PAGE ofprotein fractions obtained during purification
oftrypsin from the pyloric ceca ofhoki. Lane St., standard proteins;
Lane S-l, crude homogenate proteins; Lane S-2, Brij 35 soluble fraction;
Lane S-3, ammonium sulfate precipitation fraction; Lane S-4, acetone
precipitation fraction; Lane S-5, SBTI-affinity fraction ............................ 80
Figure 3.12
SDS-PAGE ofpurified hoki trypsin. Lane 1: standard proteins;
Lane 2: purified hoki trypsin ...................................................................... 81
Figure 3.13
Standard curve for molecular weight determination by SDS-PAGE ......... 82
Figure 3.14
Positive mode MALDI-TOF mass spectrum ofpurified hoki
trypsin. Molecular weight ofhoki trypsin is 23,791 Da............................ 83
Figure 3.15
Isoelectric focusing ofhoki trypsin in PhastGel IEF 3-9.
Lane A: pl Calibration Kit 3-10; Lane B: Purified hoki trypsin sample .... 84
Figure 3.16
Determination of pH gradient profile using broad pl calibration
kit on PhastGel IEF 3-9 ............................................................................. 85
x
LIST OF ABBREVIATIONS
A 280
Absorbance at 280 nm
APS
Ammonium persulfate
BAPNA
N-a-benzoyl-DL-arginine-p-nitroanilide
BSA
Bovine serum albumin
BT
Bovine trypsin
CAPS
3-cyclohexy-Iamino-l-propanesulfonic acid
Cys, C
Cysteine
DMSO
Dimethyl sulfoxide
EDTA
Ethylenediamine tetraacetic acid
Glu, E
Glutamate
HT
Hoki trypsin
IEF
lsoelectric focusing
Substrate turnover number
Physiological efficiency
Michaelis-Menton constant
MALDI-TOF MS
Matrix-Assisted
Laser
Desorption/lonization
Flight Mass Spectrometry
ME
2-mercaptoethanol
PAA
Polyacrylamide
pl
lsoelectric points
PMSF
Phenyl methyl sulfonyl fluoride
Pro, P
Proline
PVDF
Polyvinylidenedifluoride
Rf
Relative mobility
SA
Sinapinic acid
SBTI
Soybean trypsin inhibitor
SDS-PAGE
Sodium dodecyl sulphate - polyacrylamide gel
electrophoresis
TAME
Tosyl arginine methyl ester
XI
Time-of-
TCA
Trichloroacetic acid
TEMED
N.N.N' .N' -tetramethyl ethylene diamine
TFA
Trifluoroacetic acid
Thr, T
Threonine
TIU
Trypsin inhibitor units
TLCK
Tosyllysyl chloromethylketone
TPCK
Tosyl-l-phenylalanine chloromethyl-ketone
U
Units of enzyme activity
V max
Maximum velocity
XII
CHAPTERI
GENERAL INTRODUCTION
Digestion is a pro cess that an organism utilizes chemical and mechanical processes to
break down foods into substances that are useful for the maintenance of life. Animal
digestion is facilitated by enzymes which degrade molecules into metabolically useful
forms. The proteases (also called proteinases) are a c1ass of digestive enzymes that
degrade proteins through hydrolysis of peptide bonds and there are two basic types, the
endopeptidases and the exopeptidases. Exopeptidases function by c1eaving shorter
segments Iocated near the end of a peptide chain, into free amino acids, dipeptides, and
tripeptides. By contrast, endopeptidase attacks bonds located deep within the substrate
protein, thus transforming large polypeptide chains into shorter peptide segments.
Examples of common endopeptidases inc1ude trypsin, chymotrypsin and elastase. Trypsin
is a member of a large family of serine proteases. Many mammalian trypsins have been
reiativeiy well studied compared with fish trypsins. The amino acid composition and
primary sequence of trypsinogens and trypsins from several species are known, and the
three-dimensional structure has been determined for bovine trypsin.
Compared with mammalian enzymes, which have been well studied and used in food
processing and food production, studies of enzymes from marine fishes are still in its
infancy. Moreover, industrial scale recovery of marine enzymes is still under
experimental
stage~
The aquatic environment contains a wide variety of genetic material
and, hence represents an enormous potential for discovering different enzymes (Raa,
1990). Serving as important food source, fish and shellfish comprise the large st number
of species and the widest biological diversity. However, processing discards from these
animaIs accounts for as much as 70-85% of the total weight of the catch and these have
been generally dumped in-land or hauled into the ocean (Shahidi, 1994). Approximately
30% oftotallandings may be considered as underutilized, unconventional, or unexploited
(Venugopal and Shahidi, 1995). For this reason, there is great potential for the recovery
and use of digestive proteases from fish processing wastes. More thorough utilization of
whole fish is important not only for meat production but also for utilizing the offal from
1
which chemicals can be extracted (Wilke et al. 1986). Hence, the management of fishery
resources and new product development using underutilized species, processing discards
is very important in the world's fishing industry (Shahidi, 1994; Simpson and Haard,
1987a). Enzyme technology is being applied in the fish processing industry as fish
processing aid. Enzymes are aiso produced from waste material from the fishing industry
itself. A number of laboratories have developed commercial processes for isolating
enzymes from fish processing offal (Haard, 1992). In recent years, recovery and
characterization of enzymes from marine fish have been carried out and these have led to
the emergence of sorne interesting new applications of these enzymes in food processing.
Furthermore, extraction of enzymes from fish and shellfish processing wastes and their
utilization in the food industry may contribute significantly to reducing local pollution
problem in many places (Raa, 1997). Within the seafood industry in south-eastem
Australia alone, it is estimated that more than 20,000 tons of fish product waste is
produced annually. Sorne ofthis fish waste is rendered, but most is dumped to landfill at a
cost of up to $150/t (Knuckey, 2004). Therefore, recoveringfish enzymes is still a big
challenge and opportunity for utilization of seafood processing wastes.
New Zealand Hoki (Macruronus novaezelandiae) is a member of the Hake family
(Merlucciidae). It is New Zealand' s most important commercial fish species.
Approximately US$250 million of hoki is exported to Europe and the USA each year
from New Zealand. The total harvest is about 100,000 tons per annum. Mature hoki has
1.3 meters maximum length, average from 0.6 to1.0 meters in length. Hoki feed on small
crustacea, such as shrimps, small fish and squid, and inhabit the cold, c1ear waters
surrounding New Zealand and mainly in the middle water depths at 400-700 meters. The
hoki fishery in New Zealand produces large quantities of waste for every year. However,
this abundant material is underutilized, but is a rich source of useful enzymes such as
trypsin that may be recovered and applied for commercial use. So far, there is lack of
basic information about the biochemical characteristics of trypsin from hoki. Hoki is a
cold adapted marine fish, so the trypsin from hoki is expected to have sorne unique
properties for both basic research and industrial applications.
2
The main objectives of this project were to extract, purify and characterize trypsin from
the pyloric ceca of hoki. The kinetic properties, temperature, pH and inhibition effects on
the activity of the purified trypsin were verified. The molecular weight, isoelectric point
and N-terminaI amino acid sequence ofhoki trypsin were subsequently determined.
3
CHAPTERII
LITERATURE REVIEW
2.1 Digestive proteases from marine fishes
The hydrolysis of peptide bonds catalyzed by proteases is a common reaction in nature.
Proteases play an essential role in the growth and survival of all living organisms. In
terms of current food industry and other industrial applications, proteases are by far the
most important and most widely used group of enzymes (Chaplin et al., 1990; Whitaker,
1994). Like the proteases from plants, animals, and microorganisms, digestive proteases
from marine animaIs are hydrolytic in their action, and catalyze the cleavage of peptide
bonds. The digestive proteolytic enzymes from aquatic organisms that have been most
commonly studied include pep sin, gastricsin, trypsin, chymotrypsin and elastase. These
fish digestive enzymes can be recovered as by-products from fish processing wastes
(Haard et al., 1982; Simpson and Haard, 1987a; Stefansson and Steingrimsdottir, 1990;
AImas, 1990; Raa, 1990; Haard and Simpson, 1994). Salmon pepsin was the first fish
protease to be crystallized (Norris and Elam, 1940). Since then, recovery and
characterization of digestive proteases from fish species have been taken place and given
more and more emphasis.
2.1.1 Mechanistic classification
Proteases are classified according to their source (as animal, plant or microbial protease),
their catalytic action (as endopeptidase or exopeptidase), and the nature of the catalytic
site (as serine, cysteine, aspartic and metallo proteases). They are characterized by
common names and trade names, typical pH range, and preferential specificity.
Proteases that hydrolyze peptide bonds can be grouped into two subclasses: exopeptidases
and endopeptidases, depending on where the reaction takes place in the polypeptide
substrate. Exopeptidases cleave peptide bonds at the amine or carboxyl ends of the
polypeptide chain, whereas endopeptidases cleave internal peptide bonds. Based on the
nature of their catalytic site, proteases may be further classified into four groups: serine,
4
cysteine, aspartic, and metallo (Barrett, 1994). The name of each class is derived from a
distinct catalytic group involved in the reaction. For example, serine proteases possess a
Ser residue in the active site; cysteine proteases have a Cys residue instead; aspartic
proteases depend on an Asp residue for their catalytic activity; metalloproteases use a
metal ion (normally Zn2+) in their catalytic mechanism. The active sites of serine and
cysteine proteases use serine hydroxyl and cysteine thiol side groups, respectively, as the
attacking nucleophiles during catalysis. Reaction mechanism entails the formation of a
covalent intermediate between the nucleophile and the carbonyl carbon atom of the
scissile peptide bond, unlike the mechanism of hydrolysis for aspartic and metallo
proteases which does not involve a covalent intermediate because the nucleophile for both
of these enzymes is water molecule.
Because of the abundance and importance of protease, their reaction mechanisms have
been studied more extensively than any other class of enzyme. Proteases catalyze the
hydrolytic degradation of the peptide chain with the participation of water molecules as
co-reactant (Figure 2.1). When a protease acts on a protein substrate (see Figure 2.1), a
catalytic reaction actually consists of three consecutive reactions. This reaction
mechanism is simplified by the scheme shown in Figure 2.2 (Olsen, 1999).
protease
~
...
+
c
",caa-
+
1
RI
Figure 2.1 The hydrolysis reaction: Enzymatic cleavage of a peptide bond
E+S
42
--...
EP + H-P'
k+3
+H20
.. E + P-OH + H-P'
Figure 2.2 A catalytic mechanism ofproteolysis: E (enzyme), S (substrate), P and pl
(resulting peptide), k (reaction velocity constant)
5
The protease reaction is generally considered to have three-step kinetic mechanism (show
in Figure 2.2):
1). Formation of the Michaelis complex between the original peptide chain (the substrate)
and the enzyme (as ES, K= k-I/~I);
2). Cleavage of the peptide bond to titrate one of the two resulting peptides
(~2);
3). A nucleophilic attack on the remains of the complex to split off the other peptide and
to reconstitute the free enzyme (~3).
The Michaelis-Menten parameter can be expressed as: Km =k3 (k-I + k2) / kI (k2+ k3)
If k. I» k2, Km = k-Ik3 / kI (k2+ k3) = K k3 / (k2+ k3)
If k3» k2, Km = (k. I + k2) / kI
Before describing each protease (serine, cysteine, aspartic, and metallo) in detail, it
should be known that the reaction pathways in all four protease classes share common
elements:
• Binding of the substrate polypeptide into a channel or pocket on the enzyme surface
• Nucleophilic attack on the carbonyl carbon of the scissile peptide bond by either oxygen
or sulfur
• General base-assisted catalysis for removal of a proton from the nuc1eophile
• Stabilization of the tetrahedral transition state intermediate formed at the carbonyl
carbon of the scissile peptide bond
• General acid-assisted catalysis for transfer of a proton to the amine leaving group
2.i.1.! Serine proteases
The name serine protease refers to the nucleophilic serine residue located in the enzyme
active site. On the basis oftheir three-dimensional structures, Rawlings and Barrett (1994)
have grouped serine proteases into six clans (more than 20 families). Most of them have
in common a "catalytic triad" composed of serine (a nucleophile), aspartate (an
electrophile) and histidine (a base). In addition to the catalytic serine residue, aspartate
and histidine residues also play essential roles to form a catalytic triad together. Serine
proteases with the classic Asp-His-Ser triad are the large st class of proteases. The
catalytic serine and histidine residues in this enzyme have been identified by covalent
6
modification with active-site-specific chemical reagents (Rennex et al., 1991; Stone et al.,
1991).
The secondary and tertiary structures of the proteins vary considerably from one family to
another, yet in all families the active site serine, histidine, and aspartate are conserved and
a common mechanism of catalysis is used. AIl these enzymes catalyze the hydrolysis of
ester and peptide bonds through the same acyl transfer mechanism. A schematic of the
reaction mechanism for serine proteases is illustrated in Figure 2.3(Graycar, 1999).
Enzyme-substrate Mechaels complex
TetrahedraI transition state 1
His
His
Asp\
/0
~ e
(---
.1=1
Ser
)
0- -H-N~~-H-O
/9
~:e
Asp\
,N
O,H
<.
.~
0- -H-N@
"Ç-N-H..
..
\.-.. '<.fH-N
RI-N-C,
1
H
Ser
)
0(8"
H'
N
~\ /0- -H-
RI-N-C
l
'
H
R2
R2
N
l
Acylenzyme intermediate-Pl product complex
Acylenzyme intermediate-H20 complex
His
Asp\
/9
~\e
0-
(-
.1=1
-H-N
~N
,H
RI-N,
H
P1leaving group
Enzyme-P2 productcomplex
Tetrahedral transition state II
His
Asp
--~.. .
9
(---
L<e.~
Ser)
0-- -H-N@
"Ç-N---H-O
<r?
,N
H
H-N
R2- C- OH
P2 leaving group
Figure 2.3 Schematic of the reaction mechanism in serine proteases. Nucleophilic attack
of serine on the peptide substrate bound in the substrate binding c1eft forms an acylenzyme intermediate that is then hydrolyzed by solvent water
7
As shown in Figure 2.3, First, the nucleophilicity of serine -OH group was enhanced by
the imidazole group of histidine, so -OH group can attack the peptide bonds to fonn an
ester between the oxygen atom of serine and the acyl portion of the substrate (ES
complex), which produces a tetrahedral intennediate. After an electron rearrangement, a
molecule of amine is released and enzyme is acylated. In the next step, water molecule is
activated also by the imidazole group of histidine, and then it attacks the ester on serine.
After another rearrangement cycle, enzyme is recovered and a molecule of acid is
released. From a whole view of this process, protease helps water to break peptide bond.
The serine proteases from marine fish that were commonly studied included trypsin,
chymotrypsin, and elastase. A particular description of trypsin from marine fish will be
reviewed and discussed in later sections (section 2.2-2.6). A brief introduction about
chymotrypsin and elastase from marine fish will be given as follow.
Chymotrypsin
Chymotrypsin is a member of a large family of serine proteases functioning as a digestive
enzyme. Its inactive precursor, chymotrypsinogen, is synthesized in pancreatic tissue as a
single polypeptide chain and is converted to active enzyme by tryptic cleavage.
Compared to trypsin, less work has been done on chymotrypsins from fish species. In
1973, Ovemell first reported chymotrypsin extracted from the pyloric ceca of the Atlantic
cod (De-Vecchi and Coppes, 1996). After that, purification and characterization of
chymotrypsin from marine fish species such as spiny dogfish (Ramakrishan et al., 1987)
d
and Atlantic cod (Asgeirsson and Bjamason, 1991) l1ave been studled. Kristjânsson and
Nielsen (1992) purified and characterized two chymotrypsins (1 and II) from the pyloric
caeca of rainbow trout (Oncorhynchus mykiss). The approximate molecular weights of
rainbow trout chymotrypsin 1 and II were 28,200 (+ 1200) Da and 28,800 (+900) Da,
respectively, as determined by SDS-PAGE and their isoelectric points were about 5.0.
The cDNA encoding Atlantic cod (Gadus morhua) chymotrypsinogen B has been isolated
and sequenced by Spilliaert and Gudmundsd6ttir (2000). The calculated molecular weight
of this polypeptide was 26,500 kDa. In common with most fish serine proteases, cod
chymotrypsinogen B contained a high number of methionine residues. The presence of a
8
threonine instead of a serine residue at position 189 was a novel characteristic of this
enzyme.
Elastase
Elastase is a serine protease that specifically hydrolyzes elastin under alkaline conditions.
Elastase-like enzyme preparations were separated from several fresh water and marine
fish species by CM-cellulose column chromatography (Yoshinaka et al., 1985).
Pancreatic elastases were found to be present in all these examined fishes: bluefin tuna
(Thunnus thynnus), eel (Anguilla japonica), sea bass (Lateolabrax japonicus), yellowtail
(Seriola quinqueradiata), rainbow trout (Salmo gairdnerii), catfish (Parasilurus asotus),
carp (Cyprinus carpio), angler (Lophiomus setigerus), gummy shark (Mustelus manazo)
and red stingray (Dasyatis akajei). It seems therefore that this type of enzyme has an
important function in digestion in a wide range of fish species. Furthermore, an intestinal
elastase has been recovered from Atlantic cod (Gadus morhua) (Asgeirsson and
Bjarnason, 1993). Berglund et al. (1998) purified and characterized a pancreatic elastase
from North Atlantic salmon (Salmo salar). The molecular weight and isoelectric point of
this enzyme were estimated to be 27 kDa and over 9.3, respectively. The pH optimum
was between 8.0 and 9.5, and the enzyme was unstable at pH values below 4. Recently,
metal binding and conformational stability characteristics ofpsychrophilic elastase (ACE)
from Atlantic cod (Gadus morhua) has been investigated (Jayaraman et al., 2006).
Chelation to Ca2+ was found to be important for maintaining the biologically active
conformation and for the thermal stability of this Atlantic cod elastase.
2.1.1.2 Cysteine proteases
The functional roles of cysteine proteases are quite broad, and members of this enzyme
class are found in animaIs, plants, bacteria, and viruses. Like serine proteases, hydrolysis
of amide and ester substrates by cysteine proteases follows an acyl transfer reaction
pathway, but the attacking nucleophile is the thiol group of an active site cysteine residue
instead of a serine hydroxyl group. The plant enzyme papain is the most extensively
studied cysteine protease. Digestive cysteine proteases have been found in marine animals.
An example is Cathepsin B.
9
Cathepsin B has been isolated and purified from the digestive gland of the mussel (Perna
perna) by ammonium sulphate fractionation, molecular exclusion chromatography and
ion exchange chromatography on CM-Sephadex (Zeef and Dennison, 1988). The enzyme
had a molecular weight of 21 kDa. It was inhibited by iodoacetamide, N-ethylmaleimide,
divalent mercury ions and TPCK at high concentration, and was therefore apparently a
thiol protease. Another type of Cathepsin (Cathepsin L) was purified and characterized
from arrowtooth flounder (Atheresthes stomias) muscle (Visessanguan et al., 2003). This
enzyme displayed optimal activity at pH 5.0-5.5 and 60°C using N-carbobenzoxyphenylalanine-arginine 7-amido- 4-methylcoumarin (Z-Phe-Arg-NMec) as substrate.
2.1.1.3 Aspartic proteases
The aspartic proteases are named for the essential catalytic role of two aspartic acid
residues. The three dimensional structureS for aspartic proteases of mammalian, fungal,
and viral origin are quite comparable to each other. The catalytic site of the aspartic
protease consists oftwo aspartic acid side-chains (residues 32 and 215). These two sidechains cause the cleavage of the substrate peptide bond. The pH activity profile of
aspartic proteases is a bell-shaped curve, with activity dependent on protonation and
ionization of the carboxylate group of aspartates 32 and 215, respectively. Unlike serine
and cysteine proteases, proteolytic cleavage of peptide bonds by aspartic proteases does
not proceed via a covalent acyl intermediate between the aspartic protease and the
substrate. A water molecule bound at the active site between the carboxylates of
aspartates 32 and 215 is believed to be the attacking nucleophile (Graycar, 1999).
Pepsin, chymosin and gastricsin are three common aspartic proteases that have been
isolated and characterized from marine fish. These enzymes are active at acid pH and are
unstable under alkaline conditions. Pepsin, chymosin and gastricsin from different marine
fish species were thoroughly described in a review by Simpson (2000). A gastricsin-like
protease was purified from gastric mucosa of Atlantic cod by a single step purification
scheme on ion-exchange of Amberlite CG-50 (Amiza and Owusu Apenten, 2002). This
purification scheme has not been reported previously. The purification was very efficient
as the recovery was 205% and the purification factor was 1796-fold. The enzyme
10
preparation was homogeneous as observed by SDS-PAGE and isoelectric focusing, which
has an estimated molecular weight of 34 kDa and the pl of 4.4. Recendy, three forms of
pepsin A and a single form of gastricsin from the gastric mucosa of Antarctic rock cod
(Trematomus bernacchii) have cloned and sequenced (Carginale et al., 2004). The results
suggest that the Antarctic rock cod adopted two different strategies to accomplish
efficient protein digestion at low temperature. One mechanism is the gene duplication that
increases enzyme production to compensate for the reduced kinetic efficiency; the other is
the expression of a new enzyme provided with features typical of cold- adapted enzymes.
2.1.1.4 Metalloprotease
The catalytic activity of metalloprotease is dependent on the presence of one or two metal
ions, most commonly zinc, bound at the active site. According to the number of metal
ions,
metalloprotease
can be
classified two-metal
ion and
single-rnetal
ion
metalloprotease. Significant differences in amino acid sequence and tertiary structure
were observed both for single- and two-metal ion metalloproteases isolated frorn bacteria,
fungi and higher organisms. Crystal structures have been determined for three different
aminopeptidases having two-metal ions at their active sites (Strater and Lipscomb, 1995;
Roderick and Mathews, 1993; Chevrier et al., 1994). Like aspartic proteases, single-metal
ion metalloprotase does not form covalent intermediates during catalysis, because the
metal ion (Zn2+) provides a strong electrophilic pull to assist attacking by bounding a
water molecule. Metalloproteases do not seem to be common in marine fish. Pancreatic
metalloproteases were demonstrated only in bluefin tuna and yellowtail among ten
exanlined fishspedes (Yoshiriak.aet al., 1985). Some metalloproteases ha.ve beeri Îsolated
and characterized from fish muscle tissues but not from digestive glands. For example,
two metalloproteases (myosinase l and myosinase II) were purified and characterized
from squid mande muscle (Okamoto et al., 1993). Both myosinase l and myosinase II
gave a single band on SDS-PAGE with a molecular weight of 16 and 20 kDa, respectively.
They could be reactivated with some divalent cations, myosinase l was especially
reactivated with C02+ and II especially with Zn2+. The optimum pH ofboth activities was
7.0; the optimum temperature for both was 40°C.
11
2.1.2 Some unique properties of digestive proteases from marine fish
Sorne of these unique properties are a low Arrhenius activation energy, relatively lower
free energy of activation (~G*), a high apparent Michaelis-Menten constant, cold stability,
a low temperature optimum, a low thermostability, and a high pH optimum/pH stability
(Low et al., 1973; Squires et al., 1986; Simpson and Haard, 1987a; Guizani et al., 1991).
The structural and functional relationships of enzyme molecules suggest that proteases
from cold-adapted marine animals have more flexible structures than their counterparts
from land animaIs (Hultin, 1978; Simpson and Haard, 1987a). Marine digestive proteases
from the stomachless bonefish, cunner, mullet, and crayfish digested native globular
proteins more efficiently than homologous proteases from higher vertebrates (Pfleiderer
et al., 1967; Guizani et al., 1991; Mihalyi, 1978; Simpson et al., 1989a; Jany, 1976).
2.1.3 Applications of digestive proteases from marine fish
Proteases are by far the most studied enzymes for industrial bioprocessing. Almost half of
all industrial enzymes are proteases. The major uses of the se enzymes are divided among
many applications, all based on hydrolysis as the catalytic concept. They are mostly used
in the detergent, leather and food industries.
The food industry uses proteases as processing aids for many products inc1uding baked
goods, beer and wine, cereals, milk and fish products, and legumes plus for production of
protein hydrolysates and flavor extracts (Stefansson, 1988; Wray, 1988; Haard, 1990;
Wasserman, 1990; Simpson and Haard, 1987b; Haard and Simpson, 1994).Commercial
supplies~ of food p:rocessing proteases are presently mostly derived frOIn.variou~ pla1lts,
animal and microbial sources. Compared with these enzymes used in food processing, use
of proteases from marine fished is still in shortage. However, most proteases studied from
cold-adapted marine organisms are extracellular digestive enzymes (Haard et al., 1982),
they were applied in the food industry because their temperature and other characteristics
differ from homologous proteases from warm-blooded animals (De Vecchi et al., 1996).
They are more active catalysts at relatively low temperature compared with similar
enzymes from mammals, thermophylic organisms and plants (Simpson and Haard,
1987b). As all known, low-temperature processing could provide various benefits, such as
12
10w thennal costs, protection of substrates or products from thennal degradation and/or
denaturation, and minimization of unwanted side reactions (Hultin, 1978; Haard and
Simpson, 1994). Certain fish enzymes are excellent catalysts at 10w temperature, which is
advantageous in some food processing operations (Simpson and Haard, 1984b, 1987b;
Brewer et al., 1984). For example, cold-adopted pepsins are very effective for cold
renneting milk clotting because of the high molecular activity at low reaction
temperatures (Brewer et al., 1984). Moreover, fish digestive enzymes possess other
unique properties, such as pH stability, optimum temperature and thermal instability.
These unique properties might be make them better suited for food processing aids. By far,
the uses of marine fish proteases were developed not only in food areas, such as
fermentation and curing of fish, selective tissue degradation, production of hydrolyzed
products and extraction of pigments and so on, but also in non-food areas like enzymatic
treatment of waste water. The marine fish enzymes have been applied as processing aids
in the food and feed industries were thoroughly reviewed by Diaz-Lopez and GarciaCarreno (2000).
2.2 Trypsins of marine fishes
Trypsin is one of the most well-studied enzymes (Walsh and Wilcox, 1970). Many
trypsin and trypsinogen structures are known by X-ray crystallography (Sweet et al., 1974;
Kossiakoff et al., 1977). Many trypsins' amino acid or nuc1eic acid sequences have been
determined. In 1971, Kiel gave a definition of marine fish trypsin: "Trypsin (EC
3.4.21.4.), from the digestive glands of marine fish, is an alkaline serine protease which
catalyzes the hydrolysis of ester and peptide bonds involving the carboxyl group of
arginine and lysine, and in which a serine and a histidine residue participates in the
mechanism of catalysis" (Kiel, 1971). Over the past few decades, trypsin and trypsin-like
enzymes have been isolated and identified in a wide array of cold water as weIl as warm
water fish species. The comprehensive information about trypsin from different marine
fish species, including mechanism of catalysis, substrates specificity, general
physiochemical properties, methods of purification and assay of trypsin activity and some
practical applications will be described in later subsections. Finally, some examples of
previous research studies on trypsin from fish species will be reviewed.
13
2.2.1 Active site and mechanism of catalysis
Trypsin is a member of serine protease. No matter where trypsins were isolated from,
they all have the same catalytic function and same mechanism as the common mechanism
of serine protease described in section 2.1.1.1 and Figure 2.3. Trypsins have a triad of
amino acids (serine, histidine and aspartate) at the active sites. A serine residue acts as the
primary nucleophile for attack of the peptide bond, and the nucleophilicity of this triad is
enhanced by specific interactions of serine residue with a histidine side chain (a general
acid/base catalyst) and an aspartate side chain. The catalytic importance of the active site
serine and histidine residues has been demonstrated by site-directed mutagenesis studies,
in which replacement of either the serine or the histidine or both reduced the rate of
reaction by the enzyme by as much as 106 fold (Carter and Wells, 1988).
Trypsin catalyzes the following reaction
Ks
E+S~
ES
k2
k3
~
ES'+ Pl --7E+P2
+H20
Where ES is the enzyme-substrate complex, ES Ïs the acyl-enzyme intermediate, Pl is the
leaving group of the substrate, and P2 is the carboxylic acid (Bender and Kezdy, 1964).
The acylation step (k2) is a nucleophilic reaction dependent on two groups, one with a pKa
of 7.0 and the other with a pKa of 9.0 or 10.0. The acyl-enzyme intermediate has been
characterized as an ester of the substrate carboxyl and the hydroxyl of the serine residue
ln trypsiri.Its clecylation (k3 ) is again a nucleophilic reaction dependent on one group with
a pKa of 7 (Bender et al., 1964). Evidence for the acyl transfer mechanism was obtained
from numerous kinetic studies using activated ester substrates where deacylation is the
rate-determining step in the reaction such that accumulation and partitioning of the
intermediate could be monitored (Bender and Killheffer, 1973).
2.2.2 Substrates specificity
The substrate specificity of trypsin is directed toward the carboxyl side of lysyl and
arginyl residues in natural and synthetic substrates, thus exhibiting the most restricted
14
specificity of action of the known endopeptidases (Walsh, 1970). As shown in Figure 2.4,
trypsin has a negative charged pocket which can recognize positive charged residue of
substrate, such as arginine and lysine residue. The maximum velocity of the reaction lies
between pH 7.0 and 9.0. In this condition, the negative charged pocket can be fonned.
The length of the side chain containing the positive charge is apparently an important
detenninant ofthis specificity (Walsh, 1970). 80 arginine derivatives are commonly used
as substrates in activity study of trypsin, such as N-a.-benzoyl-DL-arginine-p-nitroanilide
(BAPNA) is an amidase substrate and tosyl arginine methyl ester (TAME) is a kind of
esterase substrate.
Negative
charged pocket
NH2+
0
r:-
o
Trypsin
H2N)lN~NÀ
H
Y
NH
r:-
Trypsin
+H3N~~À
H
yNH
Arginine residue
Lysine residue
asp-
Amidase substrate
Esterase substrate
NH
0
r
Trypsin
H2N)lN~O/
H
9.
NH
9
Tosyl arginine methyl ester (TAME)
Figure 2.4 8ubstrates specificity of trypsin
Both in synthetic substrates and in polypeptide substrates (Bergmann, 1942; Keil and
Keilova, 1964; Wang and. Carpenter, 1967; Keilova et al., 1969), trypsin prefers the
arginine side chain to the lysine side chain. In arginine or lysine vasopressin, which are
identical peptide except for the side chain of the only basic amino acid, the pH optimums
are different (8.0 and 7.2, respectively), but the arginine substrate is preferentially cleaved
throughout the whole pH range (Keilova et al., 1969).
15
There have been lots of studies in low molecular weight substrates of trypsin. Benoiton et
al. (1966) reported that if the e-amino group of the lysine side chain is monomethylated,
tryptic hydrolysis of the substrate can occur, since the positive charge still exists.
Substituents, such as €-acylated lysine which eliminated the positive charge of the €amino group, block trypsin catalysis. But the blocking of the e-amino group is not a
prerequisite of tryptic action since lysine ethyl ester is rapidly hydrolyzed (Werbin and
PaIm, 1951). The same reaction occurs for the arginine ester (Goldenberg and Goldenberg,
1950). Trypsin acts even on substrates in which a-amino group has been replaced by a
hydroxyl group (Snoke and Neurath, 1949). In low molecular weight substrates used for
routine assays, the a-amino group is usually substituted by a tosyl, a carbobenzyloxy or a
benzoyl group (Boyer, 1970).
The action of trypsin toward protein substrates can be redirected by selectively blocking
lysyl residues (Goldberger and Anfinsen, 1962) or arginyl residues (Toi et al., 1965) of
the substrate. Altematively, cystinyl or cysteinyl residues can be converted to the lysyl
homo log, such as S-(p-aminoethyl) cysteine which has the same length of side chain with
lysine. Thus, by choosing the appropriate modification procedures it is possible to use
trypsin to hydrolyze selectively lysyl, cysteinyl, or arginyl residues (Walsh, 1970).
The three-dimensional structure of native protein prevents the trypsin hydrolysis of the
basic residue. In a polypeptide chain with a random arrangement, if cysteine is an
adjacent segment, the rate of the cleavage of a peptide bond next to lysine or arginine is
reduceda lût. Moreover, the ratëof thedeavage dfopto zero if the basic residue is
followed by proline. In smaller peptides, the rate of the hydrolysis is affected by the
number of amino acid residues (Boyer, 1970).
Nowadays the genetic selection provides a powerful tool for study the elements of
substrate specificity. Evnin et al. (1990) investigated the substrate specificity of trypsin
by using a genetic selection. A negative charge at amino acid position 189 or 190 was
found to be essential for high-Ievel catalysis.
16
2.2.3 General properties of trypsin
Temperature optimum
The optimal temperature for trypsins reported to range from 30-60 oC. For example, the
optimal temperature reached for the three proteinases isolated from sardine pyloric caeca
for the hydrolysis of casein was 4SoC (Murakami and Noda, 1981). The two enzymes
isolated from capelin had apparent temperature optima of 42°C for amidase activity
(Hjelmeland and Raa, 1982). For Greenland cod trypsin, an optimum temperature below
40°C was found for the hydrolysis of BAPNA (amidase), tosyl arginine methyl ester
(TAME) (esterase) and casein (Simpson and Haard, 1984a). The trypsin fraction isolated
from cunner exhibited an optimum temperature of 4SoC for amidase activity (Simpson
and Haard, 1985). Guizani et al. (1991) reported that mullet trypsin exhibited an optimum
temperature of SSOC for amidase activity. Maximal activity of rainbow trout trypsin
(Kristjansson, 1991) against BAPNA was 60°C. This optimum temperature (60°C) is
similar to that reported for mullet (Mugi! spp) trypsin (Pavlisko et al., 1999), tambaqui
trypsin (Bezerra et al., 2001) and chinook salmon trypsin (Kurtovic et al., 1006)
hydrolysis of BAPNA. It is higher than the temperature optima for other fish trypsin that
have been reported, such as starfish (Asterina pectinifera) trypsin (SSOC; Kishimura and
Hayashi, 2002) and Monterey sardine trypsin (SSOC; Castillo-Yanez et al., 200S).
Thermal stability
The therrnostability of reported fish trypsins has been found to vary with species as well
as with incubation conditions. Thus, Greenland cod trypsin was unstable at a temperature
above 40°C (Simpson and Haard, 1984a, b), while the mullet trypsin was relatively
unstable to temperature above 6SoC and lost about 40% of its amidase activity after
heating for 30 min at 6SoC (Guizani et al., 1991). This is consistent with the fact that the
Greenland cod lives at temperatures of _2°C to 2°C, whereas the mullet live in warm water.
Starfish trypsin was stable below 40 oC, but its activity for hydrolysis TAME quickly
reduced above SO Oc (Ki shimura and Hayashi, 2002). Souza et al. (2006) reported that
trypsin obtained from spotted goatfish intestine remained fully activity while trypsin
extracted from spotted goatfish pyloric ceca lost 10% activity after incubation at 4SoC for
30 min.
17
pH optimum
The pH optima for most fish trypsins are alkaline. Murakami and Noda (1981) found an
optimum pH of 10.0-11.0 for the hydrolysis of case in and hemog1obin by trypsin from
sardine. The optimum pH for the amidase activity of the two tryptic enzymes isolated
from the pyloric caeca of the Arctic fish capelin was in the range of 8.0-9.0 (Hjelmeland
and Raa, 1982). Atlantic cod trypsin hydrolyzed N-a-benzoylarginine-p-nitroanilide
(BAPNA) at pH 7.5, and case in at pH 9.0-9.5, a more alkaline value than given by other
studies. Two distinct enzyme of cunner trypsin presented pH optima 8.5 and 7.0 for the
hydrolysis of BAPNA (Simpson and Haard, 1987b). The optimum pH for esterase activity
of Atlantic cod trypsin (Asgeirsson et al., 1989) and for the BAPNA hydrolysis of mu11et
trypsin (Guizani et al., 1991) was around pH 8.0. The optimum pH of salmon trypsin for
hydrolysis ofBAPNA was found to be 9.0 (Dimes et al., 1994), this optimum value was
similar to anchovy trypsin (Heu et al., 1995) and goatfish trypsin (Souza et al., 2006).
Proteases from a11 three tuna species had an optimal activity at pH 9.0 when case in was
used as a substrate (Klomklao et al., 2004). Castillo-Yanez et al. (2005) reported that
Monterey sardine (Sardinops sagax caerulea) trypsin had optimum pH of 8.0 on BAPNA.
pH stability
Fish trypsins differ from trypsin from mammals which are most stable at about pH 2.0.
Fish trypsins have relatively higher ratio of acidic to basic amino acid residues than those
of mammalian trypsins. This difference may explain why the fish trypsins are genera11y
stable under alkaline conditions, but unstable at acid pH values. Thus, most trypsins from
marine fish are highly unstable under acidic conditions and more stable at slightly
alkaline conditions stable at neutral to slightly alkaline conditions (Hjelmeland and Raa,
1982; Kristjansson, 1991; Kishimura and Hayashi, 2002; Klomklao et al., 2004; CastilloYanez et al., 2005). For example, Greenland cod trypsin is most stable at pH 7.5
(Simpson and Haard, 1984b) similar to mu11et trypsin which was stable in the pH range of
7.5-9.0. Cunner trypsin (Simpson and Haard, 1985) was stable from neutral to slightly
alkaline pH (6.5-8.5) and least stable at acid pH 4.0 similar to what was observed with
other fish. Asgeirsson et al. (1989) found that the trypsin l isolated from Atlantic cod was
18
quite stable in alkaline medium for esterase activity, but displayed a remarkable acid
instability. Both two isozymes of trypsin from Japanese anchovy (Engraulis japonica)
were stable between pH 6.0 and 11.0, but unstable below pH 5.0 and were stabilized by
calcium ion (Ki shimura et al., 2005). Kurtovic et al. (2006) reported that trypsin from
chinook salmon retained some activity over a broad pH range at 20°C for 30 min and was
stable from pH 4.0 to pH 10.0. The stability at relatively low pH is unusual compared
with most other marine fish trypsins which were found to be labile at low pH.
Kinetic properties
The catalytic kinetic properties property of an enzyme is determined by the affinity of the
enzyme toward the substrate (Km') and the turnover number (kcat). The catalytic efficiency
(kcaJ Km') can be increased by either increased kcat or decreased Km'.
Higher catalytic efficiency of trypsins from cold-adapted fish compared to mammalian
trypsins is caused by differences in both Km' and kcat. For example, Simpson and Haard
(1984c) found that Greenland cod trypsin had a higher turnover number (BAPNA or
TAME units per micromole trypsin) for the esterase and the amidase reaction than bovine
trypsin at temperatures of 5, 15,25, and 35°C. On the basis of the turnover number, cod
trypsin is a better catalyst than bovine trypsin at aIl measured temperatures. Mullet trypsin
(Guizani et al., 1991) showed greater affinity for TAME (Km'
than for BAPNA (Km'
=
=
0.19 mM) as a substrate
0.49 mM). The substrate turnover number was higher for the
mullet trypsin-BAPNA reaction than for the bovine trypsin-BAPNA reaction at 25°C.
Theca1:àl)ïtic effïêîency of rnùfIet trypsin was greater thanthai ofbovine trypsin at 25 6C.
However, bovine trypsin exhibited a greater affmity (lower Km' value) for the TAME
reaction at 25°C. Based on the turnover number, mullet trypsin is better suited for the
amidase reaction than bovine trypsin. Ahsan and Watabe (2001) reported that two
isoforms of anchovy trypsin (aT-I and aT-II) showed considerably higher catalytic
efficiencies (kcaJKm') than bovine trypsin as measured toward benzoyl-arginine pnitroanilide (BAPNA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT1 was 20 times and aT-II was 35 times more efficient than bovine trypsin against BAPNA,
respectively. The kinetic study for trypsin A and B from yellowfin tuna spleen for
19
hydrolysis ofTAME has been done by Klomklao et al. (2006). The Km' value oftrypsin B
was lower than trypsin A. It was suggested that trypsin B has higher affinity to TAME
compared with trypsin A. Trypsin A had a higher turnover number (kcat) value than
trypsin B. However, the catalytic efficiency value for trypsin B was higher than those of
trypsin A. This result suggested that trypsin B would be more efficient for hydrolysis of
ester.
Inhibition
The catalytic activity of trypsin can be influenced by several factors, such as temperature
and pH, molecular structural changes, chemical modifications and specifie interaction
with inhibitors (Boyer, 1970). There are two kinds of compounds forming specifie
complexes with the binding or active site of trypsin, therefore the combining of trypsin
with substrate is prevented. The other types of inhibitors are low molecular weight
compounds. These molecules are required to fit the specificity and stereochemistry of the
binding sites. p-Aminobenzamidine and benzamidine were found to be the most potent
low molecular weight competitive inhibitors of trypsin [ KI 8.2 x 10.6 and 1.8 x 10.5
(Mares-Guia et al., 1965)]. NaturaIly occurring polypeptide inhibitors are another type of
compounds which meet these structural requirements, such as soybean trypsin inhibitor
(SBTI).
Various serine protease inhibitors influence the activity of fish trypsin. Soybean trypsin
inhibitor is a competitive inhibitor of BAPNA hydrolysis by Atlantic cod trypsin
(OvemeÏl, 1973): Threetrypsin-likeeilzymes from thepyloric caeca of sardine foun.dby
Murakami and Noda (1981) were differently inhibited. The two enzymes isolated from
capelin were trypsin-like since both were completely inhibited by aprotinin, soybean
trypsin inhibitor (SBTI), leupeptin and tosyl lysyl chloromethylketone (TLCK) and
partially inhibited by phenylmethyl sulfonyl fluoride (PMSF). The purified enzyme from
Greenland cod (Simpson and Haard, 1984a) and the two enzymes from cunner (Simpson
and Haard, 1987a) exhibited inhibition by the serine protease inhibitors, PMSF, SBTI and
aprotinin. Similar results were obtained for Atlantic cod (Simpson et al., 1989a) as weIl as
for mullet enzymes (Guizani et al., 1991). Trypsin-like enzyme from tambaqui was
20
inhibited by sorne trypsin inhibitors, such as PMSF, benzamidine and TLCK (Bezerra et
al., 2001). The serine proteinase from the intestine of discus fish was inhibited by SBTI,
PMSF and TLCK while tosyl-I-phenylalanine chloromethyl-ketone (TPCK) and EDTA
showed partial inhibition (Chong et al., 2002). The Monterey sardine trypsin (CastilloYanez et al., 2005) was partially inhibited by the serine-protease inhibitor PMSF and fully
inhibited by SBTI and benzamidine, but was not inhibited by the metallo-protease
inactivator EDTA or the chymotrypsin inhibitor TPCK. The chinook salmon trypsin
(Kurtovic et al., 2006) exhibited inhibition by the general serine protease inhibitor PMSF
and also by the specifie trypsin inhibitors-SBTI and benzamidine. This result can be
conc1uded that the purified proteins are authentic trypsins.
2.2.4 Purification of trypsin
In order to obtain purified enzyme sample, several methods have been used to extract and
puri:fy trypsins from marine fishes. In principal, purification procedures always involve
with ammonium sulfate or cold acetone fraction steps, followed by chromatographie
methods. For example, Greenland cod trypsin and mullet trypsin were purified by
powdering the tissue in liquid nitrogen, making a homogenate of the powered tissue in
Tris-HCL buffer followed by ammonium sulfate fractionation, acetone precipitation and
affinity chromatography (Simpson and Haard, 1984c; Guizani et al., 1991). Ahsan and
Watabe (2001) purified two isoforms of anchovy trypsin (aT-I and aT-II) from the
visceral extracts by
(N~)2S04
fractionation (30%-70% saturation) followed by
benzamidine-Sepharose 6B affinity chromatography, gel filtration (Superdex 75 pg
coluIll) and. Ion exchange chromatographY. Crude trypsill. was p~ified from pyloric ceca
of the starfish (Asterina Pectinifera) by ammonium sulfate precipitation (40%-75%
saturation), then was applied in tum to a column of Sephacryl S-200 (3.9 x 44 cm),
diethylaminoethyl (DEAE)-cellulose column (2.2 x 18 cm), Sephadex G-50 column
(3.9 x 64 cm) and carboxymethyl (CM)-cellulose column (1.1 x 18 cm) (Ki shimura and
Hayashi, 2002). Trypsins, TR-S and TR-P, from the viscera of true sardine (Sardinops
melanostictus) and from the pyloric ceca of arabesque greenling (Pleuroprammus azonus),
respectively, were purified by gel filtration and anion-exchange chromatography. In
particular, the crude trypsin of true sardine was applied to a column of Sephacryl S-200
21
(3.9 x 64 cm) followed by a DEAE-cellulose column (2.2 x 18 cm) and then Sephadex G50 column (3.9 x 64 cm). The final enzyme preparation (TR-S) was purified 117-fold
from the crude trypsin (Ki shimura et al., 2006).
2.2.5 Assay methods for trypsin activity
The activity of trypsin in solution can be determined either from the rate of catalysis of a
specific substrate or by direct titration of its active site. Specific activity of a trypsin
preparation may be expressed as units of trypsin per milligram of protein.
The substrates of trypsin can be described by the general formula, R-CO-X (Walsh,
1970), where the specificity of trypsin is determined by the acyl moiety (R-CO-).
Trypsin possesses a very narrow specificity catalyzing the hydrolysis of bonds involving
the carboxyl of arginine or lysine, because trypsin has a negative charged pocket which
can recognize positive charged residue (arginine and lysine) of substrate. The type of
bond c1eaved (Le., amide or ester) is defined by the X group. So arginine derivatives are
commonly used as substrates in activity study of trypsin, such as N-a-benzoyl-DLarginine-p-nitroanilide (BAPNA) is a substrate for the assay of amidase activity and tosyl
arginine methyl ester (T AME) is a substrate for the assay of esterase activity .
Oider methods for determination of activity of trypsin inciuded the Casein Digestion
Method (Kunitz, 1947) and the Hemogiobin Digestion Method (Anson, 1938). Methods
of determination of trypsin activity with synthetic substrates are based on determining
ehher the amid.ase or the esterase activitY.A coniinuous, automatic meth()cl for the study
ofhydrolysis rate of peptides and amides has been described by Lenard et al. (1965).
The amidase activity of trypsin can be determined with benzoyl-L-argininamide (BAA)
and a-p-toluensulfonyl-L-argininamide (TSAA) by the method of Schwert et al. (1948).
Other substrates
such as
a-benzoyl-L-Iysinamide
(BLA),
a-p-toluensulfonyl-L-
lysinamide (TSLA), and a-hippuryl-L-Iysinamide (HLA) also have been used. The
colorimetrie procedure using benzoyl-DL-arginine p-nitroanilide (BAPNA) as substrate
provided a sensitive amidase assay method (Erlanger et al., 1961).
22
The potentiometric determination of esterase of trypsin is carried out by the method of
Schwert et al. (1948). In this method, benzoyl-L-arginine ethyl ester (BAEE) was used as
subsrate. A spectrophotometric method (Hummel, 1959) using p-toluenesulfonyl-Larginine methyl ester (TAME) had greater sensitivity and selectivity than using BAEE as
a substrate.
Nowadays, trypsin amidase activity was most widely determined by using benzoyl-DLarginine p-nitroanilide (BAPNA) as substrate according to the method of Erlanger et al.
(1961). For example, the amidase activity of an anionic trypsin from chum salmon was
determined using BAPNA as substrate at pH 8.1 (Sekizaki et al., 2000). Sarne method
used by Ahsan and Watabe (2001) for measuring Japanese anchovy trypsin amidase
activity. Trypsin esterase activity was determined spectrophotometrically by using ptoluenesulfonyl- L-arginine methyl ester (TAME) as substrate according Hummel (1959).
For example, the esterase activity of trypsins from the viscera of true sardine and the
pyloric ceca of arabesque greenling were measured using TAME as substrate (Ki shimura
et al., 2006). Ahsan and Watabe (2001) used benzoyl-L-arginine ethyl ester (BAEE) as
substrate to measure Japanese anchovy trypsin esterase activity. Proteolytic activity of
splenic extract from three tuna species were determined using haemoglobin and casein as
substrates according to the method of Kunitz (1947) and An et al.(1994) with a slight
modification (Klomklao et al., 2004).
2.2.6 Applications of trypsins from marine fishes
By far, trypsins play a key role from basic research to practical applications. They are
widely used in food industry, now they are also applied in cell culture, specific peptide
synthesis, pharmacology areas. For example, animal-derived trypsin (bovine trypsin) or
recombinant trypsins derived from a non-animal source are essential elements for use
with cell culture techniques, because cells are most commonly removed from the culture
substrate by treatment with trypsin. A recombinant trypsin derived from a non-animal
source (TrypZean™: recombinant bovine trypsin expressed in corn - a non-animal
alternative, Sigma-Aldrich Corporation, St. Louis, MO, USA) would enhance the future
23
of adherent mammalian cell culture technology in the biopharmaceutical industry.
However, the information about marine fish trypsin used in cell culture has not been
reported. The cold-adapted proteolytic enzymes from marine fish have been applied as
processing aids in the food and feed industries were thoroughly reviewed by Diaz-Lopez
and Garcia-Carreno (2000). This section will briefly discuss sorne of practical
applications in food industry and medical applications of marine fish trypsin.
Food Industry
Marine fish trypsins have two unique properties: high molecular activity at low
processing temperature and thermal instability, it is suggested that they can be used
efficiently in the food industry.
Protein digestion at low temperatures is required in the processing of fresh foods. Food
processing at low temperatures minimizes undesirable chemical reactions. So the high
catalytic efficiency of Atlantic cod trypsin is especially useful in the processing of fresh
foods (Bjarnason and Benediktsson, 2001). It is more economical as its high catalytic
efficiencies facilitate the use of smaller amounts of enzyme. On the other hand, when
enzymatic activity needs to be controlled, the Atlantic cod trypsin is easily inactivated by
relatively low heat.
Another use of cod trypsin is the extraction of carotenoprotein from shrimp processing
wastes. Cano-Lopez et al. (1987) compared the efficacy of Atlantic cod trypsin from
pyloric ceca with the bovine pancreatic enzyme for extraction of carotenoprotein from
shrimp waste at low reaction temperature (4°C). Atlantic cod trypsin was able to recover
64% of the astaxanthin and 81% ofproteins, whereas bovine trypsin recovered only 49%
of astaxanthin and 65% of proteins under similar experimental conditions. It is conclude .
that Atlantic cod trypsin was more effective.
Marine fish trypsin can be used to prevent formation of oxidation flavor in milk.
Simpson and Haard (1987a) reported that both bovine and Greenland cod trypsins have
similar effects in preventing oxidation of milk lipids. However, Greenland cod trypsin
had more thermal instability than bovine trypsin. It was completely inactivated by the
24
pasteurization process, whereas bovine trypsin retained most of its original activity after
the same pasteurization treatment. Therefore, it is better to use Greenland cod trypsin
since residual enzyme could not be present to cause subsequent hydrolysis of milk
proteins.
According to Ritskes (1971) and Orejana and Liston (1981), trypsin is an important
component in accelerating the fermentation of herring (matjes). Greencod trypsin was
exploited in the low-temperature curing ofherring because ofits higher molecular activity
at low temperatures (4°C) during the early stage of fermentation (Lee et al., 1982). In
another study, the efficiencies of bovine and Greenland cod trypsin for the low
temperature herring fermentation was compared by Simpson and Haard (1984a), the
initial rate of protein solubilization with same amounts of Greenland cod trypsin was
double that observed with bovine trypsin.
Medical Applications
Medical applications of trypsin are very broad and have been known for a long time.
Mammalian trypsins have been used for sorne major medical applications with good
results, such as wound healing (Spittler and Parmenter, 1954) and human medicine
(Leipner and Saller, 2000). Atlantic cod trypsin has already proved useful in medical
applications (Bjarnason, 2000). Bjarnason (2000) reported that the medical application of
Atlantic cod trypsin and other cold-adapted serine proteases from the Atlantic cod
includes using them for inflammation, fungal diseases, acne, wound healing, and other
dermatologie conditions (psoriasis, and eczema). Moreover, trypsin is used for the
manufacture of other pharmaceuticals and cosmetics
2.2.7 Examples of previous research studies on trypsin from fish species
During the past decades, both trypsin and trypsin-like enzymes from digestive organs of
some fish species have been isolated and characterized. These digestive and digestion
related organs such as pancreas, intestine and pyloric ceca, are common sources of these
enzymes.
25
In 1960, Bradford, may be the first one to characterize a fish trypsin-like enzyme activity,
from pylorie ceca of the Chinook salmon (Onchorhynchus tshawytscha). The enzyme was
found to have similar properties to both mammalian trypsin and chymotrypsin. This
trypsin-like enzyme had an optimal pH of 9.0 and maximum activity at a temperature of
49°C.
Reeck et al. (1972) purified and characterized one trypsin in the African lungfish,
Protopterus aethiopicus. It displayed a molecular weight of 24,000 Da and optimum
activity at pH 8.0. The lungfish trypsin amino acid composition was found to be similar to
that of other animal trypsins. However, like sorne mammalian trypsins, the lungfish
trypsin was found to be stable at pH 3.0.
Ovemell (1973) made a study of the digestive enzymes from pyloric ceca and its
associated mesentery in the cod, Gadus morhua. The trypsin-like enzyme showed a
molecular weight of 18,000 to 22,000 Da. Its pH optimum ranged from 8.0 - 9.0.
Titani et al. (1975) purified panereatie trypsins from the spmy dogfish (Squalus
acanthias) through chromatographie and related procedures. This purified enzyme
presented two bands on electrophoresis gel, one of 11,000 Da and the other of 23,000 Da.
In addition, the amino acid sequence of these trypsins was determined and compared with
bovine and porcine trypsins.
C()hen et al. (1981) purlfied pancreati.c proteolytic enzymes including trypsin from earp,
Cyprinus carpio. The enzyme showed an approximate moleeular weight of 25,000 Da.
The carp trypsin was found to be an anionie protein that is unstable at low pH.
Hjelmeland and Raa (1982) purified two trypsin-like enzymes from the aretie fish
eapelin, Mal/otus villosus. Both enzymes had a molecular weight of about 28,000 Da. The
enzymes were displayed a pH optimum of 8.0-9.0 and inhibited by standard trypsin
inhibitors.
26
In 1984b, Simpson and Haard purified and characterized trypsin from the pyloric ceca of
Greenland cod, Gadus ogac. Trypsin was isolated by ammonium sulfate fractionation
followed
by
acetone
precipitation
and
affinity
chromatography
techniques.
Electrophoretic analysis of the enzyme showed a single band with an estimated molecular
weight of 23,500 Da. Characterization of the enzyme inc1uded identifying its catalytic
specificity for amide or ester bonds involving the carboxyl group of arginine, capacity to
hydrolyze the trypsin-specific synthetic substrate tosyl arginine methyl ester (T AME), its
sensitivity to serine protease inhibitors, and the inhibition of enzyme activity when in the
presence of soybean trypsin inhibitor.
Pancreatic enzymes, inc1uding trypsin, were characterized from the sharptooth catfish,
Clarias gariepinus (Uys and Hecht, 1987). This trypsin displayed optimal activity at pH
8.2 and at temperatures ranging from 30°C to 40°C.
Purification and characterization of two trypsin-like enzymes from the digestive tract of
the anchovy, Engraulis encrasicholus, were realized through a combination of affinity
and ion exchange chromatographic procedures (Martinez et al., 1988). These enzymes
displayed molecular weights in the range of 27,000 and 28,000 Da. Their isoelectric
points were between 4.6 and 4.9. The enzymes displayed optimal activity in a pH range of
8.0-9.0. They were similar to other fish trypsins in their molecular weights, kinetic
properties, and instability at low pH.
Guizani et al. (1991) purified and characterized a trypsin from the pyloric ceca ofmullet,
Mugi! cephalus. Electrophoretic analysis determined the molecular weight of the enzyme
to be 24,000 Da. The enzyme exhibited optimal activity at a pH of 8.0 and at a
temperature of 55°C. It was stable in a pH range of7.5-9.0. This stability is similar to that
reported for most marine organism trypsins, which are highly unstable under acidic
conditions but very stable at neutral to slightly alkaline conditions.
Trypsin from the pyloric ceca of rainbow trout, Oncorhynchus mykiss, was purified and
characterized by Kristjansson (1991). The isolated enzyme had an estimated molecular
27
weight of25,700 Da. The enzyme was stable at temperatures in the range from 40°C to 50
Oc and at a pH range
of 5.4 to 8.0. However, this thermal stability was shown to be
calcium-dependent. The optimum temperature for hydrolysis of substrate was
approximately 60°C.
Comparison of trypsin and chymotrypsin from the viscera of anchovy, Engraulis
japonicus, was studied by Heu et al. (1995). The molecular weight of the trypsin was
estimated to be 25,600 Da. Maximal activity was found at pH 9.0 and 45°C for casein and
at pH 8.0 and 45°C for TAME.
Four differently charged trypsins were purified from pyloric ceca of Atlantic salmon,
Salmo salar (Outzen et al., 1996). The four isoforms of trypsin were differentiated as
anionic trypsin l, II and III, and cationic trypsin. AIl were found to have a molecular
weight of about 25,000 Da. Cationic salmon trypsins displayed optimal activity at a pH
range of 8.5 to 10.5. By contrast, the anionic salmon trypsin was optimally active at pH
10.5. This study was the first to identify, isolate, and characterize a cationic trypsin from
a marine species.
Sekizaki et al. (2000) purified an anionic trypsin from the pyloric ceca of chum salmon,
Onchorhynchus keta. The molecular weight was around 24,000 Da as determined by
SDS-PAGE. The enzyme displayed moderate activity toward artificial substrate TAME.
The main band of the anionic enzyme showed an isoeletric point of 5.1. The effect of
temperature on the hydrolysis TAME suggested that the enzyme is an efficient catalyst at
low temperature.
Purification and characterization of trypsin-like enzyme from the pyloric ceca of cod
(Beirao et al., 2001) was obtained through affinity chromatography on CHOM sepharose
4B. Characterization of the enzyme was established through both its catalytic activity on
TAME and its inhibition of serine protease inhibitors. The enzyme showed pIs of 5.30
and 5.89 and was found to have a similar amino acid composition to that of bovine
trypsin.
28
Trypsin from the pyloric ceca of the starfish (Asterina pectinifera) was isolated and
characterized by Kishimura and Hayashi (2002). The isolated enzyme had an estimated
molecular weight of approximately 28,000 Da. Optimum pH and temperature of starfish
trypsins for hydrolysis of N -p- Tosyl-L-arginine methyl ester hydrochloride were a
approximately pH 8.0 and 55°C, respectively. It was unstable at above 50°C and be10w
pH 5.0. The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was
found.
Castillo-Yanez et al. (2005) purified and characterized a trypsin from the pyloric ceca of
Monterey sardine Sardinops sagax caerulea. Electrophoretic analysis determined the
molecular weight of the enzyme to be 25,000 Da. The optimum pH for activity was 8.0
and maximum stability was observed between pH 7.0 and 8.0. Activity was optimum at
50°C and lost activity at higher temperatures. The purified enzyme was partially inhibited
by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully
inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited
by the metallo-protease inactivator EDTA.
Two trypsins, TR-S and TR-P, were purified from the viscera of true sardine (Sardinops
melanostictus) and the pyloric caeca of arabesque greenling (Pleuroprammus azonus) by
gel filtration and anion-exchange chromatography (Ki shimura et al., 2006). The TR-S and
TR-P had maximal activities at around pH 8.0 for hydrolysis of TAME. Optimum
temperature of the TR-S and TR-P were 60°C and 50°C, respectively. Both TR-S and TRP were stabilized by calcium ion. The N-terminal amino acid sequences of the TR-S,
IVGGYECKAYSQPWQVSLNS, and TR-P, IVGGYECTPHTQAHQVSLNS, were
found.
In 2006, Kurtovic et al. purified and characterized a trypsin from the pyloric ceca of
chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone
precipitation and affinity chromatography. The molecular mass of the chinook salmon
trypsin was estimated as 28,000 Da by SDS-PAGE. The chinook salmon enzyme was
active over a broad pH range (from 7.5 to at least pH 10.0) at 25°C and was stable from
29
pH 4.0 to pH 10.0 when ineubated at 20°C, with a maximum at pH 8.0. The optimum
temperature for the hydrolysis of BAPNA by this enzyme was 60°C. It was inhibited by
phenyl methyl sulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI) and
benzamidine.
We ean see from this review that trypsin is a charaeteristie digestive enzyme of fishes.
Trypsin has a moleeular weight in the range of approximately 20,000 to 30,000 Da. These
trypsins are optimally active at pH ranges of 7.5 - 9.0. In addition, at its optimal pH each
fish trypsin displayed greatest eatalytie rate at temperatures of 45°C - 60°C.
In order to better understand the digestive physiology and bioehemistry of the hoki, in
this project, trypsin was extraeted and purified from pylorie eeca tissues of hoki, and was
further identified, and characterized, at last the catalysis properties of hoki trypsin was
studied.
30
CHAPTERIII
PURIFICATION AND CHARACTERIZATION OF
TRYPSIN FROM THE PYLORIC CECA OF HOKI
(Macruronus novaezealandiae)
A trypsin was successfully extracted and purified from the pyloric ceca tissues of New
Zealand hoki (Macruronus novaezelandiae), and was further characterized with respect to
various physical and chemical properties, such as the kinetic properties, temperature, pH
and inhibition effects on the activity of the purified trypsin. The N-terminal amino acid
sequences
of purified
hoki
trypsin,
IVGGQECVPNSQPFMASLNY,
displayed
considerable homology with other fish trypsins.
The authors of the manuscript are Changying Shi, Sue Marshall and Benjamin K.
Simpson. This project was supervised by Dr. Benjamin K. Simpson and actual
experimental work and writing of manuscript were done by Changying Shi. Dr.
Marshall's lab provided the defatted sample for the study. The manuscript was edited by
Dr. Benjamin K. Simpson and this publication will be submitted to the Journal of Food
Biochemistry.
31
3.1 Abstract
Trypsin was purified from the pyloric ceca of hoki by ammonium sulfate fractionation,
followed by acetone fractionation and then by affinity chromatography on SBTISepharose 4B. The purified extract was simultaneously desalted and concentrated by
ultrafiltration, and then characterized using N-a-benzoyl-DL-arginine-p-nitroanilide
(BAPNA) as substrate.
The hoki trypsin was stable from pH 6.0 to Il.0 after incubation for 30 min at 25°C, and
its maximal activity for the hydrolysis of BAPNA was pH 9.0. The enzyme was unstable
below pH 6.0. The optimum temperature for hoki trypsin hydrolysis ofBAPNA was 60°C.
The enzyme was stable at temperatures below 40 Oc but was unstable above 50°C. Hoki
trypsin was inhibited by well-known trypsin inhibitors (SBTI, aprotinin, benzamidine,
PMSF). The enzyme showed much greater susceptibility to SBTI and aprotinin inhibition
than PMSF and benzamidine. The Michaelis-Menten constant (Km') and substrate
turnover number (kcat) for the hydrolysis of BAPNA by hoki trypsin were measured as
0.06 mM and 0.33
S·l,
respectively, while the corresponding values for the hydrolysis of
TAME were 2.08 mM and 19.0
S·l,
respectively. The turnover number (kcat) ratio for the
amidase/esterase hydrolysis for hoki trypsin was more than six times that of bovine
trypsin. The physiological efficiency (kcat 1 Km') ratio for the amidase/esterase reactions
was significantly higher for the hoki trypsin than for bovine trypsin. The fraction obtained
after affinity chromatography migrated as a signal band in sodium dodecyl sulphate
polyacrylamide gels as weIl as in isoelectric focusing gels. The molecular weight of the
isolated trypsin was determined by SDS-PAGE to be approximately 26,000 Da, whereas
the MALDI-TOF MS method ofanalysis indicated a molecular weight of 23,791 Da. The
isoelectric point was determined as 6.5. The N-terminal 20 amino acids residues of hoki
trypsin, IVGGQECVPNSQPFMASLNY, displayed considerable similarity with those of
other fish trypsins. Based on the above characteristics, it is suggested that the enzyme
isolated from hoki is authentic trypsin and a member of the trypsin family of enzymes.
3.2 Introduction
Trypsin is one of the important digestive enzymes. It belongs to the serine protease family
32
(EC 3.4.2l.X.). Mammalian pancreatic trypsins have been extensively characterized
(Walsh and Wilcox, 1970). Many mammalian trypsin and trypsinogen structures are
known by X-ray crystallography (Sweet et al., 1974; Kossiakoff et al., 1977). The
function of trypsin is catalyzing the hydrolysis of ester and peptide bonds involving the
carboxyl group of arginine or lysine. In the active site oftrypsin, there are threefunctional
residues (serine, aspartate and histidine) participating in the mechanism of catalysis.
Trypsin and trypsin-like enzymes have a molecular weight of the range from 22 to 28
kDa (Simpson, 2000). Simpson et al. (1989b) have shown that molecular weights of
trypsin from Atlantic cod, cunner and Greenland cod were 24,000, 24,000 and 23,500 Da,
respectively. Whereas Kishimura and Hayashi (2002) reported that the molecular weight
of starfish trypsin was approximately 28,000 Da. Most fish trypsins were optimally active
at pH 7.5 - 10.0 and 40 - 60°C. For example, Bezerra et al. (2001) found that the trypsin
from the pyloric caeca oftambaqui (Colossoma macropomum) had an optimum pH of9.5.
Klomklao et al. (2004) reported that optimal activity of splenic proteases from three tuna
species was at pH 9.0 and 55°C. The pH stability of trypsin isolated from different fish
species was at alkaline pH, which differs from trypsin from mammals which are most
stable under acidic conditions (De-Vecchi and Coppes, 1996; Haard, 1992). The thermal
stability of fish trypsin varies with species as well as with incubation conditions (DeVecchi and Coppes, 1996). Ahsan and Watabe (2001) studied the kinetic properties of
trypsins from Japanese anchovy and bovine and reported that the catalytic efficiencies
(kcat
/
Km') for both the amidase and esterase reactions were considerably higher for
Japanese anchovy fish trypsin compared to bovine trypsin.
Compared with trypsins from mammals, marine fish trypsins have special properties
related to the wide range of environmental conditions of their habitat. Unique properties
of trypsins from marine fish inc1ude a low Arrhenius activation energy, relatively lower
free energy of activation (AG*), high molecular activity at low reaction temperature, cold
stability, low temperature optimum, low thermostability, and high pH optimum/pH
stability. In recent years, researchers obtained the N-terminal amino acid sequences of
fish trypsins from different fish species such as Atlantic salmon (Male et al., 1995),
33
starfish (Ki shimura and Hayashi, 2003), Japanese anchovy (Ki shimura et al., 2005) and
true sardine (Ki shimura et al., 2006). As the recombinant DNA technology developing,
the limited number of amino acid sequences and structural data will be more widely
studied to explore the molecular basis of these unique properties. In this way some of the
unique properties of marine fish trypsins may be made them better suited for certain
applications in the food industry.
Trypsins and trypsin-like enzymes have been isolated and studied from many kinds of
marine fish, such as stomachless bone fish Carassius auratus gibelio (Bloch) (Jany, 1976),
capelin (Hjelmeland and Raa, 1982), Greenland cod (Simpson and Haard, 1984c;
Simpson et al., 1989b), cunner (Simpson and Haard, 1985, 1987b; Simpson et al. 1989a),
anchovy (Martinez et al., 1988), Atlantic cod (Asgeirsson et al., 1989; Han, 1993;
Simpson et al., 1990; Amiza et al., 1997), chum salmon (Uchida et al., 1984a, 1984b),
Atlantic salmon (Male et al., 1995; Schroder et al., 1998), coho salmon (Haard et al.,
1996), palometa (Parona signata) (Pavlisko et al., 1997a), Atlantic white croaker
(Pavlisko et al., 1997b), starfish (Ki shimura et al., 2003), Japanese anchovy (Kishimura
et al., 2005), Monterey sardine (Castillo-Yanez et al., 2005) and true sardine (Ki shimura
et al., 2006) an so on. However, by far no information regarding the characteristics and
properties of trypsin from New Zealand hoki (Macruronus novaezelandiae), which is by
far the most important commercial fish in New Zealand, has been reported. This study
aimed to extract and purify trypsin from the pyloric ceca of hoki, characterize some
information about its main physicochemical, kinetic properties and N-terminal amino acid
sequence and compare with other trypsins that have been studied.
3.3 Materials and methods
3.3.1 Biological specimen
Hoki (Macruronus novaezealandiae) pyloric ceca were obtained from a local fish
processing plant (SeaLord, Nelson, New Zealand). The ceca were removed from the rest
of the processing discards directly as they came off the filleting line, stored in ice, and
then packaged in plastic bags (~250 g / bag) and frozen at - 40°C until needed.
34
In the lab, the ceca were defatted successively with acetone, CHCl3/n-butanol (9: 1, v/v),
CHCh/n-butanol (8:2, v/v), diethyl ether and then dried. The defatted pyloric ceca powder
was kept at - 20°C until required for the extraction.
3.3.2 Materials and instruments
The following chemicals were aH purchased from Sigma-Aldrich Co. (St. Louis, MO,
USA):
acrylamide, ammonium persulfate, ammonium sulfate, aprotinin, benzamidine, benzoylDL-arginine p-nitroanilide (DL-BAPNA), Brij 35, bovine serum albumin (BSA),
Coomasie brilliant blue (R-250), cyanogen bromide (CNBr) activated Sepharose 4B, 3cyclohexy-Iamino-1-propanesulfonic acid (CAPS), Folin & Ciocalteau's phenol reagent,
formaldehyde, glutaraldehyde, 2-mercaptoethanol (ME), phenyl methyl sulfonyl fluoride
(PMSF), soybean trypsin inhibitor (SBTI), N.N.N' .N' -tetramethyl ethylene diamine
(TEMED), thioglycolic acid (sodium salt), and tosyl arginine methyl ester (TAME).
Acetic acid, acetone, calcium chloride, citric acid, cupric sulphate pentahydrate, ethanol,
hydrochloric acid, glycine, N.N' -methylene-bis-acrylamide, potassium tartrate, propanol,
silver nitrate, sodium carbonate, sodium chloride, sodium hydroxide, sodium hydrogen
carbonate, sodium thiosulphate, trichloroacetic acid (TCA) and Tris were purchased from
Fisher Scientific (Fair Lawn, New Jersey, USA).
Dimethyl sulfoxide (DMSO) was supplied from Anachemia (Montreal, Canada).
Standard low molecular weight markers: phosphorylase b (97,400), bovine serum
albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin
inhibitor (21,500), lysozyme (14,400) were purchased from Bio-Rad laboratories
(Mississauga, Ontario, Canada).
The amidase activity and esterase activity of trypsin using BAPNA and TAME as
substrates were determined using a Hitachi U 2000 spectrophotometer (Tokyo, Japan) at
410 nm and 247 nm, respectively.
35
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a miniPROTEIN II electrophoresis unit (Bio-Rad, Mississauga, Ontario, Canada).
Matrix-Assisted Laser DesorptionlIonization Time-of-Flight (MALD l -TO F) Mass
Spectrometry was performed on a Bruker Autoflex (Bruker-Daltonics, Leipzig, Germany).
lsoelectric focusing (lEF) was carried out on a PhastGel IEF gel 3-9 usmg the
PhastSystem™ (Pharmacia LKB, Biotechnology AB, Uppsala, Sweden).
Experimental Methods
3.3.3 Extraction and purification of trypsin
Trypsin was isolated and purified from hoki pyloric ceca powder according to the method
described by Simpson and Haard (1984b) with slight modification.
3.3.3.1 Extraction of trypsin
About 10 g of the dried powder was mixed with extraction buffer (0.05 M Tris-HCI
containing 0.5 M NaCI and 0.02 M CaCh'2H20, pH 7.8) in a ratio of 1:12.5 (w/v). The
slurry was stirred at 4°C for 3 h and centrifuged at 3,000 g for 30 min at 4°C to obtain the
first supematant(Sup 1). Brij 35 (10% stock solution) was added to the supematant to
give a final concentration of 0.2% and the mixture was stirred ovemight at 4°C and
finally centrifuged at 10,000 g for 30 min at 4°C to obtain the second supematant (Sup 2).
3.3.3.2 Purification of trypsin
Ammonium sulfate precipitation
The second supematant was fractionated with solid (NH4)2S04 up to 40% saturation and
stirred overnight at 4°C, then centrifuged at 6,000 g for 30 min at 4°C to obtain the third
supematant (Sup 3) and a solid precipitate. The solid precipitate was discarded and make
the third supematant up to 60% saturation with sorne more solid (N~)2S04 with stirring
for 3 h at 4°C, and then re-centrifuged at 6,000 g at 4°C for 30 min. The supematant
obtained was discarded and the sediment forming between 40% and 60% of saturation
was retained.
36
Dialysis
The precipitate from the 40% to 60% ammonium sulfate step was dissolved in a
minimum amount of extraction buffer (pH 7.8) to obtain the (NILt)2S04 soluble fraction.
This fraction was dialyzed in membrane (cellulose membrane. Sigma-Aldrich. St. Louis.
MO. USA) against 3 changes of6 L of the same buffer ovemight at 4°C to remove excess
salt (i.e .• NaCl and (N&)2S04).
Acetone precipitation
Three times volume of cold acetone (-20°C) were added to the dialyzed (N&)2SÛ4
fraction and kept at -20°C for 3 h. The precipitate formed was collected by centrifugation
at 6.000 g for 30 min at 4°C. The acetone precipitate was redissolved in a minimum
amount of extraction buffer (pH 7.8) and stored at -20°C prior to affinity chromatography.
Affinity chromatography
Affinity chromatography was performed on a 1.0 cm
x
10 cm column containing soybean
trypsin inhibitor (SBTI)-CNBr activated Sepharose 4B (Sigma). The basic coupling
procedure for use with CNBr activated Sepharose 4B provided by the manufacture was
followed in preparing the affinity matrix (Pharmacia LKB Biotechnology. 1988). The
column was equilibrated with extraction buffer (pH 7.8). Aliquots of about 10 mL of the
acetone fraction were then pumped into the column at a flow rate of 25 mL/h with a
microtube pump. The column was subsequently washed with extraction buffer (pH 7.8) to .
remove the unbound material until the effluent had no further change in absorbance at
280 nm with a spectrophotometer (Hitachi U 2000. Tokyo. Japan). The bound protein was
desorbed from the column by washing with 5 mM HCl at a flow rate of 60 mL/h. The
protein eluate from the column was collected in fractions of 1 mL using an automatic
fraction collector (Bio-Rad. Hercules. CA. USA). The fractions (1 mL) were mixed
immediately with 2 mL extraction buffer (pH 7.8) to avoid 10ss of enzyme activity due to
trypsin denaturation. The fraction size was 3.0 mL each for 90 total collected fractions
obtained. Protein concentration in column effluents was monitored continuously at 280
nm. The fractions showing amidase activity (toward BAPNA as substrate) were pooled
and stored frozen at -20°C.
37
The coupling procedure used for the SBTI-CNBr-activated Sepharose 4B affinity matrix
(pharmacia LKB Biotechnology, 1988) is summarized below:
1. The required amount ofCNBr-activated Sepharose 4B (2 g) was weighed.
2. The CNBr-activated Sepharose 4B was washed and swollen using 400 mL 1 mM HCI
(200 mL 1 mM HCI/g dry gel).
3.50 mg ligand (SBTI) to be coupled onto the gel was dissolved in 10 mL coupling buffer
(NaHC03, 0.1 M, containing 0.5 M NaCI, pH 8.3). The ratio of SBTI to the CNBractivated Sepharose 4B was 25 mg SBTIIg dry gel.
4. The swollen gel was washed with coupling buffer (5 mLig dry gel) and immediately
transferred to the SBTI solution. The mixture containing SBTI and swollen gel is
rotated in an end-over-end mixer overnight at 4°C.
5. The SBTI-gel was transferred to 10 mL 0.2 M glycine buffer (pH 8.0) to mix end-overend for 2 h at room temperature (20°C) to block remaining active groups.
6. Excess SBTI was washed away using the coupling buffer (50 mL) followed by 50 mL
acetate buffer (0.1 M, containing 0.5 M NaCI, pH 4) followed by coupling buffer (50
mL). This step was repeated five times.
7. The SBTI- Sepharose matrix thus prepared was ready for use and was stored at 4 - 8°C.
3.3.4 Protein determination
Protein concentration was determined by the method of Lowry et al. (1951). In this
experiment a standard curve was prepared for Lowry's assay using known amounts of
bovine serum albumin (BSA) and the protein contents of a number of unknown solutions
were determined by comparing the color produced by the unknowns with the standard
curve.
3.3.5 Enzyme activity assay
3.3.5.1 Amidase activity
The amidase activity of trypsin was determined according to the method of Erlanger et al.
(1961) using N-a-benzoyl-DL-arginine p-nitroanilide (BAPNA) as substrate.
A 200 ilL aliquot of the appropriately diluted enzyme solution was added to 2.8 mL of 1
38
mM BAPNA in 0.05 M Tris-HC1 (pH 8.2) containing 0.02 M CaCh·2H20 and the release
of p-nitroaniline was followed by the increase in absorbance at 410 nm (extinction
coefficient = 8800 M- 1 cm- 1). The concentration ofBAPNA in aIl assays was 0.3 mM. The
increase in light absorption at 410 nm was measured after 10 second intervals for a total
assay time of 100 second at 25°C using Hitachi U 2000 spectrophotometer (Tokyo, Japan).
AIl assays were performed in quartz cuvettes.
One BAPNA unit of amidase activity was defined as 1 Ilmol p-nitroaniline formed per
min from BAPNA at 25°C. It can be calculated using
~AtlOnmJminx
1000
x
3 divided by
8800, where 8800 is the extinction coefficient ofp-nitroaniline (Erlanger et al., 1961).
1mM BAPNA was prepared as foIlows: 0.0435 g ofBAPNA (M.W. 435) was dissolved in
1mL dimethyl sulfoxide (DMSO) and kept at room temperature (20°C) until needed. The
resulting solution was made up to 100 mL with the 0.05 M Tris-HCl buffer (containing
0.02 M CaCh, pH 8.2). AIl solutions used were kept at 2: 20°C to prevent crystallization
of the BAPNA from solution.
3.3.5.2 Esterase activity
The esterase activity of trypsin was determined spectrophotometrically usmg tosyl
arginine methyl ester (TAME) as substrate based on Worthington Biochemical
Corporation's (Anonymous, 1972) modification procedure of the method of Hummel
- (1959),
A 100 ilL aliquot of the appropriately diluted enzyme solution was mi~ed to 0.3 mL of
0.01 M TAME and 2.6 mL of 46 mM Tris-HCl (pH 8.1) containing 11.5 mM CaCh, and
the release oftosyl arginine was measured at 247 nm at 25°C. Absorbance readings were
obtained every 10 sec at 247 nm for a total assay time of 100 sec at 25°C using Hitachi U
2000 spectrophotometer (Tokyo, Japan).
One TAME unit of esterase activity was defined as the hydrolysis of 1 Ilmol of TAME per
min at 25°C. TAME units (U) were calculated with the following equation: U
39
=
~A247nmlminx
1000 x 3/540, where 540 is the extinction coefficient of tosyl arginine
(Anonymous, 1978).
0.01 M TAME was prepared as follows: 0.0379 g of TAME was dissolved in 10.0 mL
distilled water.
3.3.6 pH optimum and stability
The effect of the pH on the BAPNA hydrolysis rate was determined by preparing aliquots
(2.8 mL) of the substrate (l mM BAPNA) in various buffer solutions (pH range from 2 to
10) and adding 0.2 mL aliquots of the appropriately diluted enzyme solution to the
substrate. The substrate at the appropriate pH was used as blank, distilled water being
added instead of enzyme. Amidase activity was assayed according to the method of
Erlanger et al.(1961). pH was varied from 2 to 10 using the following buffers: 0.1 M
citrate-HCl, pH 2.0; 0.1 M citrate-NaOH, pH 4.0; 0.1 M citrate-NaOH, pH 6.0; 0.1 M
Tris-HC1, pH 7.0; 0.1 M Tris-HCl, pH 7.5; 0.1 M Tris-HCl, pH 8.0; 0.1 M Tris-HCI, pH
8.5; 0.1 M Tris-HC1, pH 9.0, and 0.1 M glycine-NaOH, pH 10.0.
The pH stability of the hoki trypsin was evaluated by measuring enzyme residual amidase
activity after incubation at various pH's for 30 min at 25°C. The pH of the enzyme
solution was varied in the range 2-12 using the following buffers: 0.1 M citrate-HCl, pH
2.0; 0.1 M citrate-NaOH, pH 4.0; 0.1 M citrate-NaOH, pH 6.0; 0.1 M Tris-HC1, pH 7.0;
0.1 M Tris-HCI, pH 7.5; 0.1 M Tris-HCI, pH 8.0; 0.1 M Tris-HCI, pH 8.5; 0.1 M TrisHCl, pH 9.0, and 0.1 M glycine-NaOH, pH 10.0; 0.1 M borate-NaOH, pH 11.0; 0.1 M
borate-NaOH, pH 12.0. The enzyme solution was diluted 1:1 with buffers of different pH
values ranging from pH 2 to 12. After incubation for 30 min at 25°C, enzyme residual
activity was measured at 25°C using 1 mM BAPNA in 0.05 M Tris-HC1 (pH 8.2)
containing 0.02 M CaCh'2H20 as a substrate.
3.3.7 Temperature optimum and thermostability
The optimum temperature of hoki trypsin was determined by measurlng amidase activity
(pH 8.2) at various temperatures. To do this, determine the temperature dependence of the
40
amidase reaction, 2.8 mL of the substrate (1 mM BAPNA in O.OS M tris-HCl, pH 8.2,
containing 0.02 M CaCh·2H20) was equilibrated at temperatures of 20°C to 80°C in a
spectrophotometer fitted with a peltier temperature controller for approximately S min
prior to the addition of 0.2 mL of the trypsin solution. The rate of hydrolysis of the
substrate was measured as
/).A41O nm Imin
using Unicam UV/visible spectrophotometer
(model UV4) (Thermo Electron Corporation, Waltham, MA, USA) fitted with a peltier
temperature controller.
In order to determine the thermostability of hoki trypsin, the enzyme was preincubated at
various temperatures from SoC to 7SoC for 30 min, then rapidly cooled in an ice bath for 5
min, and the residual activity was measured at 2SoC using 1 mM BAPNA in 0.05 M TrisHCI, pH 8.2, containing 0.02 M CaCh'2H20 as substrate. Residual enzyme activity after
the temperature treatment was measured as
/).A41O nm Imin
using Unicam UV/visible
spectrophotometer (model UV4) (Thermo Electron Corporation, Waltham, MA, USA).
3.3.8 Kinetics studies
The reaction catalyzed by trypsin can be represented as below, where E is the enzyme, A
is the substrate, EA is the Michaelis complex and Pl and P2 are the hydrolysis products
(p-nitroanaline and peptide, respectively).
k1
kcat
E+A ~ EA k_l
E+P 1 +P2
Under steady-state conditions, the dependence of the reaction velocity (v) (mol s-1 mg- l)
on substrate concentration can be described by the Michaelis-Menten equation
V [A]
v = ----=--=--K'm + [A]
where V is the limiting velo city, measured in mol
constant.
41
S-l
mg- 1 and K'm is the Michaelis
The Michaelis-Menten constant (K'm) and maximum velo city (V max) were determined by
the Lineweaver-Burk plot using BAPNA at concentrations ranging from 0.0156 to 1.0
mM, and TAME from 3 to 20 mM under the basic assay conditions described. BAPNA
and TAME units for kinetic evaluation were as defined previously. The turnover number
(kcaÙ was calculated by dividing V max by enzyme molar concentration
(~mol).
The
catalytic efficiency for trypsin was defined as the ratio ofkcat to K'm.
3.3.9 Enzyme inhibition studies
The sensitivity of the hoki trypsin to different specific protease inhibitors, such as phenyl
methyl sulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), the known trypsin
inhibitors benzamidine and aprotinin, was investigated according to the method of
Simpson and Haard (l984b).
Phenyl methyl sulfonyl fluoride (PMSF) was dissolved in 2-propanol to the following
concentration: 5, 10 and 20 mM. One volume of the trypsin solution was incubated with
one volume of the PMSF solution for 30 min at 25°C. After incubation, residual trypsin
activity was assayed using BAPNA as substrate.
SBTI (Type I-S, Sigma, St. Louis, MO, USA) was dissolved in distilled water to the
following concentration: 0.125 x lQ-\ 0.25 x 10-3, 0.5xlQ-3 and 1.0x10-3 mM (0.0025, 0.005,
0.01,0.02 mg/mL). The trypsin solutions were added separately to equal volumes of the
SBTI solutions and incubated in an ice bath for 30 min. After incubation, residual trypsin
activity was assayed with 1 mM BAPNA (pH 8.2) as substrate.
Aprotinin (from bovine lung, 4.0 TIU/mg solid, Sigma, St. Louis, MO, USA) was
dissolved in distilled water to the following concentration (TIU/mL): 0.0031, 0.0063,
0.0125 and 0.025 TIU/mL (where TIU stands for trypsin inhibitor units). Trypsin solution
was mixed with an equal volume of aprotinin solution and incubated for 30 min at O°C.
Residual trypsin activity was assayed with 1 mM BAPNA (pH 8.2) as substrate.
Benzamidine was dissolved in distilled water to the following concentration: 0.5, 1.0, 2.0,
42
5.0 and 10.0 mM. Trypsin solution was mixed with an equal volume of benzamidine
solution and incubated for 30 min at O°C. Residual trypsin activity was assayed with
BAPNA.
After incubation of the various inhibitors with the enzyme solution, a 200 ilL aliquot of
the mixture of inhibitor and trypsin solution was added to 2.8 mL of 1 mM BAPNA in
0.05 M Tris-HC1 (pH 8.2) containing 0.02 M CaCh·2H2û and residual activity was
measured at 25°C using Hitachi U 2000 spectrophotometer (Tokyo, Japan). Appropriate
blanks and inhibitor solvents were used as controls. Percentage of inhibition data were
calculated, considering activity in the absence of inhibitor as 100%, or inhibition in the
absence of inhibitor as 0%.
3.3.10 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
Polyacrylamide gel electrophoresis is a very widely used technique for the study of the
size and charge on biomolecules including RNA, DNA and proteins. The fundamental
principle is based on mobility of charged particles in an electrical field. The rate of
movement depends on the field strength and the number of charges. Proteins possess
surface charge due to the presence of acidic and basic amino acids.
In SDS-PAGE, electrophoresis is performed in the presence of the anionic detergent
sodium dodecyl sulphate (SDS). First, SDS binds to proteins at a constant ratio and
results in denaturation. Second, SDS swamps the native charge on the protein with
negative charges. Therefore, treatment with SDS removes effect of charge density and
shape and separation is then solely based upon molecular weight.
SDS polyacrylamide gel electrophoresis was carried out according to the method of
Laemmli (1970).
3.3.10.1 Casting of gels
A 14% resolving solution of the monomer was prepared by combining aIl the reagents in
Table 3.1 except the ammonium persulfate (APS) and tetramethyl-ethylenediamine
43
(TEMED). The monomer solution was gently mixed with APS and TEMED. Then the
monomer solution was poured between the plates and overlayed with water immediately.
The resolving gel was allowed to polymerize for 45 min. The stacking monomer solution
was prepared by combining all the reagents in Table 3.2 except the APS and TEMED.
The top of the resolving gel was thoroughly rinsed with water and dried with filter paper.
APS and TEMED were added to the stacking monomer solution and poured on the top of
the resolving gel. A well-forming comb was placed in between the plates and the stacking
gel was allowed to polymerize to sample wells.
3.3.10.2 Sample preparation and SDS polyacrylamide gel electrophoresis
The samples and standards (low range, BioRad) were diluted 1: 1 and 1:20 respectively in
complete SDS-reducing buffer (Table 3.3) and heated in boiling water bath for 5 min and
used for the electrophoresis. The electrophoresis cell was assembled and the comb was
removed from the stacking gel. The lower and upper reservoirs were filled with electrode
buffer (Table 3.3). The prepared samples were loaded into the wells in the stacking gel by
layering them under electrode buffer using a micro liter tip. The leads were then attached
to the power supply (BioRad 3000Xi, Japan) and electrophoresis was allowed to proceed
at constant voltage (120V). The running was stopped when the blue tracking dye was
about 1 cm from the bottom of the glass plates. The gels were removed from the glass
plates and fixed in 12% TCA solution (Table 3.3) at least 3 h. After fixation, the gels were
soaked in excess of the staining solution (Table 3.3) overnight and then destained with
several changes of the destaining solution (Table 3.3) until the background stain was
removed.
3.3.10.3 Molecular weight determination
The molecular weight of the purified trypsin was determined by comparison of its
mobility with those of standard maker proteins of known molecular weights which were
run simultaneously on the same gel as the trypsin fraction. The SDS-PAGE molecular
weight standards (low range, BioRad) were phosphorylase b (97,400), bovine serum
albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin
inhibitor (21,500), lysozyme (14,400).
44
The migration of each protein was divided by the distance traveled to the tracking dye
(Figure 3.1). The distance migrated is measured for the tracking dye and for each of the
protein bands. The relative mobility of the proteins (relative to the dye front) is denoted as
Rf. The Rf is then calculated as follows:
Rf = Distance migrated by protein / Distance migrated by dye
A curve was generated by plotting the log of the molecular weights of the standards vs.
the relative mobility (Rf), and used to determine molecular weights ofunknown proteins.
3.3.11 Matrix-Assisted Laser DesorptionlIonization Time-of-Flight (MALDI-TOF)
Mass Spectrometry
MALDI-TOF mass spectrometry was performed on a Bruker Autoflex (Bruker-Daltonics,
Leipzig, Germany), which uses a N2 laser at 337 nm wavelength. Experiments were
carried out using a 20 kV extraction voltage. Sinapinic acid (SA) (Fluka Chemika, Buchs,
Switzerland) was used as matrix. About 10 mg sinapinic acid were dissolved in 1 mL
acetonitrile : water (50:50) with 0.1 % trifluoroacetic acid (TFA) (Sigma, St. Louis, USA)
to make a saturated solution. Samples were prepared with the dried drop let method,
applying a volume matrix-to-trypsin (0.7 mg/mL) ratio between 1:1 to 10:1. The mixture
(0.5 ilL) was pipetted onto a stainless steel 384-well MALDI target for the trypsin
experiments. Sample spots were dried in air. A protein calibration standard (BrukerDaltonics, Leipzig, Germany) with a covered mass range of 3-25 kDa was performed in
the same condition ~o calibrate. the molar mass of the hoki trypsin.
3.3.12 Isoelectric focusing (IEF)
Isoelectric focusing takes place in a pH gradient and is limited to molecules which can be
either positively or negatively charged (amphoteric molecules), like proteins, enzymes
and peptides. Separation happens in a pH gradient which is formed by special amphoteric
buffers (ampholytes) having high buffer capacities at their isoelectric points (Pl). The pH
gradient is produced by an electric field. Before an electric field is applied the gel has a
uniform pH-value and almost all the carrier ampholytes are charged. When an electric
field is applied, the negatively charged ampholytes move towards the anode, the
45
positively charged ones to the cathode and their ve10cities depend on the magnitude of
their net charge. The carrier ampholytes align themselves in between the cathode and the
anode according to their pl, and determine the pH oftheir environment. A stable gradually
increasing pH gradient depending on the initial mixture of ampholytes is formed.
Isoelectric points (Pl) of proteins are conveniently and accurate1y measured using
calibration proteins. By measuring the distance of a sample protein from a reference point
to where it focuses, its pl can be calculated from the pH gradient profile.
Isoelectric focusing to determine the isoelectric pH of the sample was carried out on a
PhastGelIEF gel 3-9 using the PhastSystem™ (Figure. 3.2 PhastSystem manual). IEF in
the pH interval 3-9 was performed without buffer strips, the electrodes were in direct
contact with the polyacrylamide (PAA) gels. As a reference, an isoelectric focusing
calibration kit (pH range 3-10) (Amersham Biosciences, Little Chalfont Buckinghamshire,
England) containing Il standard proteins was used. The running condition for the system
was obtained from the PhastSystem separation technique file number 100. The gels were
stained using Coomassie Blue R after the running using conditions outlined in the
PhastSystem development technique file number 210.
3.3.12.1 Running conditions for IEF
The method presented in Table 3.4 gives the running conditions for one gel. The methods
for isoelectric focusing contain three steps: a prefocusing step, a sample application step
and a focusing step. The duration of each step is controlled by volt
x
hours (Vh)
(PhastSystem manual).
The pH gradient is formed in the prefocusing step. The sample applicators were loaded
during the prefocusing step and the samples were applied to the gel surface with a sample
applicator using a micro-syringe. After the programmed prefocusing, the sample
applicator was lowered and the samples were delivered to the gel. The applicators were
raised at the beginning of focusing step and the proteins migrated to their isoelectric
points.
46
3.3.12.2 Coomassie Blue staining of PhastGel IEF media
After electrophoresis, the gels were stained with Coomassie Brilliant Blue R staining
(Table 3.5 and 3.6). The gels were fixed with 20% TCA for 30 min at 20°C. The gels were
washed with 30% methanol and 10% acetic acid for 2 min. Then staining of the gels was
done using 0.1 % Coomassie Brilliant Blue R-250 in aqueous solution of 30% methanol
and 10% acetic acid overnight. Finally, gels destaining was accompli shed in aqueous
solution of 30% methanol and 10% acetic acid until the background was c1ear.
3.3.12.3 Silver staining of PhastGel IEF media
At low protein concentrations, silver staining method was used. After electrophoresis, the
gels were stained with silver nitrate (Table 3.7). The gels were fixed with 20% TCA for 5
min at 50°C. The gels were washed with 10% ethanol and 5% acetic acid for 6 min at
50°C. Sensitization of the proteins was done using 5% glutaraldehyde solution for 6 min
at 50°C. Then the gels were stained using 0.4% silver nitrate for 2 min at 40°C. The gels
were developed in 2% formaldehyde solution for 30 seconds at 30°C. Finally, gels
destaining was accompli shed using background reducer (Table 3.8).
3.3.13 N-terminal amino acid sequence analysis
The N-terminal amino acid sequence of hoki trypsin was performed by Sheldon
Biotechnology Center, McGill University. To analyze the N-terminal sequence of the
purified enzyme, the trypsin protein band was electroblotted from the non-stained gels to
polyvinylidenedifluoride (PVDF) membrane (Mini ProBlott Membranes, Applied
'--'.
,"''''''''''
"..."
.n,.'
.
".,.""
..
,
Biosystems, Foster City, CA, USA) after SDS-PAGE for 35 min at 200 mA in "XCELL
II mini cell" unit ( Novex, San Diego, CA, USA). N-terminal sequencing of the protein
bands on PVDF membranes was done with a Procise Prote in Sequencer model 492
(Applied Biosystems, Foster City, CA, USA). Free N-terminal residues were generated by
Edman chemistry and converted to stable PTH-residues. Identification of each PTHresidue was done by injection on a Spheri-5 PTH column (220 X 2.1 mm) linked to an
HPLC system. The system was calibrated using a PTH-residue standard kit containing
every amino acid (Sigma-Aldrich, St. Louis, MO, USA).
47
3.4 Results and discussions
3.4.1 Purification of hoki trypsin
3.4.1.1 Recovery of trypsin (affinity) fraction
The stepwise purification of trypsin extracted from the pyloric ceca powder of hoki is
summarized in Table 3.9. These results were obtained from lOg dried defatted pyloric
ceca powder of hoki. The levels of purification from crude homogenate sequentially to
Brij 35 soluble fraction, ammonium sulfate precipitation fractionation, acetone
precipitation fractionation and affinity chromatographic procedures were presented in
Table 3.9. The overall purification of trypsin was 44.6 fold and recovery was
approximately 24%. The yield of purified trypsin was about 41 mg protein / 10 g dried
defatted ceca powder (0.4%), based on the standard curve for the protein deterrnination in
Figure 3.3.
During this 'purification procedure, total protein decreased from the first step (initial
extract) to the last step (SBTI- affinity fraction). As expected, the specific activity
increased from the first step (initial extract) to the last step (SBTI- affinity fraction),
indicating that most of unwanted impurities were removed and the sample became more
and more pure in the course of the whole purification process. Total activity at the first
step (initial extract) was lower than that of subsequent steps. The reason for the increase
of the total activity from Brij 35 soluble fraction to acetone fraction may be due to the
activation of trypsinogen to active trypsin or the removal of some native trypsin inhibitors
.w~i<?~
are present the first step during the purification process. The total activity of the
SBTI- affinity fraction decreased sharply. A total of 75% of the activity was lost during
the last step of the purification procedure. It is suggested that this was due to partial
denaturation of trypsin by the 5 mM Hel used as eluent in affinity chromatography step.
3.4.1.2 Elution profile of the trypsin fraction on SBTI- Sepharose affinity media
The elution profile of the trypsin fraction from SBTI- Sepharose affinity column after the
application of the acetone fraction is shown in Figure 3.4. This figure shows the
absorbance at 280 nm (A280) as weIl as the BAPNA trypsin activity (Unit/mL). Ninety
fractions were collected during the affinity chromatography. There was a graduaI increase
48
in the absorbance at 280 run up to a maximum from fraction 6 to 12 and then a decrease
after fraction 15, but fraction 6 to 12 exhibited no BAPNA trypsin activity. There was a
stable base line of eluent at 280 nm for more than 30 fractions (between 25 to 59)
indicating that the complete c1ean-up of the unbounded impure protein from the column.
Thus, a 5 mM of HCI (pH 2.8) solution was applied after the fraction 60 to dislodge the
trypsin from the ligand. Based on the pH change, a second graduaI increase in the
absorbance at 280 nm was observed from fractions 68 to 75, which showed high protein
concentrations as weIl as high trypsin BAPNA hydrolyse activity. However, 5 mM of
HCI solution was observed to denature the trypsin from the hoki quiekly. Thus, a slight
modification was made to the original procedure by Simpson and Haard (1984b) to avoid
denaturation of the trypsin. The modification entailed increasing the flow rate after
adding the HCI to 60 mL/h to reduce the time of trypsin in the strong acid condition, and
collection of 1 mL fraction after HCI addition drop wise into 2 mL of the extraction
buffer (0.05 M Tris-HCI containing 0.5 M NaCI and 0.02 M CaCh·2H2û, pH 7.8) to
rapidly neutralize the acid and maintain the activity of trypsin. The fractions (from 68 to
75) showing BAPNA trypsin activity were combined together, and the specific activity
(BAPNA as substrate) ofthis enzyme was 0.5752 units/mg (Table 3.9). Then the trypsin
fractions were simultaneously desalted and concentrated by ultrafiltration.
3.4.1.3 Storage stability of hoki trypsin fraction
Purified enzymes often need to be stored for an extended period of time while retaining
their original structural integrity or activity. The extent of storage can vary from a few
days to more than a year and is dependent on the nature of the protein and the storage
conditions used (Pierce Bioteehnology Ine., 2005). Generally, enzymes are stable at low
temperatures (Schwimmer, 1981). Storage at room temperature often leads to graduaI
enzyme degradation or inactivity. The storage stability of hoki trypsin fraction at 4°C
from the modified affinity chromatography step is shown in Figure 3.5. There was no
remarkable loss of specific activity of the purified trypsin fraction (about 77% initial
activity was retained) when it was stored at 4°C for 14 weeks. This indicated that hoki
trypsin can maintain the integrity of the three dimensional structure of the active site for a
relative long time at low temperature, beeause even very slight alterations in the
49
stereochemical structure of enzymes are sufficient to destroy enzymatic activity (Juul,
1967).
3.4.2 pH optimum and stability
The effect of pH on the rate of BAPNA hydrolysis was examined and the results were
presented in Figure 3.6. The optimum pH of hoki trypsin activity for hydrolysis of
BAPNA was found to be 9.0. This optimum value was similar to those previously
reported for rainbow trout (Kristjansson, 1991), salmon trypsins (Dimes et al., 1994) and
anchovy trypsin (Heu et al., 1995). Castillo-Yanez et al. (2005) reported that trypsin from
pyloric ceca of Monterey sardine (Sardinops sagax caerulea) had optimum pH of 8.0 on
BAPNA. Proteases from aH three tuna species had an optimal activity at pH 9.0 when
casein was used as a substrate (Klomklao et al., 2004). The relatively higher activity of
hoki trypsin observed at alkaline pH range from 7.5 to 10 (Figure 3.6), is consistent with
the optimum for digestive enzyme activity generally found with most fish (De-Vecchi and
Coppes, 1996).
The effect of pH on stability was evaluated by measuring enzyme residual activity after
incubation at various pH conditions for 30 min at 25°C. Hoki trypsin was stable in the pH
range from 6.0 to Il.0, and a remarkable loss of activity was observed below pH 6
(Figure 3.7). Most trypsins from marine fish are highly unstable under acidic conditions
and more stable at neutral to slightly alkaline conditions (Hjelmeland and Raa, 1982;
Kristjansson, 1991; Kishimura and Hayashi, 2002; Klomklao et al., 2004; Castillo-Yanez
et ai., 2005).
3.4.3 Temperature optimum and thermostability
The optimum temperature for hoki trypsin amidase activity using BAPNA as substrate
was 60°C (Figure 3.8). Above 6SoC the activity decreased sharply. This optimum
temperature (60°C) for hoki trypsin is similar to that reported for rainbow trout trypsin
(Kristjansson, 1991), mullet (Mugi! spp) trypsin (Pavlisko et al., 1999), tambaqui trypsin
(Bezerra et al., 2001) and chinook salmon trypsin (Kurtovic et al., 2006) hydrolysis of
BAPNA. It is higher than the temperature optima for other fish trypsin that have been
50
reported, such as Greenland cod trypsin (30°C; Simpson and Haard, 1984b), Atlantic cod
trypsin (55°C; Asgeirsson et al., 1989), muUet (Mugi! cephalus) trypsin (55°C; Guizani et
al., 1991), starfish (Asterina pectinifera) trypsin (55°C; Kishimura and Hayashi, 2002)
and Monterey sardine trypsin (55°C; Castillo-Yanez et al., 2005).
The thermal stability data of hoki trypsin after incubation for various times from 30 Oc to
70°C are shown in Figure 3.9. The hoki trypsin was observed to be quite stable below
40°C, and approximately 80% of the initial activity of the trypsin was retained after 30
min incubation at 50°C. However, at higher temperatures, the stability decreased with the
trypsin becoming almost completely inactivated after 30 min at 60°C, and losing aU
activity in less then 5 min at 70°C.
3.4.4 Kinetics studies
The apparent Michaelis-Menten constant (Km') and maximum velocity (V max) for hoki
trypsin amidase and esterase reactions were determined using the Lineweave-Burk
procedure (Figure 3.10). The hydrolysis rates were measured at different substrate
concentrations, then a plot of IN versus 1/[S] was constructed and fitted in a linear
model to get a Lineweave-Burk equation. The V max and Km' values were calculated from
the slopes and the intercepts of Lineweave-Burk equation, respectively. The turnover
number (kcat) was obtained by V max / J..lmol trypsin.
Table 3.10 shows the results obtained from kinetic measurements at 25°C using BAPNA
and TAME as substrates. The apparent Michaelis-Menten constant (Km') and substrate
turnover numbers (kcaÙ for the hydrolysis of BAPNA by hoki trypsin were 0.06 mM and
0.33
S-I,
respectively; and 2.08 mM and 19.0 S-I for the hydrolysis ofTAME, respectively.
The catalytic efficiency (kcatlKm') of hoki trypsin for hydrolysis of BAPNA and TAME
were 5.5
S-I
mM- I and 9.13
S-I
mM-l, respectively.
A comparison of amidase and esterase activities of hoki and bovine trypsins is shown in
Table 3.11. The ratio of kcat (BAPNA) to kcat (TAME) for hoki trypsin was six times more
than that of bovine trypsin. The catalytic efficiency (kcat/Km') ratio for the
51
amidase/esterase reactions was observed 461 times higher for the hoki trypsin than for the
bovine trypsin. The kcat and catalytic efficiency for the hydrolysis of ester (TAME) were
higher than amide (BAPNA) for hoki trypsin, indicating that it facilitated the hydrùlysis
ester much easier than the hydrolysis of the amide. However, compared with bovine
trypsin, hoki trypsin showed better amidase activity than bovine trypsin.
3.4.5 Enzyme inhibition studies
The effects of various inhibitors on the hoki trypsin-BAPNA hydrolyse activity were
measured in order to confirm this enzyme's identity. As shown in Table 3.12, hoki trypsin
was inhibited by aU the three weU-known trypsin inhibitors investigated namely SBTI,
aprotinin and benzamidine. Hoki trypsin was also inhibited by the serine protease
inhibitor PMSF, which indicates that it is a serine protease like other trypsins. The data
from the inhibition studies also support the notion that the hoki enzyme belongs to the
trypsin family of enzymes.
Table 3.12 shows that hoki trypsin was more sensitive to SBTI and aprotinin inhibition
than to PMSF and benzamidine inhibition. For example, hoki trypsin was totally inhibited
by only 0.5 J-lM SBTI or aprotinin, whereas it was inhibited by about 85% with as much
as 10 mM PMSF or benzamidine.
3.4.6 Molecular weight determination
3.4.6.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE) of hoki trypsin
The purity of hoki trypsin was examined via SDS-polyacrylamide gel electrophoresis.
The SDS-PAGE data presented in Figure 3.12 shows the results for the protein standards
(lane St.) versus the fractions from the main purification step. It is evident from Figure
3.12 that most of the impurities were removed from fraction S-1 to fraction S-4, and after
the affinity chromatography, fraction S-5 only had one band in SDS-PAGE gel (Figure
3.11 and Figure 3.12).
Based on the molecular weight of the protein standards, a standard curve of relative
mobility (Rf) versus log molecular weights (M.W.) was prepared and used to determine
52
the molecular weight of the hoki trypsin (Figure 3.13). The Rf of the protein was
indicative of a molecular weight of about 26,600. This molecular weight ofhoki trypsin is
within the range of 22-28 kDa reported for trypsins from other fish species (Simpson,
2000).
3.4.6.2 MALDI-TOF mass spectrometry ofhoki trypsin
MALDI-TOF mass spectrometry has been widely used in analysis of protein molecules. It
is more sensitive and accurate than SDS-PAGE. The work of Rouse et al. (1995) inc1uded
a careful evaluation of the accuracy of mass calibration of the spectrurn. Accuracies were
found to be generally better than 0.1% above m/z 75. In the positive mode MALDI-TOF
MS analysis, inorganic or organic salts and matrix molecules, which are strong proton
donors, were used to provide positive charges to the sample molecules, and then the
analyte molecules, associated with a H+ or a metal ion, may fly in the electronic field to
the detector. MALDI is a soft ionization method, the molecules can't be fragmented
during the ionization, and therefore the molecular peaks can be detected. Aiso it is
possible to detect the double charged molecules in the spectrum. The flight time of the
analytes from the sample holder to the detector depends on the ratio of the molecular (or
complex) mass to the charge number they get (mlz). The molecules with smaller m/z ratio
fly faster. Sinapinic acid was added as a matrix molecule during the samples preparation.
The most intensive peak is a trypsin molecule plus a proton, and the following two small
peaks are a mono-charged trypsin associated with one and two matrix molecules
(sinapinic acid). And the difference between the two neighbor peaks is exactly the
molecular weight of sinapinic acid. From the MS spectrum (Figure 3.14), the molecular
weight of hoki trypsin was determined as 23,791 Da [M+H] +. There is another peak at
11,895.56 Da [M+2Hf+, which is the half value of the mono-charged trypsin peak
indicating that trypsin molecule possess two charges. These double charged peaks also
demonstrated the molecular weight ofhoki fish trypsin is 23,791 Da, which is lower than
the value determined from SDS-PAGE method. But this molecular weight ofhoki trypsin
is similar to values reported for other trypsins, such as carp trypsin 25,000 Da (Cohen et
al., 1981), Greenland cod trypsin 23,500 Da (Simpson and Haard, 1984b), mullet ttypsin
24,000 Da (Guizani et al., 1991), chum salmon trypsin 24,000 Da (Sekizaki et al., 2000),
53
true sardine trypsin and arabesque greenling trypsin both 24,000 Da (Kishimura et al.,
2006) and is in good agreement with the reported values for 23,800 -24,000 Da (Uchida
et al., 1984b).
3.4.7 Isoelectric focusing (IEF) of hoki trypsin
The results of the IEF study are shown in Figure 3.15 which shows a sl,ngle band for the
hoki trypsin. This single band from IEF further confirms the purity of the affinity purified
hoki trypsin. Two different IEF runs using two different staining agents after the focusing,
i.e., Coomassie Brilliant Blue G-250 and silver nitrate, both gave identical results. Base
on the pl values of the calibration standards (Figure 3.16), the isoelectric point of isolated
hoki trypsin was measured as 6.5, which indicated that this enzyme is an anionic protein
at neutral pH. Anionic trypsins are common in marine organisms such as sardine (pl 5.5,
5.3 and 4.9; Murakami and Noda, 1981), Gadus morhua (pl 6.6, 6.2 and 5.5; Asgeirsson
et al., 1989), rainbow trout (pl 4.9; Kristjansson, 1991), chum salmon (pl 5.1; Sekizaki et
al., 2000) and Monterey sardine (pl 5.0; Castillo-Yanez et al., 2005), while mammal
trypsins are generally cationic, e.g., the isoelectric point of bovine trypsin is around 9.3
(Keil, 1971).
3.4.8 N-terminal amino acid sequence analysis
Table 3.13 shows the N-terminal 20 amino acid sequence of hoki trypsin as
IVGGQECVPNSQPFMASLNY.
That
indicated
hoki
trypsin's
N-terminus
was
unblocked. The sequence displayed considerable homology with porcine trypsin (11
identical residues of 20), bovine trypsin (10 identical residues of 20), Atlantic salmon (12
identical residues of 20), Japanese anchovy (12 identical residues of 20), true sardine (12
identical residues of 20), Atlantic cod trypsin (11 identical residues of 20), and dogfish
trypsin (10 identical residues out of 20), but less so with the crayfish enzyme (7 identical
residues of 20). The N-terminal four amino acid sequence of hoki trypsin (IVGG) was
identical with those of the other fish mammalian trypsins (Table 3.13), such as bovine
(Walsh, 1970), porcine (Hermodsson et al., 1973), dogfish (Titani et al., 1975), crayfish
(Titani et al., 1983), Atlantic cod (Gudmundsdottir et al., 1993), Atlantic salmon (Male et
al., 1995), starfish (Ki shimura et al., 2003), Japanese anchovy (TR-I) (Ki shimura et al.,
54
2005) and true sardine (Ki shimura et al., 2006). The N-terminal amino acid sequence
further supports the notion that the hoki enzyme also belongs to the trypsin family of
enzyme.
For all the trypsins shown in Table 3.13, proline (Pro, P) residue was observed at position
13 except for the Atlantic cod (Gudmundsdottir et al., 1993). The fish trypsins except for
the crayfish (Titani et al., 1983) shared a charged glutamate (Glu, E) residue at position 6
whereas threonine (Thr, T) was most commonly found in mammalian pancreatic trypsins
(Table 3.13). Furthermore, hoki trypsin conserved cysteine (Cys, C) at position 7, which
is as the same as trypsins of mammalian and most of fish species.
3.5 Conclusion
The following conclusions are made for hoki trypsin based on the studies described in this
chapter.
1. Hoki trypsin is a single polypeptide chain with a molecular weight of about 24 kDa
based on MALDI-TOF MS analysis, whereas the SDS-PAGE method of analysis
indicated a molecular weight of approximately 26 kDa, which is consistent with that
molecular weight reported for other fish trypsins within the range of22-28 kDa (Simpson,
2000).
2. Hoki trypsin is an anionic trypsin based on its pl of 6.5, and this regards quite similar
to Gadus morhua (Asgeirsson et al., 1989).
3. The hoki trypsin was classified as authentic trypsin due to its catalytic specificity
towards two well-known trypsin specifie synthetic substrates BAPNA and TAME; its
alkaline pH optimum (pH 9.0) similar to other fish trypsins; and its sensitivity to wellknown trypsin inhibitors (SBT!, aprotinin, benzamidine, PMSF);
4. On the basis of its sensitivity to inhibition by PMSF, the hoki enzyme is identified as a
serine protease.
55
5. Hoki trypsin is acid labile, similar to trypsin and trypsin-like enzymes from other
marine organisms;
6. Hoki trypsin had a higher temperature optimum (60°C) than reported values for several
other fish species (30-55°C), but was similar to other fish trypsins such as rainbow trout
trypsin (Kristjansson, 1991), mullet (Mugi! spp) trypsin (Pavlisko et al., 1999), tambaqui
trypsin (Bezerra et al., 2001) and chinook salmon trypsin (Kurtovic et al., 2006).
7. On the basis of the kinetic properties, hoki trypsin showed higher amidase activity than
bovine trypsin.
8. The N- terminal 20 amine acids residues ofhoki trypsin, IVGGQECVPNSQPFMAS-
LNY, displayed considerable similarity with other fish and mammalian trypsins.
56
Table 3.1 Formulations ofSDS-PAGE Resolving Gel
Component
Resolving gel (14%)
Water
4.03 mL
1.5 M Tris-Hel, pH 8.8
3.75 mL
10% (w/v) SDS
0.15 mL
Acrylamide Ibis (29: 1)
7.0 mL
10% ammonium persulfate *
75 ilL
TEMED
7.5 ilL
* Prepared freshly
57
Table 3.2 Formulations ofSDS-PAGE stacking Gel
Component
Resolving gel (4%)
Water
6.1 mL
1.5 M Tris-HCI, pH 6.8
2.5 mL
10% (w/v) SDS
0.1 mL
Acrylamide /bis (29:1)
1.3 mL
10% ammonium persulfate *
50 ilL
TEMED
10 ilL
* Prepared freshly
58
Table 3.3 Solution for Running Electrophoresis and Staining Gel
Solution
Stock Sample Buffer
Composition
Distilled water (4.8 mL); 0.5 M Tris-HCl, pH 6.8
( 1.2 mL); Glycerol (1.0 mL); 10% (w/v) SDS (2.0
(purchased from BIO-RAD)
SDS-Reducing Sample Buffer *
Electrode Buffer (x5)
mL); 0.1 % bromophenol blue (0.5 mL)
2-mercaptoethanol ( 25 ilL, 5% in SDS-reducing
sample); Stock sample buffer (475 ilL)
Tris (15 g); glycine ( 72 g); SDS (5 g) in 1 L
aqueous solution, pH 8.3
Trichloroacetic acid (12 g) in 100 mL de-ionized
Fixation Solution
water
Coomassie brilliant blue R250 (0.1 % w/v) in 25%
Staining Solution
Destaining Solution
propanol, 10% acetic acid and de-ionized water
Acetic acid : propanol: water (ratio 1:2:7)
* Prepared freshly
59
Table 3.4 Pro gram for running IEF
Step
Stage
Vh
(W)
Temperature
(OC)
Voltage
Current
Power
(V)
(mA)
1
Prefocusing
2000
2
3.5
15
75
2
Sample application a)
200
2
3.5
15
15
3
Focusing b)
2000
5
3.5
15
410
a) Sample applicator down at step 2 after 0 Vh (accumulated 75 Vh).
b) Sampleapplicator up atstep 3 after 0 Vh (accumulated 90 Vh).
Note:
V, volt;
mA, milliampere;
W, watt;
Vh, volt x hour
60
Table 3.5 Coomassie Brilliant Blue staining method for PhastGel IEF media
Step
(min)
Temperature
(OC)
Time
Solution
1. Fixation
Fixing solution
30
20
2. Washing
Destaining solution
2
20
3. Staining
Staining solution
ovemight
20
4. Destaining
Destaining solution
Until background is c1ear
20
61
Table 3.6 Coomassie Brilliant Blue staining solution for PhastGel IEF media
Solution
Composition
Fixing solution
Trichloroacetic acid (20 g) in 100 mL deionized water
0.1 % Coomassie Brilliant Blue R-250 in 30 % Methanol, 10 %
Staining solution
acetic acid in distilled water
Destaining solution
30 % Methanol, 10 % acetic acid in distilled water (3:1:6)
Adapted from Amersham Pharmacia Biotech instruction manual
62
Table 3.7 Silver staining method for PhastGel IEF media
Step
Solution
Time
Temperature
(min)
(OC)
1. Fixation
Fixing solution
5
50
2. Washing
Washing solution
6
50
3. Sensitization
Protein sensitization solution
6
50
4. Staining
Staining solution
2
40
5. Developing
Developing solution
0.5
30
6. Destaining
background reducer
overnight
20
63
Table 3.8 Silver staining solutions for PhastGel IEF media
Solution
Composition
Fixing solution
Trichloroacetic acid (20 g) in 100 mL distilled water
Washing solution
Protein sensitization solution
Staining solution
Developing solution
10 mL ethanol, 5 mL acetic acid in 100 mL distilled
water
5% glutaraldehyde
0.4% silver nitrate (0.4 g silver nitrate in 100 mL
distilled water)
2% formaldehyde in 2.5% sodium carbonate
1.6 g sodium thiosulphate, 3.7 g Tris-HCI in 100mL
Background reducer
distilled water
Adapted from Amersham Pharmacia Biotech instruction manual
64
Table 3.9 Purification oftrypsin from the pyloric cee a ofhoki
Step
Initial extract
Brij 35 soluble
fraction
Total
Total
Total
Specifie
Volume
Prote in
Activity
Activity
(mL)
( mg)
(units)
( units/mg)
122
7740.90
99.83
115
7193.25
41
Recovery
Purification
(%)
(-fold)
0.0129
100
1.00
158.79
0.0221
159.1
1.71
498.15
121.57
0.2440
121.8
18.9
15
313.88
130.43
0.4155
130.7
32.2
35
41.48
23.86
0.5752
23.9
44.6
(NHù2 SÛ4
fraction
(40%-60%)
Acetone fraction
SB TI- affinity
fraction
65
Table 3.10 Kinetic properties ofhoki trypsin in the hydrolysis ofBAPNAand TAME
Note:
Substrate
Km'(mM)
kcat * (S-l)
BAPNA
0.06
0.33
5.50
TAME
2.08
19.00
9.13
kcat / Km' t
(S-l
mM- 1)
* kcat : Turnover number
tCatalytic efficiency is the ratio of turnover number to Km' at 25 oC (Pollock, 1965)
66
Table 3.11 Comparison of amidase and esterase activities of hoki and bovine trypsins
Turnover number
Catalytic efficiency
amidase/esterase xl 00
amidase/esterase xl 00
HT
BT#
HTIBT
HT
BT#
HTIBT
1.74
0.29
6.00
60
0.13
461.54
Note: HT, hoki trypsin; BT, bovine trypsin
# Data from Simpson, B. K. and Haard, N. F. (1984c) Cano J. Biochem. Cell Biol.
Vol. 62, 894-900
67
Table 3.12 The effect of inhibitors on the activity ofhoki trypsin using BAPNA as
substrate at 25°C
Inhibitor
SBTI
Aprotinin
Benzamidine
PMSF in 2-propanol
Concentration of inhibitor
o IlM (control a)
Inhibition (%) #
0
0.0625 IlM
12.5 ± 3.2
0.125
IlM
43.8 ± 0.0
0.25
IlM
85.0 ± 0.0
0.5
IlM
100.0 ± 0.0
oTIU*IL (control a)
0
1.55
TIU/L
18.8 ± 5,4
3.15
TIU/L
43.8 ± 0.0
6.25
TIU/L
81.3 ± 0.0
12.5
TIUIL
95.0± 1.1
o mM (control a)
0
0.25
mM
16.7 ± 0.0
0.5
mM
33.3 ± 0.0
1.0
mM
50.0 ± 0.0
2.5
mM
66.7 ± 0.0
5.0
mM
83.3 ± 0.0
o mM (control b)
0
2.5
mM
56.0± 6.9
5
mM
75.0 ± 7.1
10
mM
90.0 ± 3.7
* TIU: trypsin inhibitor units; SBTI: Soybean trypsin inhibitor; PMSF: Phenyl methyl
sulfonyl fluoride
#
Average ± SD from triplicate determinations
a
One volume of the trypsin solution + one volume of distilled water
b
One volume of the trypsin solution + one volume of 2-propanol
68
Table 3.13 The N-termina1 amino acid sequence of hoki trypsin compared to trypsins
from other species
Residue at position
Trypsin
Reference
1
5
10
15
20
Hoki
IVGGQECVPNSQPFMASLNY
/
Dogfish
IVGGYECPKHAAPWTVSLNV
Titani et al., 1975
Crayfish
IVGGTDAVLGEFPYQLSFQE
Tita ni et al., 1983
Atlantic cod
IVGGYECTKHSQAHQVSLNS
Gudmundsdottir et al., 1993
Atlantic salmon
IVGGYECKAYSQPHQVSLNS
Male et al., 1995
Starfish
IVGGKESSPHSRPYQV - - --
Kishimura et al., 2003
IVGGYECQAHSQPHTVSLNS
Kishimura et al., 2005
True sardine
IVGGYECKAYSQPWQVSLNS
Kishimura et al., 2006
Bovine
IVGGYTCGANTVPYQVSLNS
Walsh, 1970
Porcine
IVGGYTCAANSVPYQVSLNS
Hermodsson et al., 1973
Japanese anchovy
(TR-I)
69
Molecular weight markers
-
....--------------.
.......---------.........---............---
~
-Tracromg
dye
....
--
~~~----~----~------.---~--~
Unlmown proteins
Figure 3.1 Migration of each protein on SDS geL
70
1
Figure 3.2 PhastSYstein. (1) Sëparatiôncbmpartinent, (2) separatîon alid c6iltr61ünit.
(3) development unit, (4) development chamber.
71
0.8
0.7
El
0.6
~
0.5
1::::
0
I.r'l
~
Q)
0
0.4
-€0
0.3
-<
0.2
â
y = 2.3105x + 0.0041
2
R
a:J
~
= 0.9998
0.1
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
BSA concentration (mg/ml)
Figure 3.3 Calibration curve for protein content detennination using bovine serum
albumin (BSA) as standard. Aliquots of stock BSA concentration of 1 mg/mL were used
to prepare the standard curve.
72
4
lA
~
~
3.5
3
2.5
0
l-
1-
•
l,
- ...... - BAPNA activity
t:O
1 1
I
1
1
1
1
I~I1
2 r
1
1-
-
0.8
r
0
0
....\.....
20
~ ....
40
~
_._.
<::
:>
0
.-+
'--<
004
1
SmM
Hel
:>
~
.-+
: ~\
1
0.5
1
- 0.6
~ 1~
M
1.5
-
1 \
l-
00
<
1.2
I \
1 1
Absorbance at 280nm
0.2
l.
60
80
".........
S
S
~
'-"
0
100
Fraction Number
Figure 304 Affinity chromatography on SBTI-Sepharose 4B. Acet{)ne·fraction was applied
to the affinity column equilibrated in 0.05 M Tris-HCI containing 0.5 M NaCI and 0.02 M
CaCh'2H2û, pH 7.8. After washing with the same equilibration buffer (fractions 10 - 60),
the adsorbed material was eluted with 5 mM HCI (fractions 61-90). The effluent was
promptly neutralized with equilibration buffer (pH 7.8). The figure shows the Abs at 280
nm (-) and the BAPNA amidase activity profile (- - - ).
73
Store at 4 oC
~
eJ)
~
.1:
=
---=
.....0
......~
0.7
0.6
0.5
004
0.3
CJ
0.2
....
CJ
0.1
CJ
CIl
0
~
=
rÊ
0
2
4
6
8
10
12
14
Storage time (weeks)
Figure 3.5 Storage stability oftrypsin fraction at 4°C.
74
16
pH optimum
120
~
"-"
~
=
100
.è
.,..
80
;S
~
-<
60
;S
40
aJ
20
aJ
-=
"
0
0
2
4
6
8
9
10
12
pH
Figure 3.6 Profile of change in hoki trypsin amidase activity with various pH from 2 to 10
at 25°C. Buffer solutions are described in the Materials and Methods section. Percentage
of enzyme activity was estimated based on the highest activity detected in this assay as
100%.
75
pH s tability
-----
120
c~
100
è
.,..
.~
80
.....
~
-<
60
;5
.s
40
~
~
~
20
0
0
2
4
6
8
10
12
pH
Figure 3.7 pH stability of hoki trypsin after 30 min of incubation in various buffers pH
varying from 2 to Il at 25°C. Percentage of enzyme activity was estimated based on the
highest activity detected in this assay as 100%.
76
Temperature Optimum
-.
'$.
--t-
.,.
;,
120
100
80
~
~
-<
60
;,
.......
=
40
~
~
~
20
0
0
20
40
60
80
100
Temperature (OC)
Figure 3.8 Effect oftemperature on the BAPNA activity of hoki trypsin. Activity was
evaluated using 1 mM BAPNA as substrate at pH 8.2 and changing temperature from
20°C to 80°C. Percentage of enzyme activity was estimated based on the highest activity
detected in this assay as 100%.
77
Thermostability
~......:t:i~X
"'D"
---- ,
100
~
=
80
~
60
.....è
.........
-<
,
11.
•
\
-....=
40
~
20
o
a
1
\
30 e
-, x, -4O e
"'0-" 50 e
60 e
il
- 0 - - 70 e
D
D
D
D
,
"CI
fil
'
\
\
CJ
eo:
,
D
+-~------~--------~--------~------~
o
20
40
60
80
Incubation Time (min)
Figure 3.9 Thermostability of hoki trypsin. Residual activity at pH 8 after incubation of
enzyme extract with 1 mM BAPNA for 60 min at temperatures from 30°C to 70°C.
78
Lineweave-Burk Plot
Lineweave-Burk Plot
0.04
180
160
0.035
140
/
120
JOO
y = 2.0304x + 32.737
R 2 = 0.9997
80
60
0.03
0.025
40
, ~rf " - IN max
·20
·10
0
JO
20
40
50
60
\,,'
70
·0.2
y= O.0436x + 0.021
R2 =0.9916
Jin "-
-l/Km
30
,,"
/= O.04~6,
-<>.1
1/ (SI (l/mM)
1N ma,
0.1
0.2
0.3
0.4
1/ (SI (lImM)
Roki trypsin-BAPNA amidase reaction
1/v = KmN max
Roki trypsin-TAME esterase reaction
X
1/[S] + INmax
Figure 3.10 Lineweave-Burk plots for trypsin kinetics.
79
Figure 3.11 SDS-PAGE of protein fractions obtained during purification of trypsin from
the pyloric ceca of hoki. Lane St., standard proteins: phosphorylase b(97.4 kDa); bovine
serum albumin (66.2 kDa); ovalbumin (45 kDa); carbonic anhydrase (31 kDa); soybean
trypsin inhibitor (21.5 kD); lysozyme (14.4 kDa). Lane S-l, crude homogenate proteins;
Lane S-2, Brij 35 soluble fraction; Lane S-3, ammonium sulfate precipitation fraction;
Lane S-4, acetone precipitation fraction; Lane S-5, SBTI-affinity fraction.
80
Phosphorylase b
97.4KDa
Bovine serum albumin
66.2KDa
Ovalbumin
45KDa
Carbonic anhydrase
31KDa
Purified trypsin from hoki
Soybean trypsin inhibitor
Lysozyme
21.5KDa
14.4KDa
2
Figure 3.12 SDS-PAGE of purified hoki trypsin. Lane 1: standard proteins; lane 2:
purified hoki trypsin.
81
5.1
•
5
4.9
Y = -0.95x + 5.1266
2
R = 0.9827
4.8
4.7
~ 4.6
~
~
~
..:l
4.5
4.4
4.3
4.2
4.1
0
0.2
0.6
0.4
0.8
1
1.2
Relative Mobility (Rf)
Figure 3.13 Standard curve for molecular weight determination by SDS-PAGE.
82
:;,
23791.227
<"Ci
:400a
[M+H]+
c
lB
.s
300 a
20010
[M+H+SA]+
[M+2H]2+
10010
11895.566
,
[M+H+2SA]+
~.[M+2H+SA]2+
..IoWI
o
10000
12000
14000
16000
18000
20000
22000
l
24000
26000
28000
1
mz
Note: [M] is a trypsin molecule; [H] is a proton; [SA] is a sinapinic acid.
Matrix: Sinapinic acid (SA), FW: 224
MeO~COOH
1'-':::
HO
h
OMe
Figure 3.14 Positive mode MALDI-TOF mass spectrum of purified hoki trypsin.
Molecular weight ofhoki trypsin is 23,791 Da.
83
AB
Coomassie Brilliant Blue G-250 staining
Silver nitrate staining
Figure 3.15 Isoelectric focusing of hoki trypsin in PhastGel IEF 3-9. Lane A: pl value of
calibration standards 3-10; lane B: purified hoki trypsin sample.
84
10
9
Y = -0.15x + 9.995
2
R =0.9919
8
7
=â
6
5
4
•
3
0
10
20
30
40
50
Distance from cathode (mm)
Figure 3.16 Detennination of pH gradient profile using broad pl calibration standards on
PhastGel IEF 3-9.
85
CHAPTERIV
GENERAL CONCLUSIONS AND RECOMMENDATIONS
5.1 General conclusions
In this study, trypsin was extracted and purified from the pyloric ceca tissues of hoki and
was further characterized with respect to various physical and chemical properties, such as
the kinetic properties, temperature, pH and inhibition effects on the activity of the purified
trypsin. Based on the experimental data and related analysis and discussion, the following
interpretations conclusions are made.
i. The enzyme obtained after affinity chromatography migrated as a single band in
sodium dodecyl sulphate polyacrylamide gels as weIl as in isoelectric focusing gels,
which confirmed trypsin purity. The molecular weight of the isolated trypsin was
determined by SDS-PAGE to be approximately 26,000 Da, whereas the MALDI-TOF MS
analysis indicated a molecular weight of23,791 Da.
ii. Based on isoelectric focusing analyses, the isoelectric point of purified trypsin was
determined to be at pH value of 6.5, which indicated that this enzyme is an anionic
protein at neutral pH.
iii. The optimum pH of hoki trypsin activity for hydrolysis of BAPNA was pH 9.0.
The re1~ti~e high activity of hoki trypsin/was observëd at alkaline plI raIlge from?5 to
10, which is generally optimum for digestive enzyme activity of most fish (De-Vecchi and
Coppes, 1996). Hoki trypsin was stable in the pH range from near 6.0 to 11.0, but a
remarkable loss of activity was observed below pH 6.0.
iv. The optimum temperature for hoki trypsin hydrolysis of BAPNA was 60°C. The
enzyme was stable at temperatures below 40°C and exhibited unstable above SO°e. On
the other hand, the hoki trypsin maintained higher activity at lower temperature. For
86
example, at 30°C, relative activity of hoki trypsin was approximately 40% of the
maximum.
v. The turnover number (kcat) ratio for the amidase/esterase hydrolysis for hoki
trypsin was about 6 times that of bovine trypsin. The catalytic efficiency (kcat/ Km') ratio
for the amidase 1 esterase reactions was about 450 times higher for the hoki trypsin than
for bovine trypsin. On the basis of the kinetic properties, hoki trypsin showed higher
amidase activity than bovine trypsin.
vi. Hoki trypsin was inhibited by the serine protease inhibitor, PMSF, and by other
well-known trypsin inhibitors (SB TI, aprotinin, benzamidine). The enzyme showed much
greater susceptibility to SBTI and aprotinin inhibition than PMSF and benzamidine, since
smaller concentrations of SBTI and aprotinin totally inhibited amidase activity.
VIl.
The
N-terminal
amino
acid
sequences
of purified
hoki
trypsin,
IVGGQECVPNSQPFMASLNY, displayed considerable homology with other fish
trypsins.
Based on the above characteristics, it is c1ear that the hoki enzyme is an authentic trypsin.
5.2 Recommendations
In order to better understand the biochemistry/physiology of the hoki trypsin, further
basic srudies are recoininended to focus on the following:
The amino acid composition of hoki trypsin should be determined. From the amino acid
composition, the average hydrophobicity can be ca1culated, which gives an indication of
the contribution of hydrophobie residues to stability (Bigelow, 1967). Furthermore, a
comparison of the ratio of acidic to basic amino acid residues of hoki trypsin with other
trypsins might help to provide some insights into the pH effect on hoki trypsin activity
and/or stability.
87
More amino acid sequences should be identified via c10ning and expressing hoki trypsin in
microorganisms to explore the molecular basis of physical properties of this enzyme, such
as cold adaptation, catalytic efficiency, pH stability, temperature stability, specificities of
substrates and inhibitors and so on.
Furthermore, the practical applications of hoki trypsin also should be emphasized to
disc10se the potential utility of the enzyme in commercial applications.
88
CHAPTERV
REFERENCES
Ahsan, M. N. and Watabe, S. (2001). Kinectic and structural properties oftwo isoforms
of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus. J. Prot.
Chem. 20, 49-58.
AImas, K.A. (1990). Utilization of marine biomass for production of microbial growth
media and biochemicals. In Advances in Fisheries technology and Biotechnology for
Increased Profitability. Voigt, M. N. and Botta, J. R. (Eds.), pp. 361-372.
Amiza, M. A., Galani, D. and Owusu-Apenten, R. K. (1997). Cod (Gadus morhua)
trypsin heat inactivation: a reaction kinetic study. J. Food Biochem., 21,273-288.
Amiza, M. A. and Owusu Apenten, R. K. (2002). A single step purification of gastricsinlike proteinase from Atlantic cod (Gadus morhua). Online J. Biol. Sei., 2,591-595.
An, H., Seymour, T. A., Wu, J. W. and Morrissey, M. T. (1994). Assay systems and
characterization of Pacific whiting (Merluceius productus) protease. J. Food Sei., 59,
277-289.
Anonymous (1972) Trypsin (bovine pancrease). In Enzymes, Enzyme reagents, Related
Biochemicals. Worthington Biochemical corporation, Freehold, NJ, USA, pp. 125-126.
Anonymous (1978). In Enzymes and Related Biochemicals. Worthington Biochemical
corporation, Freehold, NJ, USA, pp. 196-197.
Anson, M. L. (1938). The estimation of pepsin, trypsin, papain, and cathepsin with
hemoglobin. J. Gen. Physiol., 22, 79-89.
89
Asgeirsson, B. and Bjarnason, J. B. (1991). Structural and kinetic properties of
chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.
Comp. Biochem. Physiol., 99B, 327-335.
Asgeirsson, B. and Bjarnason, J. B. (1993). Properties of elastase from Atlantic cod, a
cold-adapted proteinase. Biochim. Biophys. Acta, 1164, 91-100.
Asgeirsson, B., Fox, J. W. and Bjarnason, J. B. (1989). Purification and characterization
oftrypsin from the poikilotherm Gadus morhua. Eur. J Biochem., 180, 85-94.
Barrett, A. J. (1994). Classification of peptidases. In Proteolytic Enzymes: Serine and
Cysteine Peptidases. Methods Enzymol., Barrett, A. J. (Ed.), Academic Press, New York,
USA,244, 1-15.
Beirao, L. H., Mckintoch, M. L, Evanilda, T. and Cesar, D. (2001). Purification and
characterization oftrypsin-like enzyme from the pyloric caeca of cod (Gadus morhua) II.
Brazilian Arch. Biol. Techn. 44: 33-40.
Bender, M. L. and Killheffer, J. V. (1973). Chymotrypsins. CRC Crit. Rev. Biochem., 1,
149-199.
Bender, M. L. and Kézdy, F. J. (1964). The current status of the a-chymotrypsin
mechanism. J. Am. Chem. Soc., 86, 3704-3714.
Bender, M. L., Killheffer, J. V. and Kézdy, F. J. (1964). An identity of the rates of
deacylation of nonionic acyl-a-chymotrypsins and acyl-trypsins. J. Am. Chem. Soc., 86,
5330-5331.
Benoiton, L. and Deneault, J. (1966). The hydrolysis of two epsilon-N-methyl-L-lysine
derivatives by trypsin. Biochim. Biophys. Acta, 113, 613-616.
90
Berglund, G. L, Smalas, A. O., Outzen, H. and Willassen, N. P. (1998). Purification and
characterization of pancreatic elastase from North Atlantic salmon (Salmo salar). Mol.
Mar. Biol. Biotech., 7, 105-114.
Bergmann, M. (1942). A classification of proteolytic enzymes. Advan. Enzymol.,
Interscience Publishers, New York, USA, 2,49-68.
Bezerra, R. S., Santo, J. F., Paiva, P. M. G., Correia, M. T. S., Coelho, L. C. B. B., Vieira,
V. L. A. and Carvalho, L. B. (2001). Partial purification and characterization of a
thermostable trypsin from pyloric caeca oftambaqui (Colossoma macropomum). J. Food
Biochem., 25, 199-210.
Bigelow, C. C. (1967). On the average hydrophobicity of proteins and the relation
between it and protein structure. J. Theoret. Biol., 16, 187-21l.
Bjarnason, J. B. (2000). Fish serine proteases and their pharmaceutical and cosmetic use
Patent PCT, WO 00/78332 A2. December 28, 2000.
Bjarnason, J. B. and Benediktsson, B. (2001). Protein hydrolysates produced with the use
of marine proteases. Patent PCT, WO 01128353 A2 April 26, 200l.
Boyer, P. D. (1970). The Enzymes. 3d ed New York: Academic Press. 251-275.
Bradford, C. (1960). Tryptic enzymes of Chinook salmon. Arch. Biochem. Biophys., 89,
202-206.
Brewer, P., Helbig, N. and Haard, N. F. (1984). Atlantic cod pepsin. Characterization and
use as a rennet substitute. Can Int. Food. Sei. Technol. J., 17, 38-43.
91
Cano-Lopez, A., Simpson, B. K. and Haard, N. F. (1987). Extraction of carotenoprotein
from shrimp processing wastes with the aid oftrypsin from Atlantic cod. J Food Sei., 52,
503-506.
Carginalea,V., Trinchellab, F., Capassoa,C., Scudierob, R. and Parisi, E. (2004). Gene
amplification and co Id adaptation of pepsin in Antarctic fish. A possible strategy for food
digestion at low temperature. Gene, 336, 195-205.
Carter, P. and Wells, J. A. (1988). Dissecting the catalytic tri ad of a serine protease.
Nature, 332, 564-568.
Castillo-Yanez, F. J., Pacheco-Aguilar, R., Garcia-Carreno, F. L. and Toro, M. A. N.
(2005). Isolation and characterization of trypsin from pyloric ceca of Monterey sardine
Sardinops sagax caerulea. Comp. Biochem. Physiol., 140B, 91-98.
Chaplin, M. F. and Bucke, C. (1990). Enzymes Technology. Cambridge (UK):
Cambridge University Press, pp 40-79.
Chevrier, B., Schalk, C., D'Orchymont, H., Rondeau, J. M., Moras, D. and Tamus, C.
(1994). Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical
member of the co-catalytic zinc enzyme family. Structure, 2, 283-291.
Chong, A. S. C., Hashim, R., Chow-Yang, L. and Ali, A. B. (2002). Partial
characterization and activities of proteases from the digestive tract of discus fish
(Symphysodon aequifaseiata). Aquaculture, 203,321-333.
Cohen, T., Gertler, A. and Birk, Y. (1981). Pancreatic proteolytic enzymes from carp
(Cyprinus carpio)- 1. Purification and physical properties of trypsin, chymotrypsin,
elastase and carboxypeptidase B. Comp. Biochem. Physiol., 69B, 639-646.
92
Diaz-Lopez, M. and Garcia-Carreno, F. L. (2000). Applications of fish and shellfish
enzymes in food and feed products. In Seafood enzymes. Haard, N. F. and Simpson, B. K.
(Eds.), Marcel Dekker, New York, NY, pp. 571-618.
Dimes, L. E., Garcia-Carreno, F. L. and Haard, N. F. (1994). Estimation of protein
digestibility - III. Studies on the digestive enzymes from the pyloric ceca of rainbow trout
and salmon. Comp. Biochem. Physiol., 109A, 349-360.
De-Vecchi, S. D. and Coppes, Z. (1996). Marine fish digestive proteases-relevance to
food industry and south-west Atlantic region-a review. J. Food Biochem., 20, 193-214.
Erlanger, B. F., Kokowsky, N. and Cohen, W. (1961). The preparation and properties of
two new chromogenic substrates oftrypsin. Arch. Biochem. Biophys., 95, 271-278.
Evnin, L. B., Vasquez, J. R. and Craik, C. S. (1990). Substrate specificity of trypsin
investigated by using a genetic selection. Proc. Nat. Acad. Sei. USA, 87, 6659-63.
Goldenberg, H. and Goldenberg, V. (1950). pH dependence of the amino acid esterase
activities oftrypsin and chymotrypsin. Arch. Biochem., 29, 154.
Goldberger, R. F. and Anfinsen, C. B. (1962). The reversible masking ofamino groups in
ribonuc1ease and its possible usefulness in the synthe sis of the protein. Biochemistry, 1,
401- 405.
Graycar, T. P. (1999). Proteolytic Cleavage, Reaction Mechanisms. In Encyclopedia of
Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. Flickinger, M.C.
and Drew, S. W. (Eds.), John Wiley & Sons, New York, NY, pp. 2214-2222.
Gudmundsdottir, A., Gudmundsdottir, E., Oskarsson, S., Bjamason, J. B., Eakin, A. K.
and Craik, C. S. (1993). Isolation and characterization of cDNAs from Atlantic cod
encoding two different forms oftrypsinogen. Eur. J. Biochem., 217, 1091-1097.
93
Guizani, N., Rolle, R. S., Marshall, M. R. and Wei, C. 1. (1991). Isolation and
characterization of a trypsin from the pyloric ceca of mullet (Mugi! cephalus). Comp.
Biochem. Physiol., 98, 517-521.
Haard, N. F. (1990). Enzymes from food myosystems. J Muscle Food, 1,293-338.
Haard, N. F. (1992). A review of proteolytic enzymes from marine organisms and their
application in the food industry. J Aqua. Food Prod Technol., 1, 17-35.
Haard, N. F., Dimes, L. F., Arndt, R. E. and Dong, F. M. (1996). Estimation of protein
digestibility 0 IV. Digestive proteinases from the pyloric caeca of coho salmon
(Oncorhynchus kisutch) fed diets containing soybean meal. Comp. Biochem. Physiol.,
115B, 533-540.
Haard, N. F., Feltham, L. A. W., Helbig, N. and Squires, E. J. (1982). Modification of
protein with proteolytic enzymes from the marine environment. In Modification of
Proteins in Food, Pharmaceutical, and Nutritional Sciences. Feeney, R. and Whitaker, J.
(Eds.), Advances in Chemistry Series, No. 198, American Chemical society, Washington,
DC, pp. 223-244.
Haard, N. F. and Simpson, B. K. (1994). Proteases from aquatic organisms and their uses
in the seafood industry. In Fisheries Processing: Biotechnological Applications. Martin,
A. M. (Ed.), Chapman and Hall, London, UK, pp. 132-154.
Han, X. Q. (1993). Recovery of digestive enzymes from Atlantic cod (Gadus morhua)
and their utilization in food processing. M.Sc. thesis. Dept. Biochemistry, Memorial
University ofNewfound1and, St. John's, NF, Canada.
Hermodson, M. A., Ericsson, L. H., Neurath, H. and Walsh, K. A. (1973). Determination
of the amino acid sequence of porcine trypsin by sequenator analysis. Biochemistry, 12,
3146-3153.
94
Heu, M. S., Kim, H. R. and Pyeun, J. H. (1995). Comparison oftrypsin and chymotrypsin
from the viscera of anchovy, Engraulis japonica. Comp. Biochem. Physiol., 112 B, 557567.
Hjelmeland, K. and Raa, J. (1982). Characteristics of two trypsin type isozymes isolated
from arctic capelin (Mallotus villosus). Comp. Biochem. Physiol., 71B, 557-562.
Hultin, H. O. (1978). Enzymes from organisms acc1imated to low temperatures. In
Enzymes, The Interface Between Technology and Economies. Danehy, 1. and Wolnak, B.
(Eds.), Marcel Dekker, New York, NY, pp. 161-178.
Hummel, B.C.W. (1959). A modified spectrophotometric determination of chymotrypsin,
trypsin and thrombin. Can. J. Biochem. Physiol., 37, 1393.
Jany, K. D. (1976). Studies on the digestive enzymes of stomachless bonefish Carassius
auratus gibelio (Bloch): endopeptidases. Comp. Biochem. Physiol., 53B, 31-38.
Jayaraman, G., Srimathi, S. and Bjarnason, J. B. (2006). Conformation and stability of
elastase from Atlantic cod, Gadus morhua. Biochim. Biophys. Acta, General Subjects,
1760,47-54.
Juul, P. (1967). Stability of plasma enzymes during storage. Clin. Chem., 13,416 - 422.
Keilova, H., Pliska, V., Keil, B. and Sorm, F. (1969). Trypsin c1eavage oftwo analogous
polypeptide substrates containing arginine or lysine. Physiol. chem. Phys., 1, 100-108.
Keil, B. and Keilova, H. (1964). Proteins. XC. Peptic and tryptic c1eavage of the B-chain
ofoxidized insulin. Collection Czech. Chem. Commun., 29, 2206-2215.
Keil, B. (1971). Trypsin. In The Enzymes. Boyer, P. D. (Ed.), Academic Press, New York,
vol. 3,249-275.
95
Ki shimura, H. and Hayashi, K. (2002). Isolation and characteristics of trypsin from
pyloric ceca of the starfish Asterinas pectinifera. Comp. Biochem. Physiol., 132 B, 485490.
Kishimura, H. and Hayashi, K. (2003). N-terminal amino acid sequence of trypsin from
the pyloric ceca of starfish Asterias amurensis. Fisheries Sei., 69, 867-869.
Kishimura, H., Hayashi, K., Miyashita, Y. and Nonami, Y. (2005). Characteristics oftwo
trypsin isozymes from the viscera of Japanese anchovy (Engraulis japonica). J Food
Biochem., 29, 459-469.
Kishimura, H., Hayashi, K., Miyashita, Y. and Nonami, Y. (2006). Characteristics of
trypsins from the viscera of true sardine (Sardinops melanostictus) and the pyloric caeca
of arabesque greenling (Pleuroprammus azonus). Food Chem., 97, 65-70.
Klomklao, S., Benjakul, S. and Visessanguan, W. (2004) Comparative studies on
proteolytic activity of splenic extract from three tuna species commonly used in Thailand.
J Food Biochem., 28, 355-372.
Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B. K. and Saeki,
H. (2006). Trypsins from yellowfin tuna (Thunnus albacores) spleen: Purification and
characterization.
Comp.
Biochem.
Physiol.,
In
press,
available
online
at
www.sciencedirect.com.doi: 10.1016/j. cbpb.2006.01.006.
Knuckey, 1., Sinclair, C., Surapaneni, A. and Ashcroft, W. (2004). Utilisation of seafood
processing waste - challenges and opportunities.
http://www.regional.org.au/au/asssi/supersoi12004/s7/oralI1662knuckeyi.htm
Kossiakoff, A. A., Chambers, J. L., Kay, L.M. and Stroud, R.M. (1977). Structure of
bovine trypsinogen at 1.9
A resolution. Biochemistry, 16, 654-664.
96
Kristjansson, M. M. (1991). Purification and characterization of trypsin from the pyloric
caeca ofrainbow trout (Oncorchynchus mykiss). J. Agric. Food Chem., 39, 1738-1742.
Kristjânsson, M. M. and Nielsen, H. H. (1992). Purification and characterization of two
chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus
mykiss). Comp. Biochem. Physiol., 101B, 247-253.
Kunitz, M. (1947). Crystalline soybean trypsin inhibitor: II. General properties. J. Gen.
Physiol., 30, 291-310.
Kurtovic, L, Marshall, S. N. and Simpson, B. K. (2006). Isolation and characterization of
a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha).
Comp. Biochem. Physiol., Part B 143, 432-440.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assemblly of the head
ofbacteriophage T4. Nature, 227, 680-685.
Lee, Y. Z., Simpson, B. K. and Haard, N. F. (1982). Supplementation of squid
fermentation with proteolytic enzymes. J. Food Biochem., 6, 127-134.
Leipner, 1. and Saller, R. (2000). Systemic enzyme therapy in oncology: effect and mode
of action. Drugs, 59, 769-780.
Lenard, J., Johnson, S. L., Hyman, R. W. and Hess, G. P. (1965). A continuous, automatic
method for the study of rate of hydrolysis of peptides and amides. Anal. Biochem., Il, 3041.
Low, P. S., Bada, J. L. and Somero, G. N. (1973). Temperature adaptation of enzymes:
Roles of free energy, the enthalpy, and the entropy of activation. Proc. Natl. Acad. Sci.
(USA), 70, 430-432.
97
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein
measurement with the Folin phenol reagent. J. biol. ehem., 193,265-275.
Macouzet, M., Simpson, B. K. and Lee, B. H. (2003) Cloning of cunner trypsonogen
cDNA in E. coli. Appl. Bioehem. Food Sei. Poliey, 1, 243-251.
Macouzet, M., Simpson, B. K. and Lee, B. H. (2005) Expression of a cold-adapted fish
trypsin in Pichia pastoris. FEMS Yeast Researeh, 5, 851-857.
Male, R., Lorens, J. B., Smalas, A. O. and Torrissen, K. R. (1995). Molecular cloning and
characterization of anionic and cationic variants of trypsin from Atlantic salmon. Eur. J
Bioehem., 232, 677-685.
Mares-Guia, M. and Shaw, E. (1965). Studies on the active center of trypsin. The binding
of amidines and guanidines as models of the substrate side chain. J Biol. Chem., 240,
1579-1585.
Martinez, A., Olsen, R. L. and Serra, J. L. (1988). Purification and characterization oftwo
trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus. Comp.
Biochem. Physiol., 91B, 677-684.
Mihalyi, E. (1978). Application of proteolytic enzymes to protein structure studies, 2nd ed.
Palm Beach: CRC Press, pp. 215-308.
Murakami, K. and Noda, M. (1981). Studies on proteinases from the digestive organs of
sardine. 1. Purification and characterization of three alkaline proteinases from the pyloric
caeca. Biochim. Biophys. Acta, 658, 17-26.
Norris, E. R. and Elam, D. W. (1940). Preparation and properties of crystalline salmon
pepsin. J Biol. Chem., 134, 443-454.
98
Okamoto,Y., Otsuka-Fuchino, H., Horiuchi, S., Tamiya, T., Matsumoto, J. J. and
Tsuchiya, T. (1993). Purification and characterization of two metalloproteinases from
squid mantle muscle, myosinase 1 and myosinase II. Biochim. Biophys. Acta, 1161, 97104.
Olsen, H. S. (1999). Enzymes, Protein Hydrolysis. In Encyclopedia of Bioprocess
Technology: Fermentation, Biocatalysis, and Bioseparation. Flickinger, M. C. and Drew,
S. W. (Eds.), John Wiley & Sons, New York, NY, pp. 1074-1095.
Orejana, F. M. and Liston, J. (1981). Agents ofproteolysis and its inhibition in patis (fish
sauce) fermentation. J Food Sei., 47, 198-203.
Outzen, H., Berglund, G. 1., Smalas, A. O. and Willassen, N. P. (1996). Temperature and
pH sensitivity of trypsins from Atlantic salmon, Salmo salar, in comparison with bovine
and porcine trypsin. Comp. Biochem. Physiol., 115, 33- 45.
Ovemell, J. (1973). Digestive enzymes of the pyloric caeca and their associated
mesentery in the cod (Gadus morhua). Comp. Biochem. Physiol., 46B, 519-531.
Pavlisko, A., Rial, A., Vecchi, S. D. and Coppes, Z. (1997a). Properties of pepsin and
trypsin isolated from the digestive tract of Parona signata "palometa". J Food
Biochem., 21,289-308.
Pavlisko, A., Rial, A. and Coppes, Z. (1997b). Characterization of trypsin purified from
the pyloric caeca of the southwest Atlantic white croaker Micropogonias furnieri
(Sciaenidae). J Food Biochem., 21, 383-400.
Pavlisko, A., Rial, A. and Coppes, Z. (1999). Purification and characterization of a
protease from the pyloric caeca of menhaden (Brevoortia spp) and mullet (Mugi! spp)
from the southwest Atlantic region. J Food Biochem., 23, 225-241.
99
Pfleiderer, G., Zwilng, R. And Sonnebom, H.H. (1967) On the evolution of
endopeptidases, 3. A protease ofmolecular weight 11,000 and a trypsin-like fraction from
Astacusfluviatilisfabr. Hoppe Seylers Z. Physiol. Chem., 348,1319-1331.
Pharmacia LKB Biotechnology (1988). Affinity Chromatography: principles & methods.
Pharmacia LKB Biotechnology S-751 82 Uppsala, Sweden, pp.12-18.
Pierce Biotechnology Inc. (2005). Protein stability and storage. In Technical resource.
www.piercenet.com.
Pollock, M.R. (1965). Purification and properties of penicillinases from two strains of
Bacillus licheniformis; a chemical, physicochemical, and physiological comparison.
Biochem. J., 94, 666-75.
Raa, J. (1990). Biotechnology in aquaculture and the fish processing industry: a success
story in Norway. In Advances in Fisheries Technology for Increased Profitability. Voigt,
M. N. and Botta, J. R. (Eds.), Technomic Publication Company, Lancaster, PA, pp. 509-
524.
Raa, J. (1997). New commercial products from waste of the fish processing industry. In
Making the Most of the Catch. Bremner, A., Davis, C. and Austin, B. (Eds.), Proceedings
ofthe Seafood Symposium, AUSEAS, Brisbane, Australia, pp. 33-36.
Ramakrishan, M., Hultin, H. O. and Atallah, M. T. (1987). A comparison of dogfish and
bovine chymotrypsins in relation to protein hydrolysis. J. Food Sei., 52, 1198-1202.
Rawlings, N. D. and Barrett, A. J. (1994). Families of serine peptidases. Methods
Enzymol.,244,19-61.
Reeck, G. R. and Neurath, H. (1972). Pancreatic enzymes of the African lungfish,
Protopterus aethiopicus. Biochemistry, 11, 503-510.
100
Rennex, D., Hemmings, B. A. and Hoffsteenge, J. (1991). cDNA cloning of porcine brain
prolyl endopeptidase and identification of the active-site seryl residue. Biochemistry, 30,
2195-2203.
Ritskes, T. M. (1971). Artificial ripening of matjes-cured herring with the aid of
proteolytic enzyme preparations. J. Fish. Res. Board Can., 69, 647-654.
Roderick, S. L. and Mathews, B. W. (1993). Structure of the cobalt-dependent methionine
aminopeptidase from Escherichia coli: a new type of proteolytic enzyme. Biochemistry,
32,3907-3912.
Rouse, J. C., Yu, W. and Martin, S. A. (1995). A comparison of the peptide fragmentation
obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a
tandem four sector mass spectrometer. J. Am. Soc. Mass Spectrom., 6, 822-835.
Schroder, H., Willassen, N. P. and Smalas, A. O. (1998). Structure of a non-psychrophilla
trypsin from a cold-adapted fish species. Acta Crystallographica, D54, 780-798.
Schwert, G. W., Neurath, H., Kauffman, S. and Snoke, J. E. (1948). The specifie esterase
aetivity oftrypsin. J. Biol. Chem., 172,221-239.
Schwimmer, S. (1981). Source Book of Food Enzymology. AVI, Westport Conneticut.
pp.27-89.
Sekizaki, H., Itoh, K., Murakami, M., Toyota, E. and Tanizawa, K., (2000). Anionic
trypsin from chum salmon: activity with p-aminophenyl ester and comparison with
bovine and Streptomyces griseus trypsins. Comp. Biochem. Physiol., Bi27, 337- 346.
Shahidi, F. (1994). Proteins from seafood processing diseards. In Seafoods Proteins.
Sikorski, Z. E., Pan, B. S. and Shahidi, F. (Eds.), Chapman and Hall, New York, NY, pp.
171-193.
101
Simpson, B. K. and Haard, N. F. (1984a). Trypsin from Greenland cod as a food
processing aid. J App!. Bioehem., 6, 135-143.
Simpson, B. K. and Haard, N. F. (1984b). Trypsin from Greenland cod, Gadus ogae.
Isolation and comparative properties. Comp. Bioehem. Physio!., 79B(4), 613-622.
Simpson, B. K. and Haard, N. F. (1984c). Purification and characterization of trypsin
from the Greenland cod (Gadus ogae). l. Kinetic and thermodynamic characteristics.
Can. J Bioehem. Cel!. Biol., 62, 894-900.
Simpson, B. K. and Haard, N. F. (1985). Characterization oftrypsin fraction from cunner
(Tautogolabrus adspersus). Comp. Bioehem. Physio!., 80B, 475-480.
Simpson, B. K. and Haard, N. F. (1987a). Cold-adapted enzymes from fish. In Food
Bioteehnology. Knorr, D. (Ed.), Marcel Dekker, New York, NY, pp. 495-527.
Simpson, B. K. and Haard, N. F. (1987b). Trypsin and trypsin-like enzymes from the
stomachless cunner. J Agr. Food Chem., 35, 652-654.
Simpson, B. K., Simpson, M. V. and Haard, N. F. (1989a). On the mechanisms of enzyme
action: Digestive proteases from selected marine organisms. Bioteeh. App!. Bioehem., 11,
226-234.
Simpson, B. K., Smith, J. P. and Yaylayan, V. (1989b). Kinetic and thermodynamic
characteristics of a digestive protease from Atlantic cod; Gadus morhua. J Food
Bioehem., 13,201-213.
Simpson, B. K., Simpson, M. V. and Haard, N. F. (1990). Properties oftrypsin from the
pyloric caeca of Atlantic cod (Gadus morhua). J Food Sei., 55, 959-97l.
102
Simpson, B. K., Smith, J. P. and Haard, N. F. (1991). Marine enzymes. In Encyclopedia
o/Food Science and Technology. Hui, Y. H. (Ed.), John Wiley and Sons, New York, NY,
pp. 1645-1653.
Simpson, B. K. (2000). Digestive proteinases from marine animaIs .. In Seafood Enzymes;
Haard, N. F. and Simpson, B. K. (Eds.), Marcel Dekker, New York, NY, pp. 191-213.
Snoke, J. E. and Neurath, H. (1949). Structural requitements of specifie substrates for
chymotrypsin. 1. The contribution of the secondary peptide group. Arch. BioChem., 2,
351-362.
Souza, A. A.G., Amaral, 1. P.G., Espirito Santo, A. R., Carvalho Jr, L. B. and Bezerra. R.
S. (2006). Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish
(Pseudupeneus
maculatus).
Food
Chem.,
In
press,
available
online
at
www.sciencedirect.com.doi: 10.1016/j. foodchem.2005.12.016.
Spilliaert, R. And Gudmundsd6ttir, A. (2000). Molecular cloning of the Atlantic cod
chymotrypsinogen B. Microb. Comp. Genomics, 5, 41-50.
Spittler, A.W. and Parmenter, R.E. (1954). The use of proteolytic enzymes
In
war
wounds. J. Int. Coll. Surg., 21, 72-78.
Squires, E. J., Haard, N. F. and Feltham, L. A. W. (1986). Pepsin isozymes from
Greenland cod. Gadus ogac. 2. Substrate specificity and kinetic properties. Can. J
Biochem. Cel! Biol., 64, 215-222.
Stefansson, G. (1988). Enzymes in the fishing industry. Food Technol., 42, 64-65.
Stefansson, G. and Steingrimsdottir, U. (1990). Application of enzymes for fish
processing in Iceland-present and future aspects. In Advances in Fisheries Technology
103
for Increased Profitability. Voigt, M. N. and Botta, J. R. (Eds.), Technomic Publication,
Lancaster, PA, pp. 237-250.
Stone, S. R., Rennex, D., Wikstrom, P., Shaw, E. and Hoffsteenge, J. (1991). Inactivation
of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and
identification of sites of modification. Biochem. J, 276,837-840.
Strater, N. and Lipscomb, W. N. (1995). Two-metal ion mechanism of bovine lens
leucine aminopeptidase: active site solvent structure and binding mode of L-Leucinal, a
gem-Diolate transition state analog, by X-ray crystallography. Biochemistry, 34, 1479214800.
Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H. and Blow, D. M. (1974). Crystal
structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-
A resolution. Biochemistry, 13,4212- 4228.
Titani, K., Ericsson, L. H., Neurath, H. and Walsh, K. A. (1975). Amino acid sequence of
dogfish trypsin. Biochemistry, 14,1358-1366.
Titani, K., Sasagawa, T., Woodbury, R. G., Ericsson, L. H., Dorsam, H., Kraemer, M.,
Neurath, H. and Zwilling, R. (1983). Amino acid sequence ofcrayfish (Astacusfluviatilis)
trypsin. Biochemistry, 22, 1459-1465.
Toi, K., Bynum, E., Norris, E. and Itano, H. A. (1965). Preliminary communications:
Chemical modification of arginine with 1,2-cyclohexanedione. J Biol. Chem., 240, 34553457.
Uchida, N., Tsukayama, K. and Nishide, E. (1984a). Purification and sorne properties of
trypsins from the pyloric caeca of chum salmon. Bulletin Jpn. Soc. Sei. Fish., 50, 129138.
104
Uchida, N., Tsukayama, K. and Nishide, E. (1984b). Properties of two anionic trypsins
from the pyloric caeca of chum salmon. Bulletin Jpn. Soc. Sei. Fish., 50, 313-324.
Uys, W. and Hecht, T. (1987). Assays on the digestive enzymes of sharptooth catfish,
Clarias gariepinus (Pisces: Clariidae). Aquaculture, 63,301-313.
Venugopal, V. and Shahidi, F. (1995). Value-added products from underutilized fish
species. Crit. Rev. Food Sci. Nut., 35,431-453.
Visessanguana, W., Benjakulb, S. and An, H. (2003). Purification and characterization of
cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle. Comp. Biochem.
Physiol., 134B, 477-487.
Walsh, K. A. (1970). Trypsinogens and trypsins of various speCles. In Proteolytic
Enzymes, Methods Enzymol., Perlmann, G. E. and Lorand, L.(Eds.), Academic Press,
New York, USA, 19,41- 63.
Walsh, K. A. and Wilcox, P. E. (1970). Serine proteases. In Proteolytic Enzymes,
Methods Enzymol., Perlmann, G. E. and Lorand, L.(Eds.), Academic Press, New York,
USA, 19,31- 41.
Wang, S. S. and Carpenter, F. H. (1967). Kinetics of the tryptic hydrolysis of the oxidized
B chain ofbovine insulin. Biochemistry, 6,215.
Wasserman, B. P. (1990). Evolution of enzymes technology: progress and prospects.
Food Technol., 44, 118-122.
Werbin, H. and Palm, A. (1951). Trypsin hydrolysis of lysine ethyl ester. J. Am. Chem.
Soc., 73, 1382-1382.
105
Whitaker, J. R. (1994). Classification and nomenclature of enzymes. In: Principles of
Enzymology for the Food Science, 2nd ed. New York: Marcel Dekker, pp 367-385.
Wilke, H. L., Bodwell, C. E., Hopkins, D. E. and Actschul, A. M. (1986). New protein
food: A study oftreaties. Adv. Food Res., 30, 331-385.
Wray, T. (1988). Fish Processing: New uses for enzymes. Food Manufacture, 63,64-65.
Yoshinaka, R., Sato, M., Tanaka, M.and Ikeda, S. (1985). Distribution of pancreatic
elastase and metalloproteins in several species of fish. Comp. Biochem. Physiol., 80B,
227-233.
Zeef, L. A. H. and Dennison, C. (1988). A novel cathepsin from the mussel (Perna perna)
(Linne). Comp. Biochem. Physiol., 90B, 201-204.
106