Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Production of L-leucine aminopeptidase by selected
Streptomyces isolates
V. Nagy1, K.M. Nampoothiri2, A. Pandey2, R. Rahulan2 and G. Szakacs1
1 Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
2 Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIST) (formerly as Regional Research Laboratory), CSIR,
Trivandrum, Kerala, India
Abstract
Keywords
L-leucine aminopeptidase, L-leucine-pnitroanilide, shake flask fermentation,
Streptomyces, zymogram.
Correspondence
George Szakacs, Department of Applied
Biotechnology and Food Sciences, Budapest
University of Technology and Economics,
Gellert ter 4, 1111 Budapest, Hungary.
E-mail: [email protected]
2007 ⁄ 0878: received 5 June 2007, revised 26
July 2007 and accepted 26 July 2007
doi:10.1111/j.1365-2672.2007.03546.x
Aims: To screen various Streptomyces cultures producing l-leucine aminopeptidase (LAP).
Methods and Results: Twenty-one Streptomyces strains were screened for LAP
production. The best three producers were found to be Streptomyces mobaraensis NRRL B-3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis
NRRL 2364. pH optima of the three enzymes were in the range of 8Æ0–8Æ5 and
the temperature optima varied between 50 and 65C. LAP of S. mobaraensis
was stable at 60C and pH 8Æ5 for 60 min. Metal ion salts, CoCl2.6H2O and
ZnSO4.7H2O in 0Æ7 mmol l)1 concentration enhanced the relative enzyme
activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was
found to be approx. 37 kDa.
Conclusions: Streptomyces mobaraensis NRRL B-3729, S. gedanensis IFO 13427,
and S. platensis NRRL 2364 were found to be good producers of extracellular
LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable.
Significance and Impact of the Study: A good number of Streptomyces were
screened and the ability of the aminopeptidases to release a particular N-terminal amino acid along with its good thermal stability makes them interesting for
controlling the degree of hydrolysis and flavour development for a wide range
of substrate.
Introduction
Aminopeptidases are a class of proteolytic enzymes widely
produced both in prokaryotic and eukaryotic cells. They
catalyze the cleavage of amino acids from the N-terminal
position of peptides and proteins. Aminopeptidase activity is associated with many biological functions such as
protein maturation, protein degradation, hormone level
regulation and cell-cycle control. Therefore, these enzymes
play an important role in many pathological conditions
including cancer, cataract, cystic fibrosis, leukaemia and
HIV infection (Lazdunski 1989; Pulido-Cejudo et al.
1997; Selvakumar et al. 2006). Leucine aminopeptidases
(peptidase classification: clan MF, family M17, bacterial
enzyme classification: EC 3.4.1 l.l0) are exopeptidases,
380
which remove the hydrophobic N-terminal l-leucine from
peptide substrates (Kim and Lipscomb 1994) and hence
reduce the bitterness of polypeptides. l-Leucine aminopeptidase (LAP) can also be used to improve the flavour
development (Toldra et al. 2000) and for dipeptide synthesis (Arima et al. 2006a). LAP from filamentous fungi,
viz. Aspergillus oryzae and Aspergillus sojae are commercially used for flavour development. (Nakadai et al. 1973;
Chien et al. 2002; Nampoothiri et al. 2005).
Streptomyces aminopeptidases are of particular interest
for biochemical and biomedical applications, as they are
stable and have a low molecular weight, simple kinetics
and a high enzyme activity (Arima et al. 2004). Streptomyces griseus LAP is a component in the protease complex of Pronase and was extensively studied by different
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
V. Nagy et al.
authors (Vosbeck et al. 1978; Spungin and Blumberg
1989; Xueying Ni et al. 2003; Fundoiano-Hershcovitz
et al. 2004; Arima et al. 2006b,c). LAP enzyme from
Streptomyces septatus also has been described more
recently (Arima et al. 2004; Hatanaka et al. 2005). In general, the leucine aminopeptidases produced by Streptomyces spp. belong to the Zn2+ metallo-aminopetidase group,
whose activity is regulated by the presence of divalent
metallic cations (Gonzales and Robert-Baudouy 1996). To
obtain further insight into the aminopeptidases of Streptomyces, it was decided to screen a number of them.
Materials and methods
Materials
l-Leucine-p-nitroanilide, l-methionine-p-nitroanilide, 4nitroaniline, actinonin, l-leucyl-2-naphtylamide, Fast
Black K and Pronase E (from Streptomyces griseus) were
obtained from Sigma. All other chemicals are of analytical
grade and were purchased from Reanal Co. (Budapest,
Hungary). UV 1601 (Shimadzu, Kyoto, Japan) spectrophotometer was used for the enzyme assays and Mini
Protean 3 Electrophoresis Cell (Bio-Rad, Hercules, CA)
for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Micro-organisms and culture conditions
Streptomyces albidoflavus NRRL B-1271, Streptomyces
aureofaciens ATCC 10762, Streptomyces badius 252
(=ATCC 39117), Streptomyces baldaccii NRRL B-3500,
Streptomyces flavogriseus IFO 13040, Streptomyces fulvoviridis NCIB 9804, Streptomyces gedanensis IFO 13427,
Streptomyces gelaticus TUB B-110 (=ATCC 43350), S.
griseus IFO 13304, Streptomyces lavendulae ATCC 14162,
Streptomyces lipmani TUB B-112 (=ATCC 43352), Streptomyces mobaraensis NRRL B-3729, Streptomyces olivochromogenes NRRL B-1341, Streptomyces parvulus TUB
B-126 (=ATCC 43364), Streptomyces paucisporogenes
ATCC 12596, Streptomyces phaeochromogenes TUB B123 (=ATCC 43361), Streptomyces platensis NRRL 2364,
Streptomyces roseosporus TUB B-120 (=ATCC 43358),
Streptomyces sclerotialus NRRL B-2317, Streptomyces
spectabilis NRRL 2494 and Streptomyces viridosporus
T7A (=ATCC 39115) strains were obtained from the
American Type Culture Collection (ATCC), Manassas,
VA, from the Budapest University of Technology and
Economics (TUB), Budapest, Hungary, from the Institute for Fermentation (IFO), Osaka, Japan (recently:
NBRC Culture Collection, NITE Biological Resources
Center, Chiba, Japan), from the National Collection of
Industrial Bacteria (NCIB), Aberdeen, Scotland and
Production of LAP by selected Streptomyces isolates
from the Northern Regional Research Center (NRRL),
USDA, Peoria, IL, USA. The cultures were maintained
and sporulated on Petri plates containing ATCC-5 medium at 30C. Composition of ATCC-5 sporulation agar
is (in g l)1): yeast extract, 1Æ0; beef extract, 1Æ0; tryptose, 2Æ0; FeSO4, 0Æ1; glucose, 10Æ0; Bacto agar, 15Æ0; pH
(before sterilization), 7Æ2.
Production media and shake flask fermentation
Two production media (AP-1 and AP-2) were used to
screen the aminopeptidase production. The compositions
of the media are as follows (in g l)1). AP-1: soybean meal,
defatted, 10; corn meal, 20; KH2PO4, 1; (NH4)2HPO4, 1;
MgSO4.7H2O, 0Æ5; NaCl, 0Æ5; paraffin oil (antifoam), 0Æ5;
pH (before sterilization), 7Æ0. AP-2: soybean meal, defatted, 10; sucrose, 10; KH2PO4, 1; NaNO3, 1; MgSO4.7H2O,
0Æ5; NaCl, 0Æ5; paraffin oil (antifoam), 0Æ5; pH (before
sterilization), 7Æ0. Shake flask (submerged) fermentation
was carried out in 750-ml Erlenmeyer flasks containing
150-ml medium autoclaved at 121C for 20 min. Spores
of each strain were aseptically scrapped out from a fully
sporulated (5–8 days old) plates into 10-ml sterile water
and appropriate volumes were used as inoculum for each
flask to adjust 5 · 106 viable spores per millilitre medium.
The flasks were incubated at 30C on a rotary shaker at
220 rev min)1 for 24, 48 and 72 h. Crude fermentation
broths were centrifuged at 9500 g for 15 min and the clear
supernatants were used to determine the aminopeptidase
activity. All sets of experiments were carried out in triplicates and the average value is considered for presentation.
Determination of L-leucine and L-methionine
aminopeptidase activity
The enzyme assay was adopted from the paper of Tan
and Konings (1990) and used with slight modifications.
Briefly, the reaction mixture contained 1 ml of
2Æ5 mmol l)1 l-leucine-p-nitroanilide (in 100 mmol l)1
NaOH–glycine buffer, pH 8Æ5), 1 ml 100 mmol l)1
NaOH–glycine buffer, pH 8Æ5 and 0Æ5 ml of the properly
diluted supernatant. The well-mixed solution was incubated at 50C for 10 min. The reaction was stopped by
the addition of 1 ml glacial acetic acid and the absorbancy was measured at 405 nm. Assay was carried out using
appropriate substrate and enzyme blanks also. One International Unit (IU) of enzyme activity was defined as the
amount of enzyme that hydrolyzes 1 lmol of leucine-pnitroanilide per minute. Standard curve was prepared
with p-nitroaniline. In case to determine l-methionine
aminopeptidase (MAP) activity, 1 ml of 2Æ5 mmol l)1 lmethionine-p-nitroanilide (in 100 mmol l)1 NaOH–glycine buffer, pH 8Æ5) was used as substrate.
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
381
Production of LAP by selected Streptomyces isolates
Biochemical analysis
After the initial screening, the best three producers were
subjected to biochemical characterization studies, such as
temperature and pH optima, thermal stability and metal
ion dependency.
Effect of temperature and pH on the activity
The reaction was carried out at various temperatures
ranging from 30 to 80C at pH 8Æ5 and the enzyme activity at different temperature points were compared to find
out the temperature optimum in each case. Similarly, the
enzyme assay was carried out at different pH levels from
6Æ0 to 10Æ6 at 50C. Three different buffers were used
such as 0Æ1 mol l)1 phosphate (pH 6Æ0–8Æ0), Tris–HCl
(pH 8Æ0 and 8Æ5) and NaOH–glycine, pH 8Æ5, 9Æ0, 10Æ0
and 10Æ5).
Temperature stability
Thermal stability of the enzymes was determined, as it
was described by Karadzic et al. (2002). The crude
enzyme extracts were incubated at pH 8Æ5 at different
temperatures (50, 60 and 70C) for various time intervals
(0, 5, 10, 15, 30 and 60 min). After the heat treatment,
samples were cooled and the enzyme was subjected to a
typical enzyme assay at 50C and pH 8Æ5 for 10 min. To
study the role of metal ions on temperature stability,
metal ion salts such as CoCl2.6H2O and ZnSO4.7H2O
were added in 0Æ7 mmol l)1 concentration to the reaction
mixture and the stability was tested.
Influence of selected metal ions on enzyme activity
Crude supernatant was further concentrated and partly
purified using a 5 kDa Amicon Ultra Centrifugal Filter
Device (Millipore, Billerica, MA). Ultra filtration was carried out at 3500 g for 45 min. Five metal ion salts were
applied in three different concentration levels (0Æ7, 1Æ5
and 3 mmol l)1 in the reaction mixture) to study their
effects on enzyme activity. The following salts were
used: CaCl2.2H2O; CoCl2.6H2O; CuSO4.7H2O; MgSO4.
7H2O and ZnSO4.7H2O. Enzyme assay was carried
out at 50C and pH 8Æ5 for 10 min as described previously.
Effects of inhibitors on enzyme activity
The activity of ultrafiltered (5 kDa Amicon Ultra Centrifugal Filter Device; Millipore) enzyme samples were determined in the presence of 0Æ8 and 1Æ5 mmol l)1
preincubated actinonin (Sigma), an aminopeptidase
inhibitor (Banerjee et al. 1995). Diamino-ethane-tetraacetic acid (EDTA), a metalloprotease inhibitor was also
used at two different concentration levels (0Æ7 and
1Æ5 mmol l)1).
382
V. Nagy et al.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and activity staining
Twelve millilitre crude enzyme extract (supernatant) of S.
mobaraensis NRRL B-3729 was filtered through a 0Æ22-lm
filter (Millipore), then concentrated by ultrafiltration
using a 5 kDa Amicon Ultra Centrifugal Filter Device
(Millipore), and the upper (protein) part was dissolved in
240 ll 0Æ5 mol l)1 Tris–HCl buffer (pH 6Æ8). 10% SDSPAGE was performed using the buffer system of Laemmli
(1970). Detection of LAP was performed as described by
Manchenko (1994). After SDS-PAGE, the gel was washed
twice with 2Æ5% Triton X-100 solution in 100 mmol l)1
sodium phosphate buffer (pH 5Æ8) for 30 min, and then
immersed in distilled water for 2 min. The washed gel
was incubated in a staining solution containing 1 mol l)1
CoCl2, 0Æ04% l-leucyl-2-naphtylamide and 0Æ06% Fast
Black K in 100 mmol l)1 sodium phosphate buffer (pH
5Æ8) at 37C in the dark, until dark blue bands were visualized. The stained gel was washed in distilled water and
fixed in 10% acetic acid. Precision Plus Protein Standard
(Bio-Rad) was used as a molecular mass standard.
Results
Screening in submerged fermentation
Twenty-one Streptomyces strains were screened in shake
flask fermentation using two different production media
(AP-1 and AP-2). The screening results showed a great
variety in extracellular LAP enzyme production, depending
on the isolate and the medium composition. All but three
cultures, namely Streptomyces gedanensis IFO 13427, S.
mobaraensis NRRL B-3729 and S. platensis NRRL 2364
showed very low extracellular LAP production both on
AP-1 and AP-2 media. For the best three strains, the LAP
activity ranged between 0Æ3 and 2Æ4 IU ml)1. Time course
of fermentation is shown in Table 1. Both shake flask
media well supported the bacterial growth. However,
interestingly S. mobaraensis showed relatively high
aminopeptidase production only on medium AP-1, while
S. gedanensis and S. platensis favoured medium AP-2. Peak
LAP activity on medium AP-1 was 2Æ4 IU ml)1 by
S. mobaraensis NRRL B-3729 after 98 h and the maximum
production (1Æ3 IU ml)1) on AP-2 was with S. gedanensis
IFO 13427 after 98 h fermentation. All the above cultures
were also checked for MAP production. However, the
MAP enzyme activities were negligible as shown in Table 1.
Effect of pH and temperature on enzyme activity
Figure 1a shows the effect of temperature (at pH 8Æ5) on
LAP activity of the three best producers. Streptomyces
platensis NRRL 2364 LAP has a temperature optimum at
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
V. Nagy et al.
Production of LAP by selected Streptomyces isolates
Medium
Fermentation
time (h)
Final pH
LAP
(IU ml)1)
MAP
Streptomyces
gedanensis
IFO 13427
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-2
AP-1
AP-1
AP-1
AP-1
AP-1
AP-1
24
48
73
98
120
144
24
48
73
98
120
144
24
49
74
96
120
144
6Æ92
7Æ15
7Æ35
7Æ65
8Æ78
8Æ90
7Æ48
8Æ27
8Æ56
8Æ70
8Æ75
8Æ83
7Æ27
6Æ40
6Æ42
6Æ77
7Æ11
7Æ53
0Æ01
0Æ55
1Æ17
1Æ31
1Æ14
1Æ04
0Æ02
0Æ10
0Æ29
0Æ29
0Æ30
0Æ32
0Æ02
0Æ23
2Æ06
2Æ41
2Æ42
2Æ37
0Æ01
0Æ01
0Æ02
0Æ02
0Æ09
0Æ14
0Æ01
0Æ05
0Æ08
0Æ08
0Æ08
0Æ08
0Æ01
0Æ02
0Æ20
0Æ27
0Æ19
0Æ13
Streptomyces
platensis
NRRL 2364
Streptomyces
mobaraensis
NRRL B-3729
LAP, L-leucine aminopeptidase activity; MAP, L-methionine aminopeptidase activity.
Relative activity (%)
Strain
(a) 100
80
60
40
20
0
5
6
7
10
9
8
11
pH
(b) 100
Relative activity (%)
Table 1 Time course of shake flask fermentation with three Streptomyces isolates
80
60
40
20
0
20
30
40
50
60
70
80
90
Temperature (°C)
50C, while S. gedanensis IFO 13427 and S. mobaraensis
NRRL B-3729 exhibit a higher temperature optimum, 60
and 65C respectively. Figure 1b demonstrates the effect
of pH (at 50C) on the relative activity of LAP. All three
enzymes showed an optimal activity between pH 8Æ0 and
8Æ5.
Thermal stability of the enzyme
Thermal stability of the crude enzyme extracts (supernatants) was also investigated (detailed data not shown).
Incubation for 1 h at 50C and at pH 8Æ5 resulted in a
20% relative activity loss in the S. gedanensis IFO 13427
and S. platensis NRRL 2364 samples. LAP produced by S.
mobaraensis NRRL B-3729 proved to be stable in these
conditions. Incubation at 60C and at pH 8Æ5 for 1 h
practically did not influence the residual enzyme activity
in S. mobaraensis NRRL B-3729 supernatant. In S. gedanensis IFO 13427 sample c. 50% relative activity was
retained even after 1 h of incubation. However, LAP produced by S. platensis NRRL 2364 completely lost its activity. LAP enzymes produced by S. gedanensis IFO 13427
and S. platensis NRRL 2364 completely lost their activities
at 70C in 15 min of incubation. On the other hand, LAP
produced by S. mobaraensis NRRL B-3729 lost its activity
only after 30 min of heat treatment at 70C.
The influence of Co2+ and Zn2+ on thermal stability of
LAP produced by S. mobaraensis NRRL B-3729 was also
Figure 1 Temperature (a) and pH (b) dependence of activity of
L-leucine aminopeptidase produced by Streptomyces gedanensis IFO
13427 (D), Streptomyces mobaraensis NRRL B-3729 ( ) and Streptomyces platensis NRRL 2364 ( ).
investigated. In the presence of 0Æ7 mmol l)1 CoCl2.6H2O,
even after 1 h heat treatment at 70C, the enzyme
retained 61% of its relative activity, while in the case of
0Æ7 mmol l)1 ZnSO4.7H2O the retained activity after 1 h
at 70C was 66% (Fig. 2).
Effect of bivalent cations
Effect of different bivalent metal ions on the activity of
LAP enzyme was also investigated. Five metal ion salts
were tested in three different concentrations (0Æ7, 1Æ5
and 3 mmol l)1). Results are shown in Table 2. Metal
ion Co2+ enhanced the relative activity of LAP produced by S. mobaraensis NRRL B-3729 and S. gedanensis IFO 13427. Surprisingly, Co2+ addition did not
influence the activity of LAP produced by S. platensis
NRRL 2364. With 1Æ5 mmol l)1 Co2+ supplementation
the relative activities were 101, 298 and 211 (in percentage) for S. platensis NRRL 2364, S. mobaraensis
NRRL B-3729 and S. gedanensis IFO 13427 enzymes
respectively. Metal salt ZnSO4.7H2O in 0Æ7 mmol l)1
concentration also enhanced the relative activity of LAP
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
383
Production of LAP by selected Streptomyces isolates
V. Nagy et al.
Relative activity (%)
300
250
200
150
100
50
0
0
10
20
30
40
50
60
Time (min)
Figure 2 Effect of Co2+ ( ) and Zn2+ ( ) ions on thermal stability at
70C of L-leucine aminopeptidase produced by Streptomyces mobaraensis NRRL B-3729. Control (without metal ions) is also presented (n).
produced by S. mobaraensis NRRL B-3729 and
S. gedanensis IFO 13427 (126% and 117% respectively).
while actinonin did not completely inhibit the aminopeptidase activity. With the addition of 1Æ5 mmol l)1 actinonin to the reaction mixture, LAP produced by S.
mobaraensis NRRL B-3729 retained 45% of its original
activity (Table 3). The activity restoring ability of Co2+
and Zn2+ ions after actinonin and EDTA treatment was
also investigated. Four metal ion concentrations (0Æ7, 1Æ5,
3 and 6 mmol l)1), as supplementations, were used after
1Æ5 mmol l)1 EDTA treatment. The best results for activity recovery were obtained by addition of 1Æ5 mmol l)1
CoCl2.6H2O or ZnSO4.7H2O, therefore, only these data
are indicated in Table 3. The addition of Co2+ and Zn2+
ions after the actinonin treatment did not influence the
relative activities of the LAP produced by the three Streptomyces isolates, while in the case of EDTA inhibition,
the restoring ability of 1Æ5 mmol l)1 CoCl2.6H2O and
ZnSO4.7H2O addition was 57% and 62% respectively.
Effect of Co2+ and Zn2+ supplementation in shake flask
medium on LAP production with S. mobaraensis NRRL
B-3729
Effect of enzyme inhibitors and the activity restoring
ability of Co2+ and Zn2+ ions of LAP produced by
S. mobaraensis NRRL B-3729
The LAP enzymes produced by the three Streptomyces isolates were strongly inhibited by the chelating agent EDTA,
In the biochemical tests, Co2+ and Zn2+ ions enhanced
the relative activity of LAP. Therefore, the effects of these
two metal ions were also tested in shake flask fermenta-
Relative activity (%)
Control
CaCl2.2H2O
CoCl2.6H2O
CuSO4.7H2O
MgSO4.7H2O
ZnSO4.7H2O
Salt conc.*
(mmol l)1)
Streptomyces
platensis
NRRL 2364
Streptomyces
mobaraensis
NRRL B-3729
Streptomyces
gedanensis
IFO 13427
Pronase E
(Streptomyces
griseus) Sigma
–
0Æ7
1Æ5
3
0Æ7
1Æ5
3
0Æ7
1Æ5
3
0Æ7
1Æ5
3
0Æ7
1Æ5
3
100
53
38
29
104
101
53
89
41
2
25
16
12
58
13
2
100
78
67
57
256
298
239
86
56
2
93
95
77
126
70
31
100
83
86
84
223
211
77
73
33
1
57
38
30
117
15
3
100
128
103
104
445
488
231
115
37
1
81
71
67
299
149
21
Table 2 Effect of different metal ions on
L-leucine aminopeptidase activity of Streptomyces origin
Metal ion influence test was carried out as follows: 0Æ5 ml of ultrafiltered supernatant or Pronase solution (0Æ025 IU ml)1) was incubated in the presence of 1 ml NaOH–glycine buffer (pH
8Æ5, 100 mmol l)1) containing the metal ion salt in appropriate concentration and 1 ml
2Æ5 mmol l)1 L-leucine-p-nitroanilide (in 100 mmol l)1 NaOH–glycine buffer, pH 8Æ5), at 50C for
10 min. Enzyme activities before and after the salt addition were determined by the standard
method described in Materials and methods.
*Salt concentrations were calculated for the total reaction mixture (3Æ5 ml).
384
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
V. Nagy et al.
Production of LAP by selected Streptomyces isolates
Strain
Compound
Concentration*
(mmol l)1)
Streptomyces
gedanensis
IFO 13427
None
Actinonin
Actinonin
EDTA
EDTA
EDTA + CoCl2.6H2O
EDTA + ZnSO4.7H2O
None
Actinonin
Actinonin
EDTA
EDTA
EDTA + CoCl2.6H2O
EDTA + ZnSO4.7H2O
None
Actinonin
Actinonin
EDTA
EDTA
EDTA + CoCl2.6H2O
EDTA + ZnSO4.7H2O
None
Actinonin
Actinonin
EDTA
EDTA
EDTA + CoCl2.6H2O
EDTA + ZnSO4.7H2O
–
0Æ8
1Æ5
0Æ8
1Æ5
1Æ5
1Æ5
–
0Æ8
1Æ5
0Æ8
1Æ5
1Æ5
1Æ5
–
0Æ8
1Æ5
0Æ8
1Æ5
1Æ5
1Æ5
–
0Æ8
1Æ5
0Æ8
1Æ5
1Æ5
1Æ5
Streptomyces
mobaraensis
NRRL B-3729
Streptomyces
platensis
NRRL 2364
Pronase E
(Streptomyces
griseus) Sigma
+ 1Æ5
+ 1Æ5
+ 1Æ5
+ 1Æ5
+ 1Æ5
+ 1Æ5
+ 1Æ5
+ 1Æ5
Relative
activity
(%)
100
27
18
0
0
42
15
100
72
45
2
3
57
62
100
50
36
3
5
7
0
100
36
24
0
0
88
35
EDTA, diamino-ethane-tetra-acetic acid.
Inhibitor test was carried out as follows: 0Æ5 ml of ultrafiltered supernatant or Pronase solution (0Æ025 IU ml)1) was incubated in the presence of 1 ml NaOH–glycine buffer (pH 8Æ5 100 mmol l)1) containing
the appropriate concentration of actinonin or EDTA (and in some
cases the metal ion salts too) and 1 ml of 2Æ5 mmol l)1 L-leucine-p-nitroanilide (in 100 mmol l)1 NaOH–glycine buffer, pH 8Æ5) at 50C for
10 min. Actinonin solution was preincubated at 50C for 30 min.
Enzyme activities before and after the inhibition were determined by
the standard method detailed in Materials and methods.
*Concentrations were calculated for the total reaction mixture
(3Æ5 ml).
tion on the enzyme production using S. mobaraensis
NRRL B-3729 strain. Thus CoCl2.6H2O or ZnSO4.7H2O
was added into medium ‘AP-1’ applying 0–2 mmol l)1
concentration ranges from these salts. Results are indicated in Fig. 3. Compared to the screening results, addition of ZnSO4.7H2O in 0Æ35 mmol l)1 concentration into
the fermentation medium the LAP production was
increased to 6Æ1 IU ml)1 (by 2Æ5-fold to the original
medium AP-1). Supplementation of CoCl2.6H2O in
7
6
Activity (IU ml–1)
Table 3 Effect of inhibitors (actinonin, EDTA) on L-leucine aminopeptidase (LAP) activity and the effect of Co2+ and Zn2+ supplementation
on restoring of EDTA inhibited LAP activity
5
4
3
2
1
0
0
0·5
1
1·5
2
Salt concentration (mmol l–1)
Figure 3 Effect of Co2+ and Zn2+ ions on L-leucine aminopeptidase
production by Streptomyces mobaraensis NRRL B-3729. CoCl2.6H2O
( ) and ZnSO4.7H2O ( ) were added to the fermentation medium
AP-1 in different concentrations.
0Æ03 mmol l)1 concentration, the LAP production was
4 IU ml)1 after 98 h fermentation.
Zymogram
For activity staining, ultrafiltered shake flask supernatant
of S. mobaraensis NRRL B-3729 was used. For SDSPAGE, a mixture of such concentrated and partly purified
enzyme and sample buffer (containing 2% SDS and 5%
2-mercaptoethanol) in 1 : 2 ratio was boiled for 5 min
and loaded on the gel. After running, the activity staining
was performed as described. Dark blue bands appeared
around 37 kD as shown in Fig. 4.
150 kDa
100 kDa
50 kDa
37 kDa
20 kDa
Stand. Sample
Figure 4 Zymogram with 20 ll concentrated crude fermentation
extracts of Streptomyces mobaraensis NRRL B-3729 and Precision Plus
Protein Standard (Bio-Rad) as a molecular mass standard.
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 380–387
385
Production of LAP by selected Streptomyces isolates
Discussion
We have earlier studied some Aspergillus isolates (Nampoothiri et al. 2005) for LAP production. In recent work, 21
different Streptomyces strains were screened in two different production media (AP-1 and AP-2). Three promising
producers were selected for further studies viz. S. gedanensis IFO 13427, S. mobaraensis NRRL B-3729 and S.
platensis NRRL 2364. Surprisingly, S. griseus IFO 13304
showed a weak LAP production, although another S. griseus isolate (strain K-1) is used for production of Pronase
E on large scale. Pronase is a mixture of different proteases, including aminopeptidases. Spungin and Blumberg
(1989) reported that the LAP of S. griseus is a zinc metalloenzyme characterized by a high enzymatic reactivity,
high thermal stability and low (33 kDa) molecular weight.
Interestingly, S. mobaraensis NRRL B-3729 showed a high
LAP production only on medium AP-1. The reason might
be that B-3729 prefers NH4+ to NO3) as inorganic nitrogen source. Also, medium AP-1 contains corn meal,
which is complex source of carbohydrates and proteins
while in medium AP-2 the corresponding carbon source
is sucrose. On the contrary, medium AP-2 proved to be
better to S. platensis NRRL 2364 and S. gedanensis IFO
13427 for production of LAP enzyme. The pH optimum
of LAP enzymes derived from the best three Streptomyces
was found to be in the range of pH 8Æ0–8Æ5. This finding
correlates well with previous data from different authors
for other Streptomyces LAP enzymes (Vosbeck et al. 1978;
Arima et al. 2004). Similarity has been found between the
temperature optima of the LAP enzymes of the best three
isolates and the thermostability of the same enzymes. The
temperature optimum of LAP of S. platensis NRRL 2364
is approx. 50C and the enzyme is less thermostable.
Highest temperature optimum (65C) was observed for
LAP produced by S. mobaraensis NRRL B-3729. This
crude enzyme was stable even at 60C after 1 h of incubation and thermal stability could be further increased in
the presence of Co2+ and Zn2+ ions. Streptomyces hygroscopicus produced a LAP with 67 kDa, with an optimal
activity at pH 8Æ0 and 40C and proved to be stable up to
70C (Karadzic et al. 2002). The ultrafiltered enzyme
supernatant of S. mobaraensis NRRL B-3729 was strongly
inhibited by the metal chelating agent EDTA, but activity
could be restored to 62% by the addition of Zn2+ ion.
Although the presence of Zn2+ ion during the enzyme
assay in higher concentration (1Æ5–3 mmol l)1) decreased
the relative activity of LAP of S. mobaraensis NRRL B3729, addition of Zn2+ ion to the fermentation medium
enhanced the LAP production. This might be explained
with the structure of Streptomyces aminopeptidases, since
the majority of aminopeptidases belong to the M1 family
of peptidases; they are metalloenzymes (Van Wart 1996),
386
V. Nagy et al.
which require zinc for enzymatic activity and share the
zinc binding motif HEXXH (Hooper 1994). Streptomyces
mobaraensis is a known producer of transglutaminase
(TGase) enzyme (Ando et al. 1989; Nonaka et al. 1989).
TGase is secreted as a precursor protein, which is activated by the endoprotease TAMEP, a member of the M4
protease family (Zotzel et al. 2003a). A tripeptidyl aminopeptidase (SM-TAP) is able to remove the remaining tetrapeptide from the TAMEP-activated TGase (Zotzel et al.
2003b) resulting in the final form of TGase. LAP of S.
mobaraensis described by us is not identical with TAMEP
or SM-TAP because the molecular masses of enzymes are
different and SM-TAP was unable to hydrolyze Leu-pNA,
Ala-pNA or Phe-pNA (Zotzel et al. 2003b). The Streptomyces aminopeptidases like that of S. mobaraensis are very
interesting as they show higher temperature optima and
better temperature stability. They are all low molecular
metalloenzymes and offer great potential for further
exploitation.
Acknowledgements
We acknowledge the grant received under Indo-Hungarian Intergovernmental S & T collaboration through DST,
New Delhi and TeT Foundation, Budapest. Indian
authors are also thankful to the funding from CSIR task
force program under SMM 0002.
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