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Biology
M.Sc.
J
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Nicolas Velos Christou
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Suggested Short Title:\
f
Buf~er-Soluble
Cellulase in the Pea Epicotyl
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Fractionation and Purification of the Buffer-Soluble
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Cellulase from Pisum sativum
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by
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Nicolas Velos Christou
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A thesis
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sUbmi~t~to
the Faculty of' Graduate Studies and
Research in part1a1 fulfi11ment of the requirement for the
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Degree of Master of Science.
March, 1975
Departrnent o.f Bio1ogy
McG~ll
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University
Montreal, Canada
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Treatment of Pisum sativum L. var.
Alas~a
epicotyls with
the auxin 2,4-D, brings about the development of massive
increases (50 to 200-fold) i n ' -l, 4-glucan 4-g1ucanohydrolase
(cellulase EC 3.2.1.4) activi~y.
vit y is buffer-soluble (BS
Part of this cellulase acti-
cell~lase),
e.g., extraçtable in low
lonic-strength buffer (0.02 M sodium-- phosphate, pH 6.5), and
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the remainder is buffer-insoluble (BI cellulase) , e. g., extractable in ?lgh ionic strength
buf~er
(0.02 M sodium phosphate
êJ
plus 1.0 M NaCl).
Following fractionation of crude extracts by
. Sephadex chromatography, BS cellulase appears as a heterogeneous
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enzyme system VIi th apPlent
daltons to greater tha
~1y7
of maJor
70,000 daltons.
peak~ranging
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from 5,000
Further analysis of these
!
components, using centrifugation in sucrose density-gradients,
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isoelectrie focusing and elèctrophoresis, reveals that only one
ES cellùlase exists in peas, and that the Sephadex ehrornatographie profile is artifactual.
BS cellulase was purified to homogeneity using ammoniumsulfate precipitation, DEAE-cellulose chromatography, ultrafil1
trati~n,
and Sephadex ehromatography.
ran aS,a sinqle
protein'~n
iThe 'purified BS
dise electrophoresis
an~
~ellulase
showed only
Co
one peak on isoelectric foeusing. Habbit anti-serum prepared ,
ta this component was TIlonospecific as ,determined by immunoeleetrophoresis vs. crude and pure DS cellulase preparations.
The
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Abstract cont'd:
1>
antiserum
~s
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guitable for use in studies of the role(s) and
distribution of nS cellulase in
gro~ing
tissues of {he pea .. ,
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RESUME
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• Une hauSf,e massive de
'~acteur
-,
de
50'~ 200,
act~ vi ~é de 'la çellulase, par uM
est provoquée chez les
étiolés par le traitement
activité est snluble
r
~
~picotyles
~,4-D.
l'auxine
Une partie de cette
p~~t l'ext~aire,
i.e. qu'on
de pois
dans un tampon
de faiblerforce ionique (phosphate de sodium 0.02: pH 6.5), alors
que le reste y est
insoluble~
pouvqnt être extraite dans un
tampon de force ionique plus 61~v6eo(phosphate de sodium O.O~
)
plUS/du NaCI 1.0).
La cellulase solub}e semble former.
lo~squ'on
"
chromatographie un extrait gros~ier de cet enzyme sur Sephadex,
un système hèt~rogène possédant des pits de poids mo16culair~s
i
~pp:arents
allant de 5 000
analyse plus poussée
per
j~squ'à
plus de 70,000 daltons., Une
c~trifugatlon
sur gradients de concen-
(
tration de sacchprose, par analyse isoélectrique et Jar électrou
phorèse, r&vèle la
prés~nce
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d'une seule cellulase .,soluble chez le
pois. et d6montre la natur~ artificielle des r6sultats obtenus en
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chromatographié sur Sephadex.
Une préparation homogène de la cellulase soluble
par l'emploi des méthodes suivantes:
\,ût
obtenue
préc\pitation par le sulfate
,
d'ammonium
chromatographie sur
~chromatographie sur Sephadex.
DEAE-~ellulose,
ultrafiltration et
La cellulase rurifi6e consiste en
une protéine unique d'après les 6criturps suivants:
de
di~Que. l'an~lYSe
sérum. jugée par
\
l'électrophorèse
isa61ectriQue et la:mànaspéciflcit6 de san anti- \
immunoélectrophorès~nvers
des pr2parations pures
et impures de la cellulase soluble •. L'ant,isr3rum peut être utilisÉ!
pour l'étude du rôle pt de la distribution de l'enzyme dans les,
~
~
tissus en croissance du pois.
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Traduit par M. Yves Raymond
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ACKNOhTLEDGEMENTS
It,is indeed with great pleasure that l express my
sincere thanks to Dr. G.A. Maclachlan for the many ways in
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which he contributed to this work and for his guidance
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throughout
m~.studies
at~cGill.
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Thanks are'also due to
rny fellow students J.G .. Milton and H. Byrne and, Dr. D.P.S.
,
Verma for advice and discus~ons in the course of these
experiments.
To my wife, Katerina, special thanks are due for hei
pétience; technical help with sorne of the experiments as
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weIl as help in the compilation 'and typing of the initial
"manuscript.
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Thanks' are also due to the Faculty of-Medicine
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McGill University for allowing me to utilize sorne of the
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elect\ve
ti~e
periods. in their curriculum for this
work~
, ,Financial assistance was provided by a grant from the
National Research Council of Canada to G.A.M.
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To my parents a special thanks.
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PREFACE
The experimental work and compilation of this thesis
.were carried out while the 'candidate was simultaneously
studying for the
~.D.,
of McGill Univérsity.
C.M. degree in the Faculty of Medicine
Use is made of the literary style
recommended by the journal Plant Physiology,.
ALI work con-
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tained in this thesis is that o( the candidate except for
experiments
u~ing
isoelectric focusing and immunoelectro-
phoresis which were carried out together with Dr. H. Byrne
, who was carrying out sil'1u'ltaneous inve'stigations o,n
t~
extensiV~
cellulase of the pea epicotyl as weIl as more
characterization of the BS cellulase as purified by thè
....
method described in this thesis.
Part of this work has been
incorporated for publication in the J. Biol. Chem.
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et al, 1975).
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(Byrne.
The buffer-soluble antiserum prepared during
this work is being} used by other members of this laboratory
to study the role of BS cellulase in the growing plant.
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TABLE OF CONTENTS
Page
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INTRODUCTION ••••.•• "
••••••..••••••••• " ••••••••••••• 1
REVIE~'l OF' THE LITEHATURE •••••••••••••••••••••••••••• '4
1.
Definition of Cellulase, Occurrence,
Induction.
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2.
,.
III
••
III
•••••••
III
••••••
:
•••••••••••••
III
4
Multiplicity·of CelluJ.ase . . . . . . . . . . . . . . ... 7
•
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c.
A.
Fun'gal Cellulase . . . . . . . . . . . . . . . . . . . . . . 7
B.
Higher Pfant Cellulase ............... 14
Purification of Cellu'lase ............. : .. 17·
A.
B.
Fungal Celluléis-e . . . . . . . . . . . . . . . . . . . . . 17
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Higher Plant Cellulase ... ·............ 21
MATERIALS AND METHODS •••.•••••••••••••••••••••••••• 23
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Source of Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.
Extraction . . . . . . . . . . . . . . . . . . . : ........... 23
3.
Assay . . . . • . . . . . . . . . . . • . . . . . . . . . . • .
.
4. 'Ammonium Sulfate
III
III
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.....•... 25
5.
Sephadex Ge l Fractionation ..........' ....... 26
6.
DEAE-Cellulose Chromatography ......•..... 28
7.
Sucrose Density Gradient Centrifugation .. 29
8.
A~alytical
t
Gel
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Fractionation.~
III.
Discontinuous Polyacrylamide ~
Electrophores~s
................•..... 30
9.
Isoelectr~c Focusing •................. : .. 32
10.
Ultrafiltration ................. , ........ 33
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TABLE OF ÇONTENTS (continued)
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Preparation of Antiserum . . . . . . . ; .......•• 34
(
Irnrnunodiffusion ......... ;.~.~ . . . . . . . . . . . . 34
.
)2.
l.3.
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I~unoelectrophoresis
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RESULTS
•
A.
1
Fractionation of Cellulase in Crude
(
Extr acts .. : ...... ~ . : . . . . . • . . . . . . . . . . . . . . . 36,
1.
Chromatoqraphy on Seprradex Gels . . . . . . 36
2.
Suerose Uensity Gradient Profiles of
Crude and
Partia~ly
Purified BS
Cellulase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Analytical Discontinuous polyacryla-
3.
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mide Gel Electrophoresis
. . . . . . . . . . . . . 44
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'B.
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Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . • . 48
1.
Development of Cellulase Activity
.
Following Treatment of Pea Epicotyls
, \'J i th 2, 4 - D. . . . .
S>. • • • • • • • • • • • • • • • • : • • •
48
2., Ammonium Sulfate Precipitation ...•... 50
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Ultrafiltration I . . . . . . . . . . . . . . . . . . . . 55
5.
Chrorna.tography Through Sephadex (;-75.57
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Chromatography . . . . . . . .
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Ultrafiltration II . . . . . . . . . . . ~ ..•.... 59
Characterization . . . . . . . . . . . • . . . . . . . . . . ~ .. 63
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Electrophoresis .......••.........•••. 63
2.
Ouchterlony Double Immunodiffusion
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DEAE-C~llulose
6.
C.
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3.
Analys is ............................... 65
3•
Immunoelectrophoresis . . . . . . . . . . . . . .""": . 6 8
4.
Isoelectric Focusing ...•... ; .' . . . . . . . . 70
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OF CONTENTS (continued)
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.72... . .... ........ . ... ... .. .....
Heterogeneity. . . . . . .. .. . . .. . .. . .......... .72
B.
Purification.'.
DISCUSSION .•. ,
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L1TERATURE CITED ...... .
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CONTRIBUTION TO ORIGINAL KNOWLEDGE .
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L':rST O:f FIGURES
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1.
Crude
buff~r-so1ub1e
(BS)
ce11ulase activity
chromatographed Qn Sephadex G-75
2.
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Crude BS ce11ulase
chromatograph~d
on Sephadex
/
G-'SO.
3.
39
Crude BS cellulase chromatographed on Sephadex
G-50 on a separate occasion from that recorded in
40
Eiquroe 2
4.
Crudœ BS
ce~lulase
activity
chrom~tographed
on
...
Sephadex G-25
5.
. 42
Sucrose density gradient profiles of
BS'cellulase~
takeh froorn,peak fractions of 1Sephadex experirnents
45
shown in Figures l' to 4.
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Sucrose denslty gradlent profile of crude BS
.
cellulase activity
46
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Analytical discontinuous polyacrylamide gel
...
electrophoresis of crude BS cellulase açtivity,
8.
DEAE-Cellulose chroffiatography of partially
47
purifi~d
54
cellulase activity
9.
Partially pruified RS cellulase activity chromatographed on
10.
5'8
G-75
Calibration curve of the Sephadex G-75 column'
shown in
Il.
S~phadex
Fig~re
60
9
Analytical discontinuous po+yacrylamide gel
.64
electrophoresis of purified BS cellulase
12.
IIomogeneity of pure BS
c~llulase
'.
as determined
following discontinuous rpolyacFylamide gel
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electrophor:esis
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LIST OF FIGURES (continued)
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13. ' Ouch~erlony double diffusion of pure BS
c~llulase vs~ pure ES cellulase-ant~&e~~~---~---~~L-_----__
14. Immun~elec-trophoresis of pure and crude BS
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p~'11ulase vs. BS cellulase antiserum,
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I~6e1ectric focusing of crude,' ang pure BS
,15.
~e-11ulas'e
/1
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..
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LIST OF ':VABLES
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~---
---------
---_-:--..... _ - - -
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Development of BS'-Cellulase Activity
and protein Following Treatment of Pea
TABLE~I
()
Epicotyls with 0.1% 2,4-0. , _
TABLE II
Ammonium
S~1fate
BS Ce11ulase.
TABLE III
49
Fractionation of Crude
.
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51
Ultrafiltration of Partia11y Purified
. 56
BS Cellulase
TABLE IV
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Final Ultrafiltration of Pa~tia11y
'''~ :6" _1
Purified BS Cellu1asè
TABLE V
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Summary of the Purification of BS
Cellu1ase.
('; '
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==-"'="""=======, ---
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ABBREVIA['IONS
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Buf fer =-1ns-o-,1 ~-b-1-~ ·---:C=-e~1:;--:1;;-u---C1;;-a-s-e--~--~~-~~~
BI
---
Buffer,Solub1e Ce11u1ase
BS
/(
Carboxymethyl Cellulose
CMC
~
DEAE-
Die~hy1aminoethyl-
2,4-D
2,4-Dich~rophenoxyacetic
IAA
Indo1eacet'ic acid
,/.
#
ma
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Acid
r'li 11 i ampe re s
MW
Mo1ecu1ar Weight
S,A.
Specifie Activity
SDS
Sodium Oodecy1 Sulfate
V
Volts
Ve
V0
Elution Volume
Vt
Total volume
.
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----.1
~,-
---
\
Void Volume
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....
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INTRODUCTION
.r
Ce11ulo1ytic enzymes
(~-1,
4-g~ucan,
EC 3.2.1. 4) from 'fungil , bacteria and
4-g1ucanohydrolase,
~t~in
ani,ma1" species
have been studied extensively and in sorne cases have been
purified and partial1y characterized (see reviews by Gascoigne
and Gascoigne, 1960, Peese, 1963, Gould, 1969, Whitaker, 1971).
They occur in a variety of mo1ecular forms and show different
substrate specificities.
They aIl act extracèl1u1arly and
appear to function mainly in nutrition as digestive or invasive
Cellulases of'higher plants, however, are found only
agents.
locatiGTIs in tissues at particu1ar stages of
There is an increasing body of evidence to
indi~
important regulatory roles in cell growth
differentiation,
e~g.,
~e11
expansion (Tracey, 1950, Fan
Mac1achlan, 1966; Ridge and Osborne, 1969), leaf abscissi~n
"
and Osborne, 1967, Abeles,' 1969, Lewis and Varner, 1970,
Reid et al, 1974), pollen tube elonga,tion (Roggen and
fr~it
1969),
ripeninq (Hobson, 1968, Pharr and
s~an1ey~
Dicki~so~,
1973),
vascu1ar differentiation (Sasson, 1965, She1drake, 1970). None
---- ._-
-~
~_.
-
-- - . -._-
~- -.~
- ------~
has yet been p~~"ified to the -a:egree w'n,lC11their poLentil::la:"j;l,---significance in mtrphogenesis would warrant.
There is evidence from solubility studies (Lewis and
Varner, 1970, Byrne ~ al, 1972, Pharr and Diçkinson, 1973),
1
chromatography (Suzuki
~~,
e1ectrofocusing (Lewis
~
1971, Byrne
et~,
1972) and
al, 1972), ·that sorne plant tissues
2
contain more than one enzyme with cellulolytic activity.
These enzymes are associated with proteins which have different
, isoelectric points and moleeular weights, and which develop
differentially during the proeess of abseission (Linkins et al,
1973, Reid et al, 1-974).
Sorne fungi also produce multiple
forms of cellulase which differ in molecular weight or other
properties, ~ut often this is ~ result of modification of a
,; single parent species of protein by complexinq with
) eharides or
\
J_. -. ---, - ----
polypep~ides,
(Whitaker, 1971).
,
polysa~ y
.;
by glycosylation, or, by proteolysis
Thus" where multiple forms of cellulase are
indicated in higher plants, it must first be established whether
tQese are modified forms of the same protein or are distinct
enzymes.
The possibility of artifactual profiles due to non-
"
specific binding o.f this enzyme (or enz.ymes) on chromatographie
media must also be taken into consideration.
Developmental controls, such as induction by substrates
or sex hormones, may be exerted over
cellul~se
certain fungi (Thomas and Mullins, 1967).
production in
In view of i ts
limited distribution in higher plant tissues, the development
of cellulase must also be closely regulated in these organisms.
---1 n-'"S-emE7
an·t.-~- ,the.. .enzyme_app~r.â., __to __ b_e -i nduce
d by growth
--------- - ' - - - - - - ...
treatment, e.g. indoleaeetic acid, (Fan and Maclachlan,
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hormona
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1966, Fan anq Maclaehlan, 1967, Davies and Maelachlan, 1969)
or ethylene (Lewis and Varner, 1970).
~orphogenetic
Despite their potential
significance, no sueh regulated eellulase has
been fully eharaeterized or purified as a first step towards
defining aceurately sueh parameters as their synthesis,
-----,--
3
precise location and function.
Pea epicotyls (Pisum sativum) treated with auxin (lAA,or
2,4-D) develop a high level of cellulase activity which is only
,
,
partially soluble in dilute (O.02M) sodium phosphate buffer,
1
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pH~~ but completely extractable by buffer ~luslM N~Cl_J~y~ng
et al, 1972).
The present work was undertaken with the aim of
.
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purifying the buffer-soluble cellulase (BS cellulase) from the
pea epicotyl in order ~o ~~ide a source o~ pure hi~hèr plant
'.
cellulase for further
study of its properties, function,
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localization'in t~~ cell and ultimately, ihe!physiologic role
and possible applications of the epzyme.
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REVIEh' "OF THE LITERl\TURE
,
During the past 25 years, much has been written on the
,
,r
,enzyme cellulase.
For literature prior to 1960 the reader is
referred to the book by Gascoign and Gascoign (1960).
The
,
~
present
~eview
is concerned with atternpts to characterize and
purify plant ce11ulase from 1960 to
Sep~ernber
1974.
The
,
espee~l~y
reviews by Reese (1963), Gould (1969), and
the
excellent review article by Whittaker (197l) were used as
A more extensive
basic sources of lnformation and references.
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review) Gf the overall properties, 'functions', occurrence f and
•
yse of cel1ulases has been carried out by a
(1974) .
1.
colleaguef~H.
Byrne
;
Défini~ion
of Cel1ulase, Occurrence, Induction
The Commission on EnZy!es list "cellulase" as the'
J
trivia~
'1
name for a class of enzymes which have the systernatic
name ~ -l, 4-g1ucan 4-g1 ucanohydrol ase (Ee. 3.2. 1. 4.).
These
~
~m~s
are capable of hydrolysing a wide variety of
4-fucans and derivatives.
of their hydrolysis
inc~~~e:
~ -l,
Examples of substr'ates and studies
fried native fiber of high cellu-
lose content such as dewaxed co~ton (Halliwell, 1957)
i
exten-
sively hydrated cellulose sudh a& that obtained after swelling
o
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native cellulose in cold phosphoric acid (Walseth Cellulose,
Walseth, 1952)
i
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extensively degraded pellulose such as the
cellodextrins obtJinable by deacetylation of acetolyzed cellu-
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lose ,(Whi taker, 1956);
soluble deri vAtives such as carboxy-
" methyl cellulose (CMC) (Almin and Eriksson, 1968).
v~riety o~'enzyme subst~ates
proci!edures to be based on
This wide
allows for cellulase assay
the 108s of weight of native
cellulose fibers due to the solubilizing action of the enzyme,
the formation of reducing sugars from all substrates mentioned,
or the rat~of decrea~e -ln the' mean chain length
mined by viscosity loss)
(a~ deter-
of cellulose derivatives in solution.
_The latter is the most sensi ti ve method for ass"ay of endocellulase activity (Almin and Eriksson, 1967).
AIl of the assays
of pea cellula8e in this thesis have employed a reduction in
the viscosity of a CMC solution within a definite
duri~"~ich
,
~ime
period
reaction rate is 1inear and proportibnal to
amount of enzyme used :--(Perrault and Maclach1an, 1964, Fan,
1968) •
The term "cellulase complex" was first used by Reese
et al, (1950) to refer ta a qroup of enzymes, not aIl cellulolytic, which nevertheless could act synergistically to degrade
native cellulose to glucose.
It may include an enzyme
"c lt'''
to weaken the crystalline structure which renders cellulose
molecules accessiqle to enzymic hydrolysis plus one or
mo~e
endo-cellulases and/or exocell ulases ("Cx" .enzymes), as well
~
as
~glucos
confined to
id'a,ses ,. e. g., cellobiase.
~udjes
The pres en t review is
of the endo-p-l, 4-g1ucanhydrolase
•
activi ty.
Cellulase occurs in higher plants where it may act as
a morphogenic 'agent that weakens cel1ulose-containing cell
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walls in preparation for growth and differentiation (Tracey,
~1i/'
1950, Fan and Maclachlari, 1966~ Ridge and Osborne, 1969).
r
It
may also play a raIe in the process of abscission, which leads
", to severing' from the parent plant of seeds, flowers or leaves
(Abeles, 1969,
1967~,
Lew~s ~nd
Varner, 1970, Horton and Osborpe,
and in the ripening of fruit (Pharr and
1973).
Dickinson~
4
Cellulases are excreted by plant pathogens where they
act to dissolve hast wall cellulose, thus allowing the pathogen ta penetrate the tissues of its hast, as weIl as for
nutrient (Whitaker, 1971).
Ce1lulases occur in the animal
kingdam in saprophytic nematode~ (Tracey, 1952), Crustacea
(Ray, 1959), marine moluscs
(Okada et al, 1966), insects
(Lasker and Giese, 1956) cand many other
and Yasumasu, 1964).
These
~nima1s
inverteb~ates
(Yokoe
use cel1ulase as a diges-
tive agent allowing for the use of cellulose as an energy source.
Developmental controis by hormonal or substrate-induction
may be exerted over
cellu1~se
production in certain fungi
.
(Mande1s and Reese, 1957, 1960, Thomas and Mullins, 1967,
Hulme and Stranks, 1970). A ,-1, 4-q14can is usua11y an
essential component in the medium requ{red for cellulase
~
production
y ce1lulolytic micro-organisms (substrate induction).
Carb
this
cellulose or carboxyetRyl cellulose can satisfy
ion for sorne organisms (Scheneberger and Luchsinger,
,
Addition of cotton cellulose or highly ~ ...."
.
crystalline cellulose results in cel1ulase production in the
culture
es
m~ium
of Trichoderma viride (Raute1a and King,l968) .
Ce'llu1ose can be bath an inducer or a repressor for sorne
7
. :; organisms (Reese et al, 1969).
Several hormones are known to influence the production
"
>
which may then be correlated with a ~orphOgeni~
of cellulase
,
function.
..
The application of lAA to decapitated pea epicotyls
resu1ts in production of massive cellulase "activity (Fan and
Maclachlan, 1967).
"Hormone A", a steroid, stimulates ce 1'+ u-
lase production by male strains of
th~
water
~old
Achlya
1
(Thomas and Mullins, 1967).
Abscisic acid induces the cel'lu-
lase associated wi th absciss ion processes in cotton and bean
plants (Cracker and Abeles, 1969).
Ethylene appears to evoke
a particular (ellulase (basic pl) associated with the process
of absciss ~on in Phaseolus vulgaris
(Lewis et al, 1973, 1974).
Treatment of etio1ated pea epicoty1 with the auxin hor'1
mone analogue 2,4-D results in deve10pment of very large arnounts
of ce1lulase activity in a few days, as we1l as increases in
total protein and RNA synthesis (Bonn, 1970).
In general aIl
metabolic responses which occur after 2,4-D treatment also
appear ta occur after lAA treatment, a1though the extent of
t
the response may differ according to differences in auxin
stability, metabolism or, transport
(Moreland, 1967).
"\
2.
Multiplicity of cellulase
A.
Fungal Cellulase
Many authors, especially in the literature of late 1950's
and- early 1960' s, c1aim that cellulase exists in more than one
wolecular forro in the cult~re filtrates of cellulolytic fungi
(see reviews by Gaiscoigne and Gaiscoigne, 1960, Reese, 1963).
8
This was based on separations using electrophoretic and chromatographic techniques which often were not carried to theipoint
\,
~ of investigating fully the range of possible causes of the
observed diversity.
E~amples
of co~plexity encountered in cellulases fr~? a
single organism are found in the 'work by Pettersson et al
(1963 a and b) on Polyoorous~versicolor.
Culture filtrates' of
this fun gus' appeared to contain 4 main cellulase components
when investigated using a combination of Sephadex gel fil tration and
~
J
-
analytical ultracentifugatiQp.
"Cornponent A" was
identified as 'a }-glucosidase and wa$ not considered to be a
./
,
true cellulase. - "Component B" was further separable into two
peaks BI and B2 , on preparativ.e electrophoresis, and
analysis
of these on the ultracentrifuge yielded sedimentation coefficients of 4.21 and 4.26 tespectively.
The main component, BI'
had a molecular weight,of 51,000 by the Archibald method .
.
"Component C" was also separabl,e into two peaks, Cl and C ,
2
with sedimentation coefficients determined as 2.0 and 1.9
respectively.
"Component 0" yielded a sedimentation coeffi-
cient of 1.53 and a molecular weight of Il,400.
?
drate content of, D was '33% vs 1% in component BI.
The carbohyPettersson
and Porath (1963b) proposed the following hypothesis for the
pos~ble
physiologie role of these enzymes:
•
" ..... an inverse
1
relationship may exist between the molecular size 'of the enzyme
component and the substrates.
Low-molecular-weight cellulases
..
showing highest activity on glycosidic bonds in polysacharide
1
çhains, while high molecular weight enzymes preferentially
.
,
\
9
catalyze the hydrolysis of oligo-saccharides.
Thi& theory
was never verifièd experimentally and, indeed,
m~ltiplicity
~------
---~
---~-
------
of cellulases in this organism has never been corroborated
(or denied) by later work .
.,..,.
Whitaker et al (1963a,b) carefully purified the cellulase activity in culture filtrates of Myrothecium verrucaria,
using differential precipitation and chromatographie techniques.
The authors found evidence for only one cellulase
component of MW 49,000, even though Many other workers
(reviewed by Whîtaker, 1963b) had reported up to nine cellulase components produced by this fungus.
The conclusions that
there were several different cellulases in this mold was
\
criticized by Whitaker on the grounds that inadequate attention
was paid to interference by other components in
çr~nzyme
filtrates and to technical problems in the electrophoretic
methods and assays employed.
Selby and
~aitland
(1965) using Sephadex G-75 chrornato-
graphy re-examineà the cellulase activity ,in Myrothecium
verrucaria culture filtrates and found three distinct cornponents fractioned by this gel. "Cellulase 1" with an apparent
W~
.
of 55,000 (slightly more than Whitaker's single component)p
,..
"Cellulase II" with a molecular weight of 30,000 and "Cellulase
III" with a MW of only 5,300.
,
If such a low HW were verified
cellulase 1];1 woul<3 be the smal1:est enzymic'component ever
reported with cellulolytic activity.
About 90% of the activity
was associated with Cellulase II. Exposure ta cotton before
..
chromatography reduced the amount of cellulase l and III that
'f
.)
\
10
.
,
could be recovered from the column, but cellulase II was
'e
unaffected, suggesting that the components differed in
-
substrate affinities.
~J
-------
- - - - - - - ---,- - - - - - -
No synergism or
-----~-
---
1nterconvers~on
observed. when these components were rnixed in any way.
_'\_------
was
The
three components rechromatographed to tneir respective
separate loci i.e., they yielded the same Ve/Vo as compared
wi th the first run.
Tb.e authors realized that their data
suggested the existence of three separate 'enzymes, contradicting the conclusions of Whitaker et
~l
(1963).
They
proposed that differences in chromatographie technique explained the discrepency.
The disagreement has not been re-
l
solved by more recent work.
, A series of investigations has
cellulase
o~Trichode~ma
been made into the
viride, which again, raises the
question of multiplicity.
The culture filtrate from this
fungus is sold under the trade name of Meicelase.
studies,
In'early
(Niwa ét al, 1965), it was reported ehat cellulase
"--
,activity from such
-
.
prep~rations
separated into at least six
or seven components by chromatography on ion exchange resins.
Selby and Uaitland (~967) exam~ned the activity'from culture
filtrates of their mold using Sephadex chromatography and
foun~~two compon·ents of ~n'1 62,000 and 12,600.
"
In this system,
,
rechromatography of the 62,000 cellulase resulted in the
appearance of smaller components.
..
protease action, which
'
of.~ourse
This was ascribed to
could also explain the
1/
existence of the low molecular weight component (cellulase
III) which these authors (196~ found in Myrotheciumo (see
/
-
11
above).
The Meicelase preparation was re-examined
b~
v
Oka~.
et al (1968) using a series of Amnerlite CG-50,' DEAE-Sephadex
-------
-~---
A-50 and Sephadex
- - ---- - - -
G-7~,
for three components:
Il
1
---------~---------~~-
chromatography.
- - - - - - ----
This yielde-d evidence
cellulase II,1 with a .sedimentation
"
coefficient of 3.54, carbohydrate 17%;
"cellu'lasè III" with
sedimentation coefficient 3.75 and carbohydrate of 16%;
",cellulase Iy" with sedimentation coefficient 3.62 and carbo .... ,
~O%.
hydrate, pontent
il
resemb~ed
cellulases
The amine acid composition of the three
each Qther, and the enzymes showed very
1
"slight differences in their abili ty ta de;,grade different cellulose substrates'.
The. enzymes were also similar in terms of
optimum "pH for activity, and stabi'li ty at cfifferent pH, but
there was a
differen~e
in their stability ta heat denaturationl
It was concluded that,mult{plicity in Trichoderrna viride cellulase does exist que, not necessarily to apoenzyrnes, but to
diffêrences in
carbohydr~te
.-:
content.
The cellulase activity of Trichoderma koningi has bee&
analyzed ,by. Iwasaki et al (1965).
It sep~rated into "~ellulfise
l'' with a HW of 50,000, S value of 3.81, with 12% carbohydr.;tte
content, and a "ce1fu'lase II'' with a MW of 26,000,
s:Yalu~
of
3.13, wi th 2% carbohydrate "content. _ The two enzymes w.ere shdwn
to have different amino acid cornposi tion anddifferent capa-,
bi1ities for degrading native cellulose or soluble cellulose
derivatives.
Pettersson and It,aker (l968), using a commercial prepa--
/
ration from penicillium notatum opserved a cellulase system
•
/'
-
,
0
12
,
,
consisting of a single low-molecular-weight enzymic
compon~t
which, in crude soluti6ns, was associated with different inactive substanc~s.
After purification of the cellulase'activity,
only a single peak was
ob~érved
and only one prote in blnd
,
1
~as
on preparative eleptrophoresis
obvious on
analyt~c d~sc ele~tro-
The MW rof the cellu1ase was det~~ined as 34,500 by
phoresis.
~nd ~ssoci~fed
with the pure enzyme. Neverthe1ess, electroPho~slS of ine-,--,-"
crude prepa~ation -revea1ed th; ~pparent existence of severa1 : ~I
__________________c_e_n_t__r_l_·_f_u_g_a__
tion and no
carb~hydrate
could be
,
active components.
The authorsJsuqqested that the enzyme, una, , r
conditions found in a culture fi1trate, may be "bound" t():
inactive substances such as proteins or ca,rbohydrates in such a
~,
way that i t
fractionates into multiple component,s.
Eriksson et al (1967) used a commercial preparation of
, f
~spe.;gillus 'cellulase ("Cel,lulase 36"), to show that following
purification it contained onlr
~9ne
cellulase, e?en though in
1
crude preparations, it appear~d as two peaks on zone electrophoresis and Sephadex
\
chromat,o~raphy.
Adsorption of the parent
enzyme t'o othêr proteins 'in the\ crude preparation was ~i ven as
the reason for wultip1icity.
~
.
Eriksson et al (1968 a',b, c,) studied intens i vely the
--
cellu1ase of the rot fungus Stereum sanguinolentum using a
combinatioI\' of BiO-get P-150 chromatography,
1
DE~-sePhadex
'chromato~raphy, Sephadex G-75 ehromatography and zone electro\
pRoresis.
They obtained two discreet
cel1ul~se
terms of physical propertïes (S values, wiV).
components in
However, they
p
/1
,.
\
,.
,
.
13
_-.
."
/
state that:
"During the course of characterization of the
two cellulases ..••. a striking similarity in aIl (chemical)
properties was found-between the enzymes".
By this they
meant similar substrate specifities, inhibitor studies, pH
, '
t~mperatpre
optima for activity and stabi1ity, and
for activity.
-.
optima
They postulated that both cellulases were
carbohydrate complexes of a single enzyme (1968b).
- r
The
II pur ified" enzyme contained up to 7% carbohydrate but this
was removeable by dialysis.
enzymes
w~re
When the c~rbohydrate-free
rechromatographed on
DE~Sephadex,
\
they ran as
\
a single peak which di,ffered from the l'Ç)ci of ooth of the
1.
\
origin~l cellu1ases:
Q
The same observati~n
was made using
,
/.
e1ectrophoresis.
These authors a1so poin ed out that Sephadex
shou1d not be regardeJ as a tota11y inert s pport for the
unfractionated enzyme solution.
Enzyme yiel s decreased
\
\
gr~at1y
\
after the use of Sephadex gels during purification
procedures. '
".
Wood (1969) stuoied the cel1ulase complex of Fusarium
soiani using DEAE-Sephadex chromatography followed by
Sephadex G-IOO chromatography.
The crude
"Cx"compon~nt
when
chromatographed on Sephadex'G-100 appeared smeared through_out the whole elution profile.
.
On further investigation, (Wood,
.
1971), afte! purification by ammonium sulfate and
DE~E-Sephadex
chromatoqraphy, the IICX" cellulase yielded only one peak on
Sephadex G-IO O.
However, this peak could be separated into
to
,
1
.
three components by isoelectric focuslng
with pl values of
,
4.75, 4.80 and 5.15.
These components maintained the same
14
substrate,~p~~ifities
relative
(using CMC and Walseth cellu-
,
lose) as the parent
enz~me.
or '-glucosidase activities.
They synergized equally with Cl
This led to the proposal that
the three Cx enzymes may have been carbohydrate complexes of
.
one enzyme, like the Stereum cellulase (Eriksson, 1968 b),
and the~ author concludes that there'is no reason for sug~est---
j
---~-
ing functional hcteroqeneity for these cornponents.
Yoshikawa et al (1974) distinguished five celfulise--components (peaks 1, II, III, IV, V) on Biogel P-l50 chromatography from culture filtrates of Pseudornonos fluorencens.
Each peak contained two electrophoretically separable components, A and B, in varying proportions;
nent B was highest in the highest
M\'l
the amount of compo-
fraction (1), decreased
progressively through peaks II, III, IV and was absent from
peak V.
As culture age
incq~ased,
peak l
steadi ly decreased
and eventually almost disappeared, while the ratio of total
A to total B increased.
cultures.
Only peak l was found in very young
It was concluded that aIl the other cornponents are
derived from the higher ~1 enzyme ~peak I} .
From these studies it appears to be an open
whether any genuine mul tiplici ty
.
dl
questi~n
pf cellulase enzymes exists
i
in fungi.
B.
Higher P;ant Cellulase
1
Although cellulase from higher plants has rrot been st~died
as extensively as that of cellulolytic rnicroorganisms, the
q~stion
of heterogeneity of cellulase has
~gain
been
15
,
considered.
,
In a preliminary report on the 2,4,-D-evoked
ce11ulase ac-l:ivity in crude extracts"of growing régions of
Pisum sativum epicotyls,' Mifton et al (1971) described
several cellulolytic components
chromatography.
fract~oned
by Sephadex gel
Further investigation over the past 2 years
by the present author and in studies by Byrne (1974), reveals
that there are, similar difficulties in fractionating pea
;
l
cel1ulase on Sephadex columns as those experienced by workers
on the fungal enzymes.
The question is, whether the cel1ulase'
multiplicity observed after chromatography with certain media
(especially Sephadex gels and cellu10se-based ion exchangers) ,
indicates the existence of many separate cellulases or whether
it
i~
in part or tota1ly artifactual.
With fungal enzymes,
artifacts have been observed ànd attributed to carbohydrate
comple~ing
of a parent enzyme, adsorption to chromatographie
;
media or proteolytic enzyme action on a parent
~olecule.
'In
an effort to overcome binding artifacts (Byrne et al, 1972),
total crude cellulase extract from peas was chromatographed
on Sephadex G-iOO using high salt concentrations (1 M NaCl
in
the~elution
buffer).
,the Sephadex profile.
The result was to completely alter
Two distinct enzyme activities appeared
with elution volumes corresponding to MW of 20,000 and 70,000.
The 20,000 component was soluble in
low-ionic~strength
buffer,
whereas the t 70,000 component was soluble only in the presence
of 1
M
NaC1.
Lewis and Varner (1970) have a1so shawn the existence
of two differentially soluble cellulases in the abscisson zone
\
·~
16
of Phaseolus vuE9aris.
One was buffer-soluble and the other
•
buffer-insoluble (residual) but extractable with high salt
buffer (0.02 M phosphate pH 6.1, ~lus 1.0 M NaCl).
activity altered in response to treatments
with various hormones.
.,..
o~
The latter
abscissidn zones
Lewis et al (1972) and Reid et al
--
--
(1974) further charadterized these cellulases, using isoelectric focusing.
The bulk of the cellulase activity was in the
regions corresponding to pl values of 4.2-4.6 and pl values
of 9.2-9.6, with "minor" components at pl values of 5.0, 6.5
and 7.5."Cellulase 4.5" was the component soluble in 0.02 M
sodium phosphate buffer and -"Cellulase 9.5" was the residual
cellulase soluble in buffer plus 1.0 M NaCI.
These enzymes
were equally stable to heat denaturation and they had the
same pH optima for hydolytic activity against CMC.
The molecu-
'"
lar weight of "cellulase 9.5" was estimated ~24,000 and that
of "cellulase 4.5" at 73,000, using Bio-gel P-150 chromatography.
Lvidence for at least two cel1ulase components has also
been recorded in the "cellu1ase complex" of tomato fruit,
Lycopersicon esculentum,
using ammonium sulfate precipitation.
Pharr and Dickinson (1973) reported that enzyme extracts of
tomato fruit locules contained "Cx" cellulase and cellobiase
activity.
Sobotka and Stelzig (1974) identified a nonspecific
1-glucosidase, an
in these extracts.
exo-~-l,
4-g1ucanase and two endocellulases
Together these enzymes were capable of
\
\
completely degrading insoluble cellulose to short chaîn cellodextrins and glucose.
The greatest multiplicity in cellulase activity from a
-------~~-----
17
higher plant has been reported in a commercial bromelain
preparation from pineapple stem (acetone-dried powder' of the
juice of pineapple stem tissue).
a
co~ination
.
Suzuki 'et al
(1971) using
of Sephadex G-75 and DEAE-Sephadex, column
chromatography (emphazing sequentia1 e1ution with increasing
buffer-salt concentration), identified four ce1lulase peaks
(narned cellula~e I-IV).
When tested for CMC saccharifying/CMC
liquifying activities, these cornponents appeared to differ
only s lightly.
Al though the MW of "cellulase l'' was
rneasured on Sephadex G-100 as 50,000, no such data was given
for the other cellulases.
There were no reported differences
in pH optima or other enzyme properties.
.
Furthermore, the
Sephadex profile shows cellulase
activity throughout the
.
{
/
'
colurnn and ~he authars thernselves state:
"It was evident from
the figure that cellulase l still contains sorne minor constituents of higher and lower rnolecu1ar weight
one."
t~àn
the major
The data from these tests cannot be considered.to prove
the existence of more than one Cx cellulase in pineapp1e
bromelaln.
3.
Purification of Ce1luiaBe
A.
Fungal Cellu1ase
/
A number of ce1lulases from fungal organisms have been
(
extens~ely
purified, sorne te homogeneity.
Whi taker et al
(1963) described two separate procedures for purification of
the
~e1lulase
secreted by Myrotheciurn verrucaria in its
culture medium.
The first involved fractienations with
1
18
ammonium sulfate, elution through Sephadex G-25 followed by
Sephadex G-75, precipitation of.tlle eluted cellulase with
polymethacrylic acid, and finallY,e:1:ution through
~mberlite
1
CG-50.
The second method involved ammonium sulfate precipi-
tation, elution through Sephadex G-25, precipitation with
-
polymethacrylic acid, elution through DEAE-cellulose followed
by elution, on Sephadex G-25 and
Se~hadex
G-75.
Both methods
,
gave final products that were homogeneous on starch gel elec_ _ _ _ _t_r_op,horesis'- haSl
t~e
s ame acti vi ty
0I(
CMC, and represented a
twelve-fold increase in specifie activity .
....
. Iwazaki et al (1965) purified the cellulase activity
from Trichoderma koningi culture filtrates using ammonium
sulFate fractionation (30-80% saturation range), elution
through DEAE-Sephadex A-25 (water first then 1 M NaCl), concentration of appropriate fractions, chromatography on Amber] ite IRC-50 (elution with pH step g,radient) fo11owed by chromatography on a co1umn of hydroxylapatite (e1ution with ionic
step gradient of phosphate).
The last step gave two enzyme
fractions, one e1uting at a phosphate concentration of 0.001
M (cellu1ase 1), the other at a phosphate concentration of
0.1 M (cellulase .11).
The specifie activity increased thirty
seven-fold and recoveries were 78%.
Both enzymes were homo-
geneous based on electrophoretic and ultracentifuga1 criteria.
Okada et al (196a) have purified the cellulase from T. viride
using chr~matography on Amberlite CG-50
and pH step gradient on1y).
(e1uting with ipnic
"
The final step involved filtra-
tion through gauze to remove P-glucosidase and amylase.
~hree
19
cellulase peaks were obtained after DEAE-Sephadex chromatography two of which were homogeneous on ultracentrifugation
although the third peak needed further purification through
..
,
Sephadex G-75.
The specifie actlvlty of the final preparation
increased about
twel~e-fold.
~"
No comparisons were attempted
between these ce1lu1ases and the t?o from
_T~.__k_o_n_l_·_n_g~i_l_·_.
The most recent study of Trichoderma celluloy1ytic
activity was carried out on T. viride filtrates by
and Pettersson (1973).
was very active
~gairlst
Berghe~
These authors purified an enzyme which
cel~odextrins,
crystalline cellulose and
producing ce11obiose, but had no hydro1ytie activity tawards
CMC or ce11obiose.
is understood today,
They ad opte d' the term "Cl cellulase" as it
(i.e. "a ce1lu101ytie enzyme which either
alone or together with "Cx" enzymes is active towards high1y
ordered cellulose", Pettersson et al 1972).
The enzyme
(" ce11ulase Onosuka SS") was purified using, in series, chromatography on Biogel P-10 to
~emove
sma11-molecular-weight
material, DEAE-Sephadex A-50 chromatography to separate 4
"Avicelase" components, and isoeleetric foeusing in a pH
gradient of 3.0-4.2 followed by chromatography on Biogel P-60
1
to further purify a "Ce1lulase IV:D" from the DEAE-Sephadex
ste,p. No mention is made of the fate of the other three
components which show "Avicelase" activity.
was homogeneous as determined by
centrifugation.
Cellulase IV:D
e~ectrophoresis
and ultra-
The specifie activity inereased 14-fold
after purification and the yield was 108 mg of pure "eellulase
20
..,;
IV:D" from 75 9 of crude Onosuk'a S5 powder, with 6% of the
initial activity retained at the end of the purification
procedure.
Eriksson and Pettersson (1968) have purified two
cellulase components from the rot
leutum.
fu~gus
Stereum sanguino-
They concentrated the' culture filtrate by DEAE-
Sephadex A- 25 followed,. by ammonium sulfate precipi tation at
-~~
·V
90% saturation which in'turn was followed by
o~
Biogel P-1S0.
The single
cellu~ase
chroma~o9raphy
peak was then chroma-
~
tographed'on DEAE-Sephadex
~-50.
This separated the cellulase
into two peaks that were further purified on Sephadex G-75.
,
'
The final purification step was preparative zone electrophoresis.
Enzyme yields were very low (26
~g
senting 0.00002% of the initial activity)
in peak l was homogeneous by
of enzyme repreand the ce11ulase
elec~rophoretic
methods.
Pettersson (1968) has also purified the cellulase of
Penicillium notatum using/DEAE-Sephadex A-25 chromatography
1
followed by precipitation of peak cellulase fractions with
ammonium sulfate at 80% saturation.
This was followed by
(
Sephadex G775 chromatoqraphy and ?reparative electrophoresis
.....
on a Sephadex column.
The final product was homogeneous on
'1
analytical polyacrylamide discontinuous gel electrophoresis
using amido black for detection of protein bands.
The increase
in specifie activity was 70-fold with 39% of the initial
enzyme activity'recovered in the purified preparation.
This
is a marked improvement in enzyme recovery over that of the
21
,
Stereum cellulase purification reported earlier (Eriksson
and Pettersson
196~).
The DEAE-Sephadex step gave the
greatest increase in specifie activity.
1
\.,
B.
Higher Plant Cellulase
To the author's knowledge, no higher plant cellulase
has been fully purified to date, except for the buffer-insoluble pea cellu1ase studied in this laboratory (Byrne, 1914)
concurrently with the present study •. There have been a few
reports of fractionations or concentrations of plant ce11ulases by acetone and/or ammonium sulfate precipitations
(Perrault and Maclachlan 1964, Pharr and Dickenson 1973), by
isoelectric focusing (Lewis et al, 1972) and by combinations
of Sephadex G-75 and DEAE-Sephadex chromatography (Suzuki et
al, 1971), but none of these efforts resulted in more~than a
'.
few-fold increase ,in specifie activity.
The major difficulty encountered in using higher plants
as a source of cellulase is the relatively low level of
cellulase found in most
tissues~~nd
the fact that the enzyme
is usually highly localized in tissues that are difficult to
obtain in quantity.
The viscometric assay used to detect
cellulase,is very sensitive and gives a deceptive picture of
the actual quantity of cellulase proteine
As little as l ng
of Aspergillus cellulase is readily detected by this assay
-
-.- -- -
" .
~
cellulase activity found after treatment of pea epicotyls
.
\
'~~
D
with auxin (Fan 1968; Bonn, 1970), the convenience of growing
/J
-~
22
peas in large numbers and the ease of harvesting tissue
~
, suitable for cellulase extractions, make this system an
appropriat~
one -fÇ'}r the purposes o.f isolation and puri fi-
~
cation of higher plant cellulase.
D
Its localization in
growing regions (Perrault and Mac1achlan,
1964) adds
,
special interest to the properties of cellulas8, although
it also means that very high levels of other proteins are
.' ,
al~ extracted a10ng with it and purification is ~[eceà
ter*? more difficu1t.
1.'
23
MATERIALS AND METROOS
1.
,
Source of Enzyme
'Pea seeds (Pisum ,sativum L. var. Alaska) were soaked
for 20 minutes ln 0.5% sodium hypochlorite.
,.
After 8 h
imbibition in tap water, seeds were p1anted in moistened
vermicu1ite and grown in darkness at room temperature. When
the third internode was more than 1 cm long (7-8 days) the
intact seedlings we,re sprayed wi th 0.1% 2,. 4-D in 0.1
NaCl (pH adjusted ta 7.0) plus 0.1% Tween 80.
Approximately
10 ml of spray was.used per 500 seedlings, and they
left in darkness for 4-5 days.
M
wer~
This treatment results in the
cessation of cell elongation and marked swelling in the subapical region of the epicotyls, and is accompanied by
l~rge
increases (50-100 fold) in total cellulase activity in this
tissue. / The 2, 4-0 apices (plumule, hook, and swollen tissue)
were excised(approximately 200 mg fresh wt/apex), cooled to
. approximately 4 C and used for extraction of crude buffersoluble cellu1ase.
2.
Extraction
A typical enzyme extraction is described.
7----
e
AlI procedures
for this and subsequent purification steps were at 4 C unless
otherwise noted.
Approxirnate1y 100 9 of 2, 4-D-treated apices
were homogenized for 5 min in 2 volumes of "extraction buffer"
24
/
(20 w1 sodium phosphate, pH 6.7,
1% glucose,
~
th~n
squeezed tnrough
a resil:ié~.
glycerol) with
1
a Waring blender (400 ml capacity)
'was
?%
nylo~
.....
at tep speed.
The homogenate
~
cloth te obtain a filtrate and
The ~iltrate was céntrifuged at 13,.000 x 9 fOl::'>--+l~O-­
min (Sorvall Re-2X, rotor capacity 3,20 ml).
-The 5uper;nat~~t·
obtained af'ter careful deçantation serves as crude buffersoluble (BS) cellulase.
The precipitate after centrifugation
is discarded but the residue retained by the nylon after
squeezing
~hrough
it the initial homogenate can be used to
prepare crude buffer-insoluble (BI) cellulase (Byrne, 1974).
•
1
3.
Assay
Cellulase activity was assayed viscometrically (Fan and
Hac1achlan, 1966) as fo11ows:
Carboxymethyl-cellulose (CMC,
Hercules PowdeJ? Co.), 0.6% 7LP-0.2% 7IISP in 20 mM sodium
phosphate buffer pH 6.0, 0.2% sodium fluoride, was used'" as
J
substrate.
Semi-microviscometers
used in a water bath
mai~tained
water pump (Fisher Scientific
(C~nnon 200, vol l
ml) were
at 35 C by a Haakke circulating
~o.).
Under these conditions the
viscometer flow-through time for water was 7-10 sec.
A 0.9 ml
aliquot of stock CMC solution was placed in the viscometer,
allowed to equi1ibrate for at least 20 min, and 0.1 ml of an
appropriate enzyme dilution was ~dded.
The enzyme and substrate
were thoroughly mixed by blowinq air through the viscometer,
care
bei~g
glass rod.
taken to disrupt aIl remaining air bubbles with a
The assay was begun after 5 min incubation, taking
o
10 min readings to the nearest 0.1 sec.
time for this solution was approx.
Initial flow-through
70 sec.
/
5
.1
25
,
.
\
The assay
measures
endocellulase acti~ity lPerraulE and
o
,
,
(1
C"
Maclachlan 1 1964). and remai.ns .linear up to 20% 10$5 in viscosi ty of
S::~C_
(Fan, 1968)
~
One unit of enzyme acti vi ty, is
"'
'-1.
defined here as the amount of enzyme required to
caùs~
in viscpsity in 2 h und e r" the above conditions~
Empirically,
1% 10ss
the most 'convenient enzyme concentration was'one that gave
2-3% 1055' in yiscosity of CMC per 10 min
readin~ ~~O
units/ml.
\>
4.
Arnmoniu~
Sulfate Fractionation
....
ft
Approximately
400 q of 2,4-D-treated apices were used to
<J
prepare 1,000 ml of crude BS cellulase, and 194 g of ultrapure
crystalline ammonium su:fate
(~artz-Mann)
was
add~d
with
constant stirring, 50 as to obtain 35% ammonium sulfate saturation.
.,was
After 30-45 min', constant stirring, the precipitate
(P35)
collected by centrifugation at 13,000 x g for 15 min
(Sorvall Re-X, rotor capacity 2L).
~e
supernatant, 1,100 ml,
.,
was brought to 60% ammonium sulfàte saturation by the addition
t
of 168 g of ammonium sulfate, stirred for 30-45 min and the
precipitate (P60)
collected as before.
The same
procedur~
<J •
was,repeated for the 80% ammonium sulfate saburation (Pao'
.
volume 1,180 ml, add 152 g ammonium sulfate) and the 100%
ammonium'sulfate
a~onium
saturatio~J(~o"
sulfate).
volume ,1,250 ml, add 174 9
The precipitates were washed'with 10 ml of
.
extrac.tion buffer so as to remove excess aRlItlonium sulfate
o
..
----~~.
_. fiôm - the centr
7Iùge
tube ii/aIls. ana thé surface of the precipitates
an9/then
they were redissolved
\
. in the minimal amount of extrac(
,-.
,.-------c
...
-
-~
•
26
"
tion buffer necessary to obtain·complete solubilization.
,
The
resulti11;ij---s-elutions werE;' cent:r'ifuged at 13,000 x g ,t,d'r 10 min
------------------
50
as to remove any gross contciminants (particulate debris,
\
etc.).
A clepr yellow-brown supernatant resulted which was
.
used for further'puFification.
~
'~
for>cellulase and
p~otein
,After several such
Aliquots of this were assayed
levels after appropriate dilution.
experim~nts,
it was found repeatedly
that the major part (70-80%) of BS cellulase activity was in
the 35-6~% ammonium sulfate fractionation cut.
Thus, P60 was
used for further purification.
5.
Sephadex Gel Fractionation
l
Although various column sizes were employed in these
experiments, the sawe method of packing, loading, developing
and calibrating the columns was used in aIl.
For example, in
a typical experirnent, a ]acketed column (e.g., 2.5 x 40
c~,
K25 column,Pharmacia Corp.) was secured on a refrigerated
fraction collector with provisions for circulating coolant
through the outer jacket of the column {Buchler Instruments).
The co1umn was packed with,Sephadex (e.g., G-75, fine, as per
manufacturer '"s instructions) using a peristal tic pumE.
(LKB-12000) to draw Quffer through the outlet of the column.
In arder to,avoid subsequent shrinkage of the column, the rate
of flow of buffer was about twice the rate to be used later
to develop
. ,- the column.
A 'sample appli.cator was placed on top
of the colurnn and the column was le~t until use, wit~ puf~er
.
çontinuously
f~owing
th~ough
it at a low rate •
27
Colpmns were calibrated by loading 5 ml of a solution
co~taining blue dextran (1 mg/ml, Pharmacia) plus a rnarker
----_
.
protein at concentration of l mg/ml.
tests included ovalalbumin (ffi~ = 45,000) ,~-chymotrypsinogen
./ (Mw
(MW
= 25,000), myoglobln (MH = 17,800) or cytochrmntr-c
=
12,400).
.
ât
Columns were developed overnight
a constant
flow rate (e.g., 24.0 ml/h) and fractions were collected at
particular time intervals (e. g., 3.6 ml/9 min).
Blue dextr'an
1
and marker proteins were detected spectrophotoroetrically at
O.D. 280 nm or O.D. 410 nm.
The columns were then ready for
chromatography of cellulase.
Columns were loaded with 5 ml of crude BS cellulase and
1
developed exactly as described above.
Fractions were collected
and maintained at 4 C until assayed viscometrically for cellulase activity, and for protein concentration by the method of
Lowry ~ al (1951).
Certain minor modifications of this
technique' were occasional1y ne~essary.
For example, the very
large co1umn (10 x 90 cm, KlOO, Pharmacia) was run using
\,
Sephadex G-25 (coarse) and flow adaptors were required in order
to a110w use of a pump (Buchler Instruments) ta load the sample
as well as ta develop the column.
l~se
samples through
~lumns
Also, after running cellu-
which were intended for repeated
use (e.g., during purification, where two 90
"
cmr~
,
2.5 cm
columns packed with G-75 were run simultaneously), the columns
./
were washed with approximately on~ column volume (V t 500 ml)
,
,
of extraction buffer plus 1.0 M NaCI, followed by at least 2
4
}.
column volumes of extraction buffer.
.'
This step was necessary
~--_.
-
-------~,
.....
28
- e ----
in order to remove impurities that bound to the gel or
precipitated~__ the_tQP
..
10
cm of - --column and e1uted slow1y,
-- - - - - - ~
---~-
contaminating subsequent samples.
6.
DEAE-Cellu1ose Chromatography
DEAE-cellulose (Cellex-D, anion exchange cellulose,,
Bio-Rad Laboratories) was treated as fol1ows before packing
in a column.
Approximate1y 200 g DEAE-cellulose was repeatedly
suspended in 6 1 of deionized water an~refined
(e.g., 5 times)
by decantation after standing undisturbed for 30 min.
It was
then washed three times with 4 1 of 1 N NaOH, three times with
4 1 deionized wa
.
once with 2 1 of 0.1 N HC1, and finally
until aIl acid
wa~
removed.
The DEAE-
----------
cellulose was resuspended in extraction buffer (approx. 5
g/lOO ml)., and a:l1owed 24 h for equi libration.
It was packed
in co1umns as described above for Sephadex.
Concentrated partially purified BS cellulase (e.g., 50 ml
of P60) was loaded on a DEAE-ce11ulose column (e.g., 2.5 x 30
~)
using a pump (Buch1er Instruments)
and the column was
washed with one Vt of extraction buffer.
~ith
2 to 3 Vt
of 0.4 M NaCl in extraction buffer until OD 260
was low and constant ( 0.1 OD).
3 Vt of 1.0
~1
This was followed
~he
final wash was with 2 to
NaCl in extraction buffer.
Appropriate fractions
were collected and assayed for cellulase and protein levels.
The column was dismantled after use, and the DEAE-ceIlu1ose
was washed as befare, equilibrated with buffer and used to,
....J
repack the column.
_
e
\
29
~.
Sucrose Density Gradient Centrifugation
Linear sucrose density gradients were prepared using a
manual density gradient former <ST s=t.andard désign:
'Ple mixlIi.g
chamber was filled with 1.8 ml of 30% sucrose in extraction
buffer, and the stopcock was opened in order to fill the outflow tubing (0.1 ml volume), and closed again.
The other
chamber \vas filled wi th 1.7 ml of 10% sucrose in extraction
buffer.
Mixing was accomplished py a small magnetic bar in
the mixing chamber after both stopcocks were opened, and the
gradient was allowed to form in polyallomer centrifuge tubes
(10.9
K
54.7 mm, '4 ml capacity).
A 1 mm band of nai1 polish
was ·.painted on the inside of the tubes running from top to
bottom so
~s
to create a hydrophilic surface, allowing the
·gradient to pour as· a slngIe column down- the slde -6r---uiet.ube,
rather than drop by drop.
This gave minimum'disturbance to
the forming gradient.
A 0.1 ml sample containing DS cellulase in extraction
buffer to which myoglobin (1 mg/ml) was added as a marker,
was carefully layered oh the gradient using a micropipette.
The tubes were centrifuged (SB405 rotor, I~ternation prepara-
.
tive ultracentrifuge,
for
. MQdel B-60), at 300,000 x g average
.
12 h:
Tubes were fractionated into 0.25 ml fractions using a
4
density gradient fractionator (ISCO, Model 0).
Cellulase was
assayed visc0metrically and myoglobin spectrophotometrically
at 00408 nm.
Sedimentation coefficients were determined
using the method of Martin and Arnes (1961).
30
,
8.
4It
Analytical Discontinuous Polyacrylamide Gel Electrophoresis
Essentia1 stock solùtions were prepared at concentrations
4 C.
AlI (except Solution F) are stable for at
1eas~Tsix
months
,
under these conditions (Solution F must be fresh1y prepared
immediately be'fore use).
Cornponents per 100 ml aquaeous solution
...
Solution A
(pH 7.5)
--
-- ---- - - -
1 N
Hel
T'ris
Temed
l'
Solution B
(pH 5.5)
l M H- PO 4
Tris 3
Temed
----.,.,So1.ution-C
- --Acrylamide
Bis
Solution D
Solution
E
ml
6.85 9
0.46 ml
48.0
39.0 ml
-4.95 9
0.46 ml
-----
---
: 30___Q____ g
-
0.8
\...
9
Acrylamide
Bis
10.0
2.5
9
Rîboflavin
4.0
mg
g.
,
Solution F
Sodium Persulfate
Solution
Sucrose
G
0.14 9
40.0
Solution H
Bromphenol Blue
Electrode' Buffer
(pH 7.0)
Diethyl Barbituric Acid
Tris ';.'
Water to
9
0.001 9
'. !
.
5.52 9
- 1.0 9
1.0 l
The above solutions were mixed as follows in order to
obtain the gels
requireti~for
standard electrophoretic procedures.
For the "stacking" gel, i:e., a 2.5% gel at pH 5.5, solution
tatios were B:D:E:G::l:2:l:4.
For the "separation" gel, i.e., a
7.5% gel at pH 7.5, ratios were A:C:H20:F::l:2:l:4.
)
s
The
31
separation gel was polymerized first at 20 C in 7.5 x 0.5 cm
glass tubes to a h~ight of 5 cm.
----rnèï'i~ed
The stacking gel was poly-
on top to a height- of- -1. 5- cm.
The gels were
ç.Q9l~d__________ _
to 4 C and placed 'in the electrophoretic cnamber (Buchler
The lower "electrode buffer" reservoir" was
Polyanalyst).
kept at 4 C.
The top electrDde buffer reservoir was filled;
trapped air bubbles were removed and the sample (0.1 ml,
10-200 pg protein/gel in 20% sucrose)
the buffer
(Hamilton).
1
on~o
was loaded underneath
the stacking gel using a 100 pl syringe
Bromphenol blue (2 ml of Solution H) was added
to the top buffer reservoir.
The electrodes were connected
r
with cathode (-) at the top reservoir and anode (+) at the
bottom.--~(Electrophoresis was carried
out at 2 - ma/gel
uSirig-a
-
-~.=..
constant current-regulated
Hodel 3-1155).
power~supply
(Buchler Instruments,
Electrophoresis was complete when the brom-
phenol blue migrated to within approx. "5 nun of the bottom of
the gel.
The gel columns were removed from the glass tubes,
. plaoed in aluminum molds (0.6 cm x 7 cm, 0.05 mm thick) and
fr~zen in 'dry ice.
gel
.~licer,
r
Gels '/lere s liced into l mm sections (Hickle
Brinkman) starting from the top of the ,separation
"
,
gel a;d stopping at the slice containing bromphenol blue.
Each slice was placed in l ml of extraction buffer and after
~l h incubation at 35 C, 0.1 ml aliquots were assayed for
cellulase activity.
In order to visualize the location of
protein bands in the gels, duplicates of
gel~
examined for
cellulase activity were placed immediately after stopping
)
32
electrophoresis into a solution of 1% coornosie blue in 7%
)
acetic acid.
After 20 min~ destaining was carried out over----
-----
-
night in 4 1 of 7% ace tic acid.
- - - - - - - - - - ---.-- ----
t
9.
Isoelectric Focusing
The rnethod employed here was identical to that described
by Haglund (1967), using 110 ml capacity column (LKB) with
inner and outer cooling jackets.
Experiments were conducted
at 2 to 4 C, using 11 Ampholine, pH 3-10, in a 0-50% (anticonvection) sucrose gradient.
a
The gradient was prepared from
50% sucrose (w/v) sofuticn containing 1% Ampholine, in water,
(solution H) and a 1% Ampholine solution in water, (solution
L) " in 24 4.6 ml portions as follows;
portion nurnber 1
contained 4.6 ml solution H, portion number 2 contained 4.4
.'
ml of solution H and 0.2 ml solution L.
In each subsequent
portion the volume of solution H was decreased by.O.2 ml and
that of solution L increased correspondingly
number 24 contained solution L only.
50
that portion
The anode (bottom of
column) and cathode (top of column) were protected by 1%
monoethanolamine, .in 50% sucrose, and 10% phosphoric a~id
respecttvely.
The enzyme extract, in 20 mM sodium phosphate
buffer plus 1% Ampholine replaced solution L in portion number
12 of the gradient.
Focusing was carried out for 72 h at 300 V.
The initial
,
currEmt fl:ow ·was 3-5 ma, dropping to less than O. 5·....ma by
end of the rune
the
When focusing was completed, the column was
fractionated into 22,_ 5 ml fractions, each fraction was mixed on
33
the vortex, and the pH was measured immediately at 2
(Ampholites in the higher pH range tend to take up CO
! - - ---
~--~--~-----eeeerne~-mGr-e-
aGidicr---hence-plL_meas.urement _ffiUSt_ be_Jllade
~
c.
2
and
~as~ ____________ _
r
soon as possible after fractionation).
The cellulase acti vi ty
in each fraction was measured in the usual manner.
/
10.
\
Ultrafiltration
Concentration of cellulase activity after DEAE-cellulose
chromatography was performed using two ultrafiltration cells
~of
2 1 capacity (Model 2000, Amicon)
nitroqen gas.
in series, at 40 PSI of
Membranes (PH-10) with a retention MI'J
daltons were used.
=
10,000
Pressure was released when the concentrate
"
•
_
H
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
volume was about 50 ml, the concentrate was removed and the
membrane surfaces were washed with 10 ml of extraction buffer
containing 1.0 M NaCl.
The concentrate and washings were
clarified by centrlfugation at 13,OOOxg for la min, transferred to a smaller ultrafiltration cell
200 ml) and ultrafiltration
~as
(Model 202, capacity
continued at 20 PSI until the
concentrate volume was about 8 ml.
This "vas removed, the
membrane surface \Vas washed with 2 ml salt buffer solution and
jJ
the concentrate and washings were clarified by centrifugation.
Aliquots were assayed for BS cellulase and protein levels
befare further
ptl~ification
(e.g., on Sephadex G-75).
Concentration after Sephadex G-75 chromatography was
done similarly using a
200
~l
cell followed by a 10 ml cell.
The final concentrate was clarified by centrifugation for la
'"
min at 13,OOOxg and assayed for cellulase and protein leveis.
34
e.
This final product is referred ta as -" purified" BS ce11u1ase.
/
Il.
-Preparation of, Antiserum
A 5 Kg albino male rabbi t was injected subcutaneously
with 1 mg of purified BS cellulase emulsified with Freund's
complete adjuvant.
This was followed in 2 weeks wi'th 3 bi-
weekly injections of 1 mg cellulase emulsified with Freund's
\
incomplete adjuvant.
Six days after the last injection 40 ml
of blood were collected from the marginal ear vein into
1
vase1ine-coated tubes.
They were incubated at' ,37 C for 1 h
in order to ènhance clot formation,
night to obtaj.n clot retraction.
decantation and
15 min.
clarif~ed
and placed at 4 C over-
Antiserum was co1lected by
by centrifugation at 13,OOOxg for
.
This yie1ded 21 ml of antiserum, part of which was
freèze-dried and part was frozen at -20 C for future use.
The presence of BS cellulase antibodies was initially
detected using the "ring te'st".
12.
Immunodiffusion
'
)
Ouchter1ony double immunodiffusion was carried out on
(,
microscope slides in 1% Ionagar in borate-buffered-saline
(6.184 g boric acid, 9.536 9 sodium tetraborate, 4.384 g'
sodium chloride per l of H 0).
2
1;vel1s of 1 mm diameter with a
center-to-center dis~an~of 7 mm,were cut in the agar,
fil1ed with appr.opriate reâctants and immunodiffusion was
carried out at 37 C in a moist chamber for 24
h.
Slides were
washed for 5 h in 3% NaCl followed by 24 h in borate-bufferedsaline.
They were dried at room temperature for 24 h and
35
stained for 20 min with 0.2% acid fuchsin in 50% methanol-10%
acetic acid-40% water.
Destaining was carried out in the acid
fuchsin solvent.
13.
Immunoelectrophoresis
Immunoelectrophoresis was carried out on microscope
slides.
These were coated with 2% agarose and air dried.
A thick layer of 1% agarose (2 ml/slide) in sodium barbital'
buffer pH 8.6, ionic strength 0.05 was spread on top of this
and slides were transferred to a moist chamber at 2
c.
Wells
2 mm in diameter w~re punched l cm from one end and approximately 5
rl
of sample (plus bromphenol blue as marker dye)
were added to each weIl.
Electrophoresis was carried out at
l~ V/cm for 120 min, with the anode connected on the end
farthest from the wells.
After electrop~oresis was completed,
a 2 mm wide strip of filter paper impregnated with 75 ~l of
antiserum was placed on the slida between the wells and parallel to the path of electrophoretic migration.
Slides were
incubated for 24 h at 2 C and then treated as per immunodiffu,
sion (see above) for precipitate visualization •
•
36
e
- - -- -- -
~sums
----------------~--------------------I
A.
Fractionation of Cellulase Activity in Crude Extracts
1.
Chromatography on Sephadex gels
"Figure 1 s]1ôws a cellulase activity profile following
chromatography on Sephadex G-75, of a crude extract of pea
epicotyl protein soluble in 0.05 M sodium phosphate buffer,
pH 6.5, (referred to as. phosphate buffer) •
Approximately
1
5 9 of 2,4-D-treated peas were homogenized with 10 ml of cold
phosphate
b~flr
centrifu~ed
usinq mortar and pestle.
The homogenate was
at 37,OOOxg for 20 min and 5 ml of the supernatant
containing 1,780 units of cellulase and 23.5
loaded on the column (2.5 x 40 cm).
~g
protein was
Thls was developed with
•
phosphate buffer at 28.3 ml/h and 3.6 ml fractions were
collected.
.
Cellulase activity was measured viscometrically
and protein was measured by the method of L~ry et al (t95l).
Recoveries in this experiment were 1,480 units of cellulase
(83%) and 20.2 mg"protein (90%).
Four cellulase peaks were observed:
,
volume of the column (Va):
V e =72
. 0 ml',
one in the void
0
a second with elution volume,
a third with Ve =lOO.8 ml: and a fourth with
From a ca"libration curve using well-known
protein markers on this same column (similar to that of
Fig~
10), the molecular weight equivalents of these cellulase
activity peaks corresponded ta approx. 38,000, 19,500 and
37
.'
~
Fig. 1:
-----
~-
~----
-
Crude buffer soluble (BS) cellulase activity chromatographed on
S~phadex
G-75.
Approximately 5 g of 2,
4-D-treated peas, (swollen region of epicotyl without
j
plumule or hook) were homogenized wit~ 10 ml of cold
1
,~~o mM sodiwn phosphate buffer pH 6.7 'Using a mortar
and pestle.
The homogenate was centrifuged at 37,OOOxg
for 20 min and 5 ml of supernatant) (crude BS cellulase"
1,780 units, 23.,5 mg protein) was ;Loaded on a Sephadex
G-75 colurnn (2.5X40 cm). This was developed with
1
pha~hate
buffer, 3.6 ml fractions were collected and
cellulase activity in aliquôts of each fraction was
measured viscometrically. protein
methodrof
Low~y ~
wa~
,
""""by the
measured
âl. Recoveries in this experiment
1
were l,480 unitrs ce1lulase (83%) and 20.2 mg protein
_ (90%). Values inserted abave peak fractions represent
.
approxima te mo1ecular weights, as deter-mined from
-calibrati~n
a
curve using marker proteins (similar ta
Fig. 10). Protein levels beyond fraction 20 were very
low (0-20 r-g/m1) and J:pve been drawn as zero in this
and subsequent figftes for simp1icity.
,~,
ID
•
o
...
.
,
?...
p
G-75
...-
JJ180
600
-
()
s
~
....
135
19.500
z
400
::::J
u.J
z
90
V")
e
--J
:;:,
--J
--J
LI.J
c:..:>
.
.
.
.
....
..5
0
"'0
.
-"
.
......__________
-.'~s
~~
NUMBER
FRACTION
()
r
.....
&.I.J
CC
____
~
.
......
.. 8
200
0
ct::
c..
0
.-
i
1
L..
\
38
5,500 respeetively.
The moleeular weight of the eellulase
activity appearing in the
of the eolumn can not be estio
"
rnated from a calibration curve but would be ~reatér than
V
7,0,000.
This experi:ment
was repeated five times using the 'saÎne
'-'v
eolumn and a different erude 'extraet.
The relative level of
1)
eellulase activity in the Vè varieq and
t
th~
....
38,000 and 5,500
\
peaks were sornetimes missing, but the maj;rity of aetivity was
always in the 19,500 peak.
Figure 2 shows a ~ellulase activi~Y~file obtained
using erude enzyme extract and Sephadex G-50 column chromatograp~y.
loadi~g
.
The column size and proce~ure for preparing and
sa~e
of sample was the
as that in Figure 1.
Cellulase
activity loaded on the eo(uMn wa~ 7,750 units and protein was
'22 mg.
Tl).e column was deve);oped w;Î.th phosphate buffer at 32.0
mljh and 4.0 ml fractions were collected and assayed as before.
Cellulase recovery was 7,730 units
100%).
(100% afid protein 23.5 ~g,
The profile i5 very diffèrent from that using Sephadex
\
G-75 (Fig. 1).
Aetivity was irregularly distributed through-
out the elution profile with the bulk of it close to the V t
peak of this column, with Ve =128 ml (centered at fraGtion 34)
corresponding to a HN of 5,000.
In seme repeti tions of this
/
experirnent (Fig. 3), performed on a separate day with a
differe/nt erude enzyme preparation but the same column and
developing conditions, (except iV Fig. 3 fraction size was 4.7
, '
ml, and flow'rates 31.6 ml/h), the results showed up to five
aparent cellulase peaks.
,
The main compone nt oftent'but not
39
"
.
i
,
.
.
~
1
,.
'.
\
\
______
~
l_~~~~
.
\
Crude BS
Fig. 2:
CellUlaSe'~activi tyO chroIr}itograPhe~
e
1
Sephadex G-50.
•
Cellulase was extracted as in
.
•
,F1gure
1; 5 t ml (7,750 units) were
Sephadex G-50 ?s before
(~lurnn
~eveloped
size : .. 4 .. 0 ml/fr.
,
,
/
1
,
.
.
on
size 2.5X40 cm)
-and. recovery was 7,730 units (100%).
·e
à
~raction
,
•
\
",
.., .....
.....
.-
~
il<
,
<lJ\
l-
-···
-5,000
160
""
"
·
Vo
0
12
,
:1
....
:lE
::»
80
&..I.J
U)
.....
cC
::)
-J
-J
w
u
..
···
···
··
··
···
···
···
·'.
z:
""'-
.0
u.J
cc:
0-
...........
~...t' '
16
2
FRACT InN
1
...
.J
~
-'
..........
CI)
·
32
NUM/BER
40
40
,
Fig. 3:
Crude BS' cellulase actl.vl.ty chLomatograpRGQ on ,
Sephadex G-SO on a separate .occasion from the
experiment recorded in Figure 2.
extracted as in
(5,O~O-ünits
Fig~r~_~;
5
~l
Cellulase was
of supernatant
cellulase) were developed on
Sephadex G-50 (2.5X40 cm) with recovery being
3,500 units (70%).
Fraction size: 4.7 ml ..
Note that the initial 'enzyme preparation and
the column used in fractionation were identical
to those in Fig. 2, but the activity profile
was not reproduced.
_.
.
.r_.s......________ ___ . __
~~
.,
.' .
)
r
~
-t
-
.J
2
G-SO
Vo
-- 4,500
,
",
60
"
'.
,"
··.,.
,,' ,.
,· ,.,
....
""
en
-=
:::t
LW
-;
--
•
L--
\
800,
·
··,
600~
2
,~
./
.. 0
.. 00
~
(/)
Z
cC
--l'
L&J
::::»
--1
--1
u.J
t-
200 ~
20
0..
(.)
,
O~~
10
'.
________'_'_"_'~'~~"_"_'_"_'_"_'_"_'_"_"_"~~~~~~~~~~----~
2
30
FRACTION HUMBER
1-
.. 0
4°
41
always, corresponded'to a molecular weight of 4,50? to 5,500 •
The minor peaks were of mo1ecular
~eights
.
which were completely
different from those of the Sephadex G-75 experiment (Fig. 1).
In these experirnents, varyil1-g rue
-céllul~
activity
which was loaded on the column from 1,000 units to 10,000 units
per,5 ml, gave no set pattern in the observed profile, i.e.,
the prèsenée or absence of subsidiary activity peaks did not
r
appear ta be dependent on total protein or enzyme concentration,
--
-~-
-
-
~----_-=---------:.-- -~
-=-
x --
The variability and random nature
at least within this range.
of these profiles indicated clearly that this method of chromatography of cellulas~ activity was subject to artifacts.
,
1
~
~he
repeated observation of an enzymic component with an apparent
molecular weight as low as 5,000 (referred ta here as "minicellulase") , was particularly questionable, although there
was one report in the literature
(Sel~y
and Maitland, 1965) of
, the existence of such an extraordinarily srnall cellulase in the
fungus Myrothecium verrucaria.
Figure 4 shows an attempt ta obtain this "rnini-cellulase"
co~ponent
in large quantities
"
f~r
further investigation.
A
10 x 90 cm column with Vt =7,000 ml was packed.with coarse
Sephadex G-25, and 200 ml of crude
pea epicotyl
1
extract con-
taining 45,000 units of cel1ulase activity and 800 mg protein
was loaded on this column.
J
\vas develqped wi th phosphate
buffer at a rate of 1620 ml/h and 150 ml fractions were
col1ected and assayed for cellulase and protein content.
Recoveries were 44,000 units.of cellulase (9B%) and 780 mg
protein (98%).
Most of the cellulase activity was in the void
.
,-
42
'.
r
<,
-
J.
c
Il
Î
Flg-q:
~
cruae
on Sephadex G-25.
,
L.<
~~ ....r;m~~"' .... ~nhAr'I
Approximately 100,g of 2,
r
4-D-treated'peaS were used to prepare 20Q ml
.
~--,--_.
of crlJdEL~.supernatant (45,000 units
of
-- --.---------~-----II
cellulase, 800 mg protein).
This was loaded.
on ~ Sephadex G-25 column ( 10 X 90 cm ) and
,-
developed with
phosp~ate
buffer.
Recovery
was 44,000 units cellulase (98%)'. and 780 mg
prote in (98%).
Fraction size: 150 ml; flow
rate: 1,620 ml/ho
1
"/
...
:..
(
'0,
--
100
---.-·-1
~
••
••
•
••
••
·
~
••••
••
•
•
•••
· .•..
·••••
••
·•••
400
'Z
"
••
•
La.I
00 ....
••
••
·
...0
0::::
0..
••••
••
••
••
10
15
20
FRACTION NUMBER
'\
43
volume but a significant amount,shoulder fractio~ 13 to 16)
-'1
was delayed and fractionated by this gel, which has an
L
exclusion'limit of ml approx. 5,000.
preted~im~l~i~at -the activity observed in fractions
------
13 to 16 has a MW of less than 5,000.
- - --------
An aliquot from
fraction 15 was concentrated by p1acing it in cellulose
~--------..{:au:c;Ee~tI:-aar.t.ee~dLiLJa;uly~_~ubing
.
Tnis is norrna11y inter-
and~covering
thi,s wi th
sol~d
poly--;---------
_--- ~~--~- ethylene g±jTGO-l..- -- ThL~L GQI1cerli:~_~.!:~~
t~~~arnple
-
rechromatographed on a 0.9 x
~o
=--_
-""L~
______
~
~hen
~_~
... ----,,-.... "-----"----~~-
cm co1umn packed with
G-50 and calibrated in the usual manner.
was 92%.
~
which was
Sephad~x
RecQvery of activity
Two ce11uLase peaks were observed (data not shown),
one in the void volume of the column and the main peak (70%
total activity) at an e1ution volume corresponding to MW
-----
------
19,000.
. 2.
No activity reappeared at a MW of 5,000.
Sucrose density gradient sedimentation profiles of crude
and partially purified BS cellulase
Further investigation of the
on Sephadex G-25
(Fig. 4)
~rcellulase
fractionated
or G-50 (eg., fractions 30-36, Fig.
J
2 and 30-35, Fig. 3) as well as void volume cellulase from
Sephadex G-50
(eg. fractions 14-18, Fig. 2) wàs undertaken
after pooling and concentrating fractions with polyethylene
glycol.
Velocity sedimentation was carried out in 10-30%
sucrose density gradients prepared as in Materials and Methods:
A
~.l
ml aliquot of:cellulase activity (20-30 units) was
layered on gradients which were centrifuged at 300,000 x 9
\,.
(
44
,~
1average) for 12 h with myoglobin included as internal
reference at a concentration of l mg/ml.
The distribution
",.r
in gradien,ts of cellulase acti vi ty and myoglobin concentration
----
--
was assayed.
-~-
As can be seen from Figure 5, all the seemingly
-----
different cellulase fractions from Sephadex gel chromatography
migrated to the same locus on this gradient corresponding to
a
sedi~entation
coefficient
~elative
to myoglQbin .9f 3.4 S.
Figure 6 shows the results of sedimenting a 0.1 ml aliquot of
/
crude BS cellulase under
Figure 4.
ide-ntic~af-condIITons
as Ehose
~~
Again, only one cellulase peak is observed with a
sedimentation coefficient relative to myoglobin' of 3.4 S.
_.1
___ _
-----
3.
-----
--
1
----- ----------
Analytical discontinuous polyacrylamide gel electrophoresis
There can be no doubt, therefore, that the "mini-cellulase"
(,~
in peas is a Sephadex art4fact.
Figure 7 shows results of
electrophoresing crude BS cellulase activity in an 'anionic
system using
discon~inuous
polyacrylamide gel electrophoresis.
Crude BS ce11u1ase extract was made 20% with solid sucrose and
0.1 ml aliquots were placed on top of columns of polyacrylamide
gel (0.5 x 5 cm) prepared as in Materials and Methods.
gels were e1ectrophoresed at 2 ma/gel for 120 min.
blue was used as a "front" marker.
The
Bromp~nol~
At the end of the electro-
phoresis one gel was treated for celiulase detection and a
duplicate gel was stained for proteine
As can be seen from
Figure 7, on1y one ce11ulase peak was observed, with Rf =0.37
relative to the marker brompheno1 blue.
45
.-
..."i~
Fig. 5:
,f
~
Sucrose àepsity gradient profiles of BS
cel1u1ase taken from peak fractions of Sephadex
\
>,
.
experiments shown in Fisures" 2 to 4.
~-~~'~--~----------Aft~±~~-of
A 0.1 ml
ce11ulase activity (20-30 units), was
1ayered on 10-30% 1inear sucrose gradients in
phosp~ate
buffer.
The tubes were centrifuged at
300,OOOxg for ]2 b with myog1obin (Img/ml)
included as internaI reference (arrows). Gradients
~e
~was
separated into 0.25 ml fractions, ce1lul::e
assayed viscometrléaI1y and myoglobin spectro-
photometrica11y at 408 nm.
a's fo11ows:
Sources of enzyme were
a=fractions 14 to 18, Figure 2; b=
fractions JO te 36, Figure 2; c=fractions 30 td 35,
Figure 3i d=fraction 14, Figure 4.
Values inserted
above peak fractions are sedimentation coefficients
deterrnined relative to myoglobin (s=1.9).
7 7
,
'\0
.
<.
/
- .
.
&
.
•
e
,
,
,
.
-
.
.;;
,JL;---..,
.
,
,
...,
_..
16 1--
.,,,--
.
-
,
.
J,
'
'l:J:
-
1
.
,
a
•
16
-
---
~
b
~
r
~
,...
...-~-...,,-
,
,
~
2
.~
81--
ci
......
.ut
-a:
0~
12 ~
l.&.J
c.I')
.
\
0
--- i
c
r
~
cc
~
~
1
1
~
"
~
.
1ft
N
'\
_&
1
2(] ~
1
d
~
,
-
.,.J
~-
~
l.&.J
6
.
Ir
::;)
J"
(
.--
""
1
.
Q
10 1-
~
,
(.)
.
1
0
...
0
- ...
1
1
1
1
~
8
12
~
NUMBER
FRACTION
1
,
...
-.
...
1
•
r)
t
...
12
•
1
~
1
,
e
-
-
,
..
--
r'
1
~
.c
•
46
/
Fi'g. 6:
Sucrose densi ty gradiè'n t profile cof crude BS
~elJlllase
activity.
Crude enzyme, was prepared
as in Figure l, and 0.1 ml (72 units cellulase
and 0.45 mg protein) was carefully layered on
the sucrose gradient wjtb myoglobin included
as internal reference (arrow).
The tubes were
centrifuged and fractionated as in Figure 5.
Recovery was 61 units of cellulase (82%) activity.
The value inserted above the peak fraction is the
sedimentation coefficient of cellulase determined
relative to myoglobin.
"
.1"-
.
\.
,
s.,. =3.4
",yol'obin
~
30
S
}.:
"
CI)
.-
,
4
-
.:
Z
.
:::::»
20 -
LI.J
en
cs:
,....J
::;,
,-.
1
-'
-'
if,
L.A.J
U
8
4
12
FRACTION HUMBER
,
.'
i
J
47
•
./
•
-~---""'~-----------~----.-~
--0"
Fig. 7:
Analytical discontinuous pOlyacrylamide gel
electrophoresis of crude BS'cellulase activity.
Enzyme preflar-ed as- in Figure l was made 20%'with
solid sucrose, and 0.1 ml was placed on top of a
column of gel (0.5X5 cm) prepared so as to give
a stacking gel, pH 7 and a separation gel pH 8.0
The g~l was developed at 4 C with a current of
2 ma/gel for 120 min.
Bromphenol blue was used
as a front rnarker (arrow).
At the end of the 4un
the gel was frozen and sliced into l
ànd cellulase activity was elute? and
mm
fractions
assaye~
-(
viscometrically as described in Materials and
Methods.
.
.,
\,
Recovery was
ap~roximately
20%.~
j)
..
.,
Rf
"
=0.31
60
-'
~
C1
~
Z
:;,
n
~O
L.&.J
(/)
ç
<
:=;)
--1
--1
20
Q
(,.)
12
1
FRACTION
----------
----. -
--
30
NUMBER
.
----
48
The recovery of cellulase activity was only about 20%
using this method of fractionation.
Cellulas~
was probably
lost "due to heat generated during t~e process of current
application since recoveries from polymerizing cellulase
~rectly
into gels were quantitative (eg., by substituting
crude BS cellulase for water in polymerizing the separAtion
gel, letting this stand for 1 to 2 h at 4 C and then extracting 1 mm slices as described in Materials and Methods).'
It appears from repeated,Sephadex fractionatioIl, elecLro"
phoresis and density gradient centrifugation that cellu1ase
activity in crude BS cellulase extracts is associated with a
--
single entity of substantia1 size i.e., a protein of MW 20,000
and S value of 3.4. The multiple components observed using
Sephadex gel chromatography are undoubtedly artifacts.
c~clusion
B.
This
was supported by results of purification (below).
Purification
----------1.
Development of cellùfase activit_ fo1fowing treatment of
pea epicotyls with 2,4-D
Table l shows the development, of BS ce11ulasè activity
and protein with respect to time following treatrnent of pea
epicotyls with 2,4-D.
Tissue was treated, harvested and
extracted as in Materials and Methods.
Maximal ce11ulase
t.
activity and specifie activity was attained after 4 to 5
days following 2,4-D treatment.
/
... "
,
49
Table l
.
Deyelopment of BS cellu1ase'activity and protein
4-D.~
following treatment of pea epicotyls with 0.1%, 2,
1
(p1umu1e,~hook
Apical regions of epicoty1s
plus 1-2 cm of
third internode) were homogenized in 2 volumes of 0.02M
sodium phosphate, pH 6.2, 5% gly:erol, l%jglucose and the
mixture was centrifuged at 13,000 xg for 20 min. Ce11ulase
activity was assayed in the supernatant.
2, 4-D treatment
led to a visible swelling of the epicotyl apex within 1 to
2 days.
Î
~
?
,
Tifne
.BS cel1ülase
Protein
(units/ml)
(mg/ml)
(days)
I------------=~------------.:.-._----------------
58
0"
140
l
.
- -
J
..
-~---
-
"
----
-
,3
664
4
752
5
848
6
504
-
.
--
•
--------------2.2
3.5
40.0
t
•
- --- - -- --
4.9
..
153.5
5.1
166.3
.
1
t
!
.
-
100.8
~
v
.
•
95.3-- -
-~3~.5
4
.
--
4.9
5.0
-
(units/mg)
- - --
2.7
.___ Â_7 -'--
_-448
.-
.
-Sp-ec. -Act ~ -
,
-
50
)
2.
Ammonium sulfate precipitation
Table II shows a typical experiment where different
concentrations of sulfate were used to precipitate cellulase
from a crude extract.
The extract was made 35% saturated
with crystalline ammonium sulfate and after 30 min stirring,
the precipitate
(P35) was collected by centrifugation.
The
suppernatent was made 60% saturated'with ammonium sulfate
and after 30 min the precipitate was collected.
was repeated to obtai? the precipitate at 80% (P
The procedure
SO
) and 100%
----------------~(IP~l~O~O~)~aurnmlliThoonnI~~umm~S?,UUjl~f~anL~ee_~s~artt~u~r~a~tdi~o~n~__~Ajfjt~e~r wash~ng the surface
of the precipitates with extraction buffer to remove excess
ammonium sulfate, Ehey \.. ere redi-ssolved ill the minimal amo.yu+;n. .t. _ _ _ __
of extraction buffer required to obtain complete solubilization.
An aliquot of each sample was
levels.
It can be seen that
assayed~Qr_cell~~9se
cellulas~
and protein
activity is found to
precipitate at every saturation level used, but most (75%)
precipitates in the ammonium sulfate saturation range of 35
ta 60%.
Furthermore this ammonium sulfate .range gave the
- - - - - -_____ -larg~_§t increase (4-fold) in 'the specifie activity of BS
cellulàse over values in the crude extract.
This
fractiona~
tion range was used routinely for concentration and partial
.... ~
purification of the enzyme.
It was possible to obtain up ta 90% of the'cellulase
activity in the P60 fraction if the crude BS cellulase
obta~ned
after centrifugation at 13,000 x 9 for 10 mln was ceIlLrifuged
further at 105,000 x g for 45 min ln order to remove micro-
[m________ _
somes etc.
_
.
This gave a IIcleaner" precipitation but was
51
Table II
Ammonium Sulfate Fractionation of Crude BS Cellulase.
BS
cel1ulase extracted from 500 9 of 2, 4-D-treated pea
epicoty1s was brought to 35% saturation with crystalline
ammonium sulfate and after 30 min stirring, the precipitate
was co11ected by centrifugation (P3S)'
The
supernata~t w~s
1
made 60% saturated with ammonium sulfate and after 30 min
the
was collected (P 60 ). The procedure was
repeated to obtain the precipitate at 80% (P 80 ), and 100%
precip~tate
\
(Prou) saturation.
The precipitates were washed with
---
extraction buffer (no stirring) to remove excess ammonium
sulfate and redisolved in the minimal arnount of extraction
.
buffer required to obtain complete solubilization.
An
aliquot of each samp1e was assayed for cellulase and protein
levels.
,
Fraction
Cellul"ise (Uni ts)
---Crude
720,000
Protein (mg)
4,680
Spec. Act.
(lAnits/mg)
153
~-----
(
l\,
P 3S
26,000
-----
110
-------
-~
233
--
P60
520,000
880
591
Pao
68,000
1,770
39
PIaO
25,000
1,GGQ
25
629,000
3,760
Recovery
----
52
7
impractical to use for large scale purification.
\
Centrifuging aliquots of cellülase activity that precipitated at the above ammonium su\fate levels on
density gradients showed
_with the same S value
th~t
sucro~e
thl activities aIl sedimented
relati~o--mYOg10bin
i.e., -3.4 S.
Chromatography of the P35 and P60 fractions on a Sephadex G-50
column (0.9 x 10 cm) gave the faMiliar pattern of one peak
~
found in the void volume with the majority of cel1ulase
activity (80%)in the 19 to 20,000 and MW region.
~---------------------------------------
3.
,i)DEA~-ce1lu1ose
- - - - - - - -----
~
.
chromatography
Further purification of BS cel1ulase following ammonium
sulfate precipitation was ach~eved using DEAE-ce11ulose chro,
matography.
The reasons why this ion exchanger was chosen
over DEAL-Sephadex were fourfo1d.
First, the sephadex ion
exchangers bound ~ess ce1lulase per unit ion exchanger that
"-
DEAE-cellulose, (DEAE-Sephadex A25 bound 5,200 unit~ P 60
cellulase/g exchanger and DEAE-Sephadex A50 bounq"l13,200 uni ts
.\
P60 c~lltt1ase/g 8xChang.eL-Jls....~~ units P60~cellula~~/g _______ _
,1-.
exchanqer bound less protein/ry
50
that the specifie activity
of the bound c~llulase was higher (145 units P 60 ce11u1ase/mg
protein per 9 ion exchanger (dry) for Sephadex A50 vs 320
unlts
II
per g dry DEAE-cellulose) . .!-
--------
Thirdly, the DEAE-Sephadex co1umn shrinks with increasing
cOncentration of NaCI (due to change ln part~cle properties)
yie1ding marked1y altered flow rates even with the use of a
pump, thus making chromatographie procedures relative1y more
53
(
difficult than with DEAE-cellulose, which does not alter its
,
~.
.
particle properties with high salt.
prelirninary experiments with a P60 cellulase prepara-
\
tion loaded on a DEAE-cellulose anion exchange column and
r--
-----~-
eluted with a continuous gradient of NaCI in extraction buffer
----showed that rnost of th~~-p~o~n--T85-95%) was---removed when the
~
1
~-~-
1
il
salt concentration eluting the column had reached about 0.4 M.
In contrast to this, cellulase eluted continuously from the
column up to 1.0 M NaCI, wher~ it 'reached peak levels.
After
- - ------------------~__passi-ng--approximat_e_ly l Vt-0W to L.M 'JaCl gradient throp.gf,l _______ _
the column, it was found that only 40% of the initial cellulase activity had been eluted.
with approximately 2 V
t
Further washing of the column
of l M NnCl in extraction buffer gave
a continuous elution of cellulase from the column \vi th
of the initial activity recovered.
~1
wi th -1
70-80~
Continuing the washing
NaCl eluted still more cellulase but the uni ts per
ml ,,,Jere lQw.
Fig
shows the results of such an elution modified
____ to obtain a h gh specifie activity and yleld of ce).lu1ase.'
--~-=------~--,.,
,
r--------rr~::-~6nO----->j~aeB:-&l~GbLu..ne.éL.s;aufE.:tt:e~r ammoni um sul fa
tf pre c i pi t êl- --- -
tion (Tnble II) was loaded onto a DF,AE-cellulose column
(2.5 x 30 cm) and the column was washed with a step gradient
of
~O%
~aCl
in extraction buffer.
of the protein pnt
acti vi ty.
only
The buffer wash rernoved about
-about 10% of the cellulase
A subsequent el~lion wi th 0.4 H NaCl in ~xtraction
,
buffer _removed nearly dll remaining protein but little of the
adsorbed
cellulas~
activity.
-
54 \
.-
1------- - - - - - - - - - - - - - -
•
DEAE-cellulo~e chromatography of partiallY
ase
~." ___ .~ .pY~ifieà ce)) nlase activi ty. ,.----------------------'Cellul
of
,
Fig.\. 8:
relatiyelY hi
ammonium sulfate
fractionatio~ (P60~ Tab~e
II)
was loaded onto a DEAE-cellulo se anion exchange
column ( 2.5 X 30 cm ) ~nd the column was
.
washed with a step gradient of NaCl in extracAPproximately, 100 ml fractions
tion buffer.
-*------- were
collected anC: aliquots were tested for- - ~-
~-----------
~
360,000 units cellulase ( 70% ) and 920 mg
$
p:rntBl. n ,llOO% ).
J
---~. - --------
_a.
,
_
.
t:
..
_--
~._---_.
,-
.. 1
1.0 M NaCI
1000
- - '\
100
~
"li.
:::
NaCI~1
Il
.
e
600
0
0
-
........
t:lO
E
~
z
::E
~
UJ
~
t0
z
;:)
cc:
~
UJ
(/) 20
. -<....1
:::>
....1
.....
UJ
u
600
~bUME (ml)
800
55
SubEequent elution with 1.0 M NaCl in extraction
buffer removed eellulase of specifie qctivity at least 10fold higher than the P60'
Cellulase collection suitable
for further purification was from V =400 ml to Ve=lOOO ml
e
in the column shown in Figure 8, or from a corresponding
region in a much larger colurnn used for large scale puri fication.
Cellulase reeoveries from such experiments were
approx. 70% of which 40% was suitable for further purificatian.
The specific aetivity of the usable cellulase was in
the range of 2,800 ta 3,200 units/mg protein with 18 to 24fold incréase in specific aetivity over erude enzyme.
4.
Ultrafiltration l
The volume in which the cellulase is found after DEAE-
\r
1
·~cellulose ehromatography was large and a method for concen""'"
trating the enzyrre was required.
p
1\t first, ammonium sulfate
at 60% saturation was used but this was abandoned for two
\
reasons.
First the technieal aspects of concentrating up to
4 1 of solution contairing only 500 mg total protein made
/'
this not feasible:
-
the preeipitate could not be reeovered
quantitati ve)y.
Secondly,--using ultrafiltration through- ______
D~afl0 lttelidnalle'J
tG
-------~~----------Goncentrate the enzyme
was easier, gave
,
high recoveries of cellulase activity and a much greater
increase in specifie actiyity of cellulase than did concentration by ammonium sulfate.
Table III shows the typical
concentration effect:s of "J trafiltration.
It was found that
as the volume of the eoncentrate decreased, more protein
o
56
.r-
I
Table III
--------------~-- --~-------
Ultrafiltration of partially Purified BS Cellulase.
,
FOllowing ammonium s'ulfate fractionation
Table II
and DEAE-cellulose chromatography ( Fig. 8 ), BS cellulase
in a large volume was concentrated by ultrafiltration
uSing the Amicon
Mod~ls
2000 and 202 equippSd with
Pfo1
10
membranes.
o
Voluœ
Initial
Final
~---------
Protein Concentration
ml
rrg
g%
3800
450
0.012
10
90
~llulase
units x 10- 6
Spec. Act._
units/mg
1.4
3,110
1.2
13,300
57
precipitated from solution.
Cellulase lost in the filtrate
was no more than 5% of the initial cellulase activity.
Two
types of membranes were used, either PM-IO or PM-30 and
both gave
~ilar
results, except that the PM-30 membrane
gave bett~r flow rates allowing for faster concentrations.
"
Recoverie of cellulase activity in the concentrate were in
______________ .__t=h~e~rang~of 80 to ~~% with a~ approx~mately 4-fold increase
1
---------
in specific activity over the enzyme eluted from DEAE'. ,,
;
.cellulose columns.
,
5.
Chromatography through Sephadex G-75
)
Following fractionation wi th ammonium sUlfatercnromatography on DEAE-cellulose and ultrafiltration through Diaflo
membranes, the cellulase preparation was chromatographed on
(
Sephadex G-75.
Two columns (90 x 2.5 cm) wer~ used simultan-
eously in order to speed up the procedure.
\
Each column was
loaded with 5 ml of the cellulas~ concentrate which was still
in the presence of l M NaCl in extraction bufter, and
1--- -
developed ,vi th extraction buffer only.
of such a column rune
Figure 9 shows resul ts
Duplicate columns yielded identical
t---------------~~.~H'~~';~,~o~,~~--~~'~he fractions in the reqion of the void column
I:"
"'lnnt:>::lr,::>r1
-Je
mi.lkv and it was possible to detect the void volurne-
L
of the column by scanning the tubes visually,
ce the cloudy
'tubes wére superimposable on the void volume protein peak
observed using the Lowry method,or absorbance at 00280 nm.
The c"lllJlase m:tivity <eluted
(ais
a single sharp peak_w_i__t_h___________
elution volume correspondinq to a MW of 38,000 as determined
\
58
,
-------
~
---
---~-
PartiallV purified BS cellulase activity
ch~omatographed Qn Sephadex G-75. Following
fractionation with ammonium sulfate, DEAE-
r-__________________c_e-l-l-u--lO~7-e__c_h_r_o_m_o_t..o_graPhy,
and ultrafiltration
(sample prepar~ti6n data in table III), a 5 ml
aliquot was placed on top of a Sephadex G-75
1
colurnn ( 2.5 X 90 cm ) which was developed
•
wit. extraction buffer.
The protein and
cellulase activity was determined and yields
_ were as follows: cellulase 440,OOO.units
protein 88 mg ( 98~~.
75 % ),
See Figures l to 3 for
comparison of elution profiles of crude BS
-------------- -
---- -----"
<)
e.
. '0
- - - - - - -_ _, _ _ _ --1
'-'
/
1
_
...
-l
v
·...:.
.
G-75
O
(l
10
~________
'-----~,
·
C")
M
,...
X
/
8
:
.
· ··
··
··
....J
:E
'-.
....
CI)
z
~
E
........
00
u.J
en
~
10
<C
e
:z:
.....J
UJ
::::>
1-
.....J
.....J
u.J
U
.
~
.'
0
l~O
,-
-
---
...
....
.
"
0
Cr::
a..
....
•••••
'. e ••
-...
-- --------2
ElUJJ1lN__ ~O lUME (ml)
-
o
0
"
---------------------------------------------------
-- - -
59
fro~
a calibration curve of thlS column shawn in Figure 10.
During this chromatographic' sttp very little ES cel1u-
;r*·e
fractlo~
1ase é:::Cti'll ty was located in
c,orresponding to haif
of this HW, even though a 20, 000 component was always the
major peak in simllar
FlgS. 1-4).
fractionatio~s
of crude enzyme (cf.
The simplest exp1anation for
is that es cel1u1ase tends ta
Byrne, 1974).
dimeri~e
th~se
observations
upon purification (see
The Sephadex p;rofile for purified BS
cel~u~ase
(Fig. 9) was reproduciblp to the point where in routine puri,
fication,
the fractions -EroP'i the co1umn corresponding to this
regi0n, i.e. ,-V e IV c, of 1.45 to 1.50 were Dooled and monitored
~
for total
cellulase~and
protcln levels without the need to
,
,
as~ay
individu~l
fractions WhlCh
del~yed
~
procedur~.
the
Re-
coveries of initial cellulase activitics loaded on the co1umn
were lisual1y"in the range of 80% and protcin recovcry 95-100%.
_ , _ , 4 _ _ __
6.
(
Ultrafiltration II
The final step in this purification procedure involved
concentration
o~
the pooled cellulase activlty from the two
1
Sephadex G-75 columns using again,
ultrafll~ration
through .
\
DiiPlo ~embran~s.
as described in
'The ultrafiltration process was performed
~aterials
and Methods and the results were
similar to those obtained after the first ultrafiltration
step.
The filtratlon rate was much faster duo ta the lower
prote in concentration.
tion are shawn
--- --
-
.1-
---
--~-
j
2
-
..
~
Table IV;
.~ small p;e~ipi t_~t-~
.t
e.
-
ii
•
Data for a typical final ultrafiltraAt the end of the concentration
was- rerrovedby
cenir-iiûgation--a~---_
tainkd no extractable cellulase adtivity
(by either buffer or
~
r
60
)
.
{
Fig. 10:
~
Calibration curve of the Sephadex G-75 column
shown in Figure 9.
of ovalbumin and
"
A 5 ml aliquot of a solution
my~globino
( l mg each/ml, ) was
loaded on" the column and developed
w~th
extraction-
butter. Ovalbumin concentration was determined at
•
OD 280 nm and myoglobin at OD410 nm.
~
-chymotrypsinogen and cytochrorne-C were run and
a.
assayed
sim~larlY
on a separate occfrsion.
"
,
~,
o
..
-.
BSC~lIulase
3:
-"-chymotrypsinogen
MW =38,000
4.4
Myoglobin
:E.
f
3.6
160
~200
240
ELUTION
280
320
VOLUME (ml)
\
(
,
e
2 g
\
"
61
,.
D
Table IV
Final Ultrafiltration of Partiall
BS Cellulase.
The fractions containing the cellulase p
which eluted
between 225 ml
a~d
280 ml from the Sephadex column in
•
Figure 9, plus the same peak of an identical column developed
o '
simulta~eously,
filtra~ion
were pooled and concentrated by ultra-
using the Amicon> Models 202 and 12 equipped with
PM-IO membranes.
t
Volume
f
protein
ml
110
6.9
0.0063
Final
1.5
r.9
0.12
r
./
Cellulase
units x
0.88
'~"'O
<'
- ---===:::::----
1
/
g%
Initial
'f
e--r
Concentration
:86
10- 6
Spec. Act.
unitsjmg
127,500
450,700
62
~.
Table V
Surnmary of the Purification Procedure of BS Cellu1ase.
Fràctionation
.
rSequence
Total
Tot<111
Specifie
Recovery
Fold
activi ty prote in activ'ity
~
purification
li!
'müts x 10- 3
,
m:J
...
units/rrg
%
protein
4
6,600
36,900
180
3,800
8,700
430
2
420
3,llO
17
90
13,300
6.9
880
------ - - - - ----\
Amicon PM-10
850
1.9
,Crude
extract
DEAE-ce11u1ose
1,400
Amicon PM-10
1,200
G-75
f
>
58
74
18
128,000
709
13
447,000
- 2,500
4:
-, .... ""~
-
'I.~)'lfl'iSCj
~...,
" ",
M'
...
-e
13
Pl ,•
,....;ot~
. . ,I,f....,
','
.~
.,
.
é3
.J
saI t-buffer).
100%.
Cellulase recoveries were in the-- range of 90-
After this proc~dure, the specifie activity increased
approx. 3-fold over the Sephadex G-75 step and 2,500-fold
ini~ial
from the
crude extract.
The cellulase obtained after
this ultrafiltration step is called "purified BS cellulase".
TapIe V summarizes the steps and typical results of this
purification procedure •
..
C.
CharaGterizatiorl
1.
Electrophoresis
Analytical polyacrylamide discontinuous gel electro.A
phoresis was used to determine the d~gree of purity of the
BS cellulase obtained after the second ultrafiltration step
in the purification proçedure described above, i.e., the
last AJnicon PM-ID -fraction __of ':t'able V.
purified
us
and 12.7
rg
cel1ulase
of~protein
sontaini~g
A 10
Y' l
aliquot of
5,700 units of cellulase
(S.A.=499,OOO
V/mg, cf. Table IV)
was added ta 0.1 ml of 20% sucrose anà electrophoresed as in
-----~-
~
-
--
---
Figure 7 except that the time required for el~ctrophoresrs-- .,.
was only 70 mjnutes due ta the lower protein concentration.
The results of the experiment are shawn in Figure Il.
The
cellulase profile obtained is identical to that of crude BS
cellulase electrophoresed in the same iystem and shawn in_
Figure 7.
Recovery of enzyme activity was only 15% for this
.-: run, i. e., comparable to that of the crude BS ce1lulase run.
-- .. _ - - - - -
i1
/
•
64'
---1
1
- 1
Fig. Il: _Analy.-tical_disc0I1tinuous polyacrylamide gel
electrophoresis of puriffed BS cel1ulase. A
10
r- l
,---,--~:..,
."
(iquot of pure BS cellulase ( Table IV
containing 5,700 units cellulase and 12.7
\A-g
- added to 0.1 ml of 20% sucrose and
protein was
,run .on polyacrylamide gels as in Figure 7,
..,
except the time require~ was only 70 min.
7 which
shows crude.BS cellulase fractionated in this
. - system.
\
\
•
fi',
.
F
...
~-
.9
--e
o
,
Bromph.
blue
-
600/
-
.4001u.J
(/)
<
-::j--'
ü
200-
LLJ
U
o
~~T~
_A
1
---i)-~-~ ~ -~
o
"
<
~1
-IL
"~T""·~l"~-~-~·~'·~r-·'··~-~··~l~~~I
\.
--
18
2.4
30
36
42
-
FRACTION6-~UMBËR-· ~-~~---
65
~
•
Figure 12 shows
the results of staining
for p
,
.
electrophoresis
,
.., gels containing purified
~S
the
cellulase
ponding to bhe activity profiles shown in Figure Il)
'ns cellulase (cf. Figure 7).
crude
Only a single band is
en~yme
gel with a Rf of 0.37 corresponding ta the
cellulase activity.
The concentration of cellulase protein in
the purified
"
the crude extract was too low ta give a distinct protein band
upon
electrophore~is.
,\
2.
Ouchterlony Double Immunodiffusion Analysis
i
ns
Rabbit antiserum ta the pure
cellulase was prepared
as described in Materials and r1ethods.
The presence
~f
anti-
bodies to pure B&.cellulase were monitored in trial bleedings
using the interfacial rinq test.
The presence of such anti-
bodies were detected after the 3rd injection.
more inJection was given to
àssar~
Cl.
However one
good antibody tlter.
\Jhen the antlserum ob lai lleo after the 4 th in J ecti on was exarnined
,
for jts ability to
precipit~te
1
pure BS cellulase using the
Ouchterlony double irnmunodiffusion technique as described in
Materials and f.lethods, it was founù ta give
optimal precipita,
The optimal precipitatlon conditions were used to
obtain Figure 13 which shows results of Ouchterlony double
----------------~iMmafiedi~·tusio~
anti-serum.
of_nuxified BS cellulase
~
ns
cellulase
In, order to rlinimize background sta}-_ni12_g. due to
pr0t--ein in the
-------
se:r;-~m,--it
was 115ed at a dilution of 1/8 and the
pure BS cellulase was diluted 1/32.
A
singl~
precipitatiQn. line _____
(
66
J~
fi
..
f
~igure 12:
Homogenèity of pure BS cellulase as de~ermined
following discontinuous polyacrylarnide gel
electroph&resis.
The gel on the left corresponds
to a gel used to obtain Figure Il.
The gel on the
;--------------_riQ1:t.:t__ iS_9_ gel
run simultaneeusly with a gel used
---~.~~
~-
te obtain Figure 7.
Both gels were
sta~ned
for 30
min with 1% coornasie briliant-blue in 7% acetic
acid, fol1owed by destaining·overnight in 4 l of 7%
acetic acid.
On1y one protein band with Rf of 0.37
(cbrresponding~to
Figure Il)
the cel1ulase activity Rf of
is evident in. the pure BS
c~llulase
•
preparation.
7
...................
------------------~
~
\
67
\\
/
.
,1
".
."
'-
r
4-
1
.,
,
1
,
!
?' '.
,.
,
T
"
,
."l
\
Fisure 13:
-.."
.
~
Ouchterlony double immunodiffusion of pure BS
lides
were prepared with l i Ionaga~~in Borate-bufferd"
saline (BBS). Wells 1 and ~ contain i/32 dilution
o~e .
of pure BS cellulase, and
center well cantains
1/8 dilution of BS cellulase antiserurn. The rest
- -- --- -- - - - of the tellS are buffer bïanks~ Immunodiffusion
-~---
~
was allowed to proceed for 24 h in a hurnid chamber
at 37 C.
The slide was washed in 0.3 M NaCl for
6 h followed by 18 h in BBS~
.Dried sl~dEs w~~--------~
stained with 0.2% acid fuchsin ~n 50% methanol-lOi
acetié' aCl
r.
Dë's"E'à1n~ng wa~
ln the acid fuchsin ·solvent.
-----------------------~-------------------
.carried
out
,
68
was found between the center weIl
contai~ing
1 and 2 containing pure BS cellulase.
lines are evident.
the As and wells
No other precipitation
To allow for the possibility that a conta-
,
~
minant, may Qé present but not at optimal preclpitation ratios
numb~r
with the antiserum, a
were set up reacting As
~
of double irnmunodiffusion slides
0, 1/2, 1/4, 1/8 .•••••.••. 1/1024
dilutions of pure BS cellulase and Vlsa versa.
At no time
was there more, than one precipitation line visible between any
1
two wells.
Precipitation lines were seen with Aq/As ratios
of 1/4 , 1/8" and 1/16 wi th the denser, one being that of Ag/As
1
of 1/4.
3.
Immunoelectrophoresis
,.
;
Figure 14 shows further analysis of'the pure BS cel1uIase
~
using immunoelectrophoresis. Slides were prepared as in Materials
and Methods.
"-
One weIl was filled with 5
pure BS cellulase (P).
rI
of 1/4 dilution of
The other weIl (C) cOIlLail1ed three 5
rI
applications of crude BS cellulase in order to ensure enough
cellulase units to obtain a precipitate. Electrophoresis, immu,
,
noprecipitation and staining of the slide is described in
Materials and Methods.
Only one precipitation line 15 seen
-------~~----------:--~
with pure' BS cellulase and the antiseruJ1l preclpltates anly one
1
Ag froPl the crud1 ES
prepara:~on, .i. ~.
1
the Psnis
<
T~e
~onospecific.
•
implication is that the purified BS ce11ulase was indeed
pur:e.
'" 69
i
1
•
---
p
)
+
"
f.,..'
-Figure 14:
Immunoe1ectrophoresis of
p~re
and crude BS ce11u1ase
vs. pure BS ce11u1ase antiserum.
"
pure
\
A 5 pl
ali~uot
of
ce11u1ase was applied to the well marked
BS
an
,
0
--
-~
P
pl applications of crude DS cellulase
r
, were applied to the well marked C.
.
r
Electrophoresis
was carried out at 10 V/cm for 120 min.
a 2
mm
st~ip
Afterwards
of filter paper impregnated with 75
pl
of antiserum wa§ placed on the slide parallel to the
1\
,~
path
o~
electropholesis alld -the---slide was-
for 24 h in a moist chamber.
,
'v
Slides were stained
1-
with acid fuchsin for visualization.
1
incubated~
70
4.
Isoefectric focusing
Figure 15 shows, resul ts of isoelectric focusing of the
~.
',
o
crude and pure B~'cellulase activities.
Experiments were
carri~d out as described in Materials and Methods.
To get a
somewhi3.t concentrated crude B,S cellulase with as little pro-
,
tein -as possible, aliCJuot~ of ammonium sulfat-e fraction P ~O
were used in 1 ml of 20 mM sodium phosphate buffer pH 6.2.
,
For the pure enzyMe, the Amicon P~1-l0 fraction was used.
Only bne ,peak of enzy~e activity was observed for these runs
with pl values of 5.2.
No difference in pl of crude or pqre
BS ce~l~lase ~ere detected indicating that the-pure BS
1
enzyme maintains the spme qverall distribution as the crude
enzyme, thus it must be the same proteine
.,
,'
f,
o
...
r,
,
~,
1
1•
._ - - - - , - '----_._---
_ _ _ _- - a
--------~--------
._----------.--------
r
~---~--=----~---~-~--;.~ - - - - - - - - - - - -
7
71
)
.
J
Figure 15:
Isoelectric fQcuiing of crude and pure BS ce11ulase.
The procedure was identical for both,enzyme runs.
Crude BS cellulase {aliquots of P60
~ble
f~action,
II, containing 5,000 units of ce11ulase
~g.
activity and 8.6
protein) or pure BS cellulase
(a1iquots of Amicon PM-IO, Table V, containing
~
'4,500 units of cellulase activity pnd approx. l ug
pratein) VIere lO~~d in the middle of the electrofocusing column i
1% Ampholine pH
supporned by a 0 to
tian grqdient.
ran~
3 to 10,
0% (w/v) sucrose anticonvec"-
Isaelectric focusing was carried
out for 72 h at 300 , V, and afterwards the column
•
was separated into 5 ml
fract~ons.
~The
fractions
1
were mixed immediately by a brief ;artex agitation,
and the pH was measured, at 2 C using.a pH-meter.
Cellulase activity wàs measured as before and
recoveries for bath runs were aver
90~
1
. -..,
"
'
1
F'
1
ç."
}
.
.
1
'
",
\
•
.
Crude BS
()
60
,
)
,-
#
40
.."
~
"
.(1
..
c::
0
20
..
~
(.)
co
.....
L.&...
.....
,
0
cu!
c..
pH
~
11
9
.
>..,..
.-
90
:>
......
u
Pu r·e ~s
,<C
-c
.....
Cl!)
Q.)
,-
60
:>
0
u
Q.)
Cé::
~
30
-{
.
sa
72
.
DISCUSSION
'(
Heterogen~ity
A.
~
Many authors have claimed thàt cellulase produced by
\
fungi and higher plants exists in
form (s~e literature review).
more~than
one molecular
Upon closer examinati6n, the
evidence to support this conclusion was generally equivoeal.
In particular, work on fungal cellu~ase showed that the U$e
of sorne chromatographie and electrophoretie techniques
yielded artifa~tual cellulolytic eomponents which' did ~ot
necessarily indieate true
heterogeneity of the cellulase
.."
,
" -'
speeies. - The same observ?tion was made in the present work
with eellulase from a higher plant.
The heterogeneity of
crude pea'epicotyl BS cellulase aetivity after fractionation~
by
S~phadex
gel chromatography (Figures 1-4) was not main-
tained following f~ther experimentation and purification.
,
It appeared to be due to a single protein eomponent which
,
'was subject to binding and/or agglutination
i~
those,prepar-
ations.
A speeral affinity of cellulase for Sephadex gel is
readily understa~dable 'sinee the "gel is a pdrtymer o'f glucose
with 90%
~-l,
6-glycosïdic bonds.
It is conceivable that
,s6me of the cellulase could bind loosely and reversibly to
such a moleeule.
As 'the chromatographie process oecurs, this
bound enzyme would be retarded in random fashion on the
sePha~olumn
leading to an elution pattern that resembles
.
. . . . . . . . . ._ _ _ _ _ _ _ _~s~~~
"
73
r
, \
Il
that of a heterogeneous molecular weight enzyme system.
RaRdom retardation is irnp~ied because the éhrornatOgraphiè
~
~
pattern uding crude enzyme is not reproducible in' any ,two
or more sucçe~sive attempts, eve~ using the saroe ~nzyrne
preparation and the same column (cf., Figures 2 and 3). ,
.
)
,
,X
-- ~
When, ~~e 'vé3fious cellulase êCJ4.vi"ty pea~which
separate~ on
a Sephadex column
were\cen~ifU9~d"}hrOUgh
a
sucrose density qratlient, they aIl yi~de~th€ same sedilj
mentation coefficient (approx. 3. 6S "Figure 5)'~ suggesting
:that they are the same enzyme.
It is ,possible that different "
molecular weight proteins have the same or very similar
"
sedimentation coefficients not discernible'on sucrose density
.
~
gradlents.
How~yer
.
the observation that sucp varied apparent
MW as 5,000, 20,000 and greater trhan 70,000 daltons
~ame sedime~ion
h~ve
the
coefficient, makes i t unlikely tha't the
MW €qu'i valents on Sephadex gels a.re indeed true values.
•
•
fact that polyacrylamide,gel electrophoresis (Figure 7)
The
and
•
isoelectric focusing' (Figure 15) show only one BS cellulase
'\
co~ponent
-
in crude extracts of pea epicotyl supports the
centrifugation findings.
The conclusion to be drawn from those expe~iments is
•
t1!.at only one BS cellulase ynzyme ~of MW approxim'ately 20,00b
daltons, exi~ts in crud~ extract~ of 2,4-D-treated etiolated
pea epicotyl::;.
1
•
74
"
B. Purification
'"
.
The p~Tificatibn of crude BS ~llulase activi~ des,
'*
cribed ln this thesis 9upports the observation that only one
BS cellulase exists iJ,the pea plan~.
The purification'
method as described, yielded pure BS cellulase of relatively
h~9.h
high yield and very
specifie
activi~y,*
with approxi-
mately 2,500-fold increase in S.A. over the crude enzyme.
DEAE-~ellulose
The step involving
chromatography gave the
greatest increase in S.A. in this procedure.
This chromato-
graphie step procably depends on specifie affinity of cellulase for the cellulose support rather than ion exchange
chromatography.
In ion exchange ehromatography, eellùlase
woulà have been expected to have eluted \Ji th a sharp peak at
a specifie ionic strength bebveen zero and 1 M NaCl.
At a
1
salt level of l r·1
N~l,
aIl ions bound ta t'he quaternary
ammonium qroup of the exchanger should be displaced by CI-.
(eg.f60~)
This implies that' most
of the cellulas"e activity'
which is stlll bound to the column after washing with . one
~
V of l n NaCl (See Figure 8), is bound tà the cellulose
t
part of the exchange=.
1 M NaCl is required
The fact that more
~o
le~ch
th~
one V of
t
.
out the cellulase from the
*The activity per unit protein of the purified pea cellulase
was 'comparable to that of purified cellulase preparations
1
..
1
\ . . . '
f;om Trichoderma viride' (QM 6a) and Streptomytes (QM B8/4)
supplied by Dr. E.T. Reese and measured by Dav.ies and
Maclachlan (1969).
"
1
ï5
"
•
column, supports the hypothesis that th~s chromatographie
step is .not of the ion exchange type.
During the purification procedure, the artifactual
pr6fl1e seen 'on Sephadex chromatography when\irude BS
,
1~4),
cellulase is used (cf. Figures
fs not abS@rvedvhèn
,\
~
partially 'pure BS cellul.ase is usea Ccf. Figure 9).
This
lt
could be dqe to the fact that.a constant,
~aximal'amount
of
-.
~ulase is retarted çn the COLumn (due to saturation of a
constant number of available b~hding sites),
and"app~ars as
t
vsrnaller
~1
cornponents.
The amount
boun~
,
may be a signifi-
.
canto fraction ,of the totalqwhen relatively low levels of
~
cellulase are used on the column, ~.g<., 5,000 uni'ts.
"100
How-
l'
ever,
6
wi~h large amounts of c~ll~la~e
, e.g.,
,
.lxl0 units,
the retarted cellulase would be sa low in compafison with
the major peak,
t~at ~t
appears as background noise.
(
The single MW
com~onent
of 38,000 observed after
Sephadex fract~onation of the ultrafiltra~ion l fr&ction
(Figure 9), was shown ta be due to dimerization of the
parent BS cellulase molecule.
Byrne (1974)
,
de~onstrated
that ultracentrifugation of pure BS cellulase in the presence
HC1~
'and ahsence of guanidine
respectively.
yielded the monOIller and dimer
Moreover, electrophoresis in SDS buffer also
1
dissociated the dimer to a single com']?onent 'of low MW.
r
i
Sorne
.
evidence for-a dimer can be seen in Seph~dex p~ofiles'of the
crude enzyme (e.g. Figure 1), but very little of the purified
1
BS cellulase behaves as a monorner unless conditions are )
oesigneà ta dissoci~te it.
FE
2
.
In this study, the pure ~S ceIIuIase was shawn to~bè a
.ft
homogeneous entity by disc electrophoresis, ouchterlony doubl~
immunbdiffusion;,
j~o~lectiic' fOCUSi~g .and irnrnuno~lectrophoresis.
In further studie~ by ~yrne (lg74), this pure BS ~ellulase,was
ana~ytical
shown to be homdgeneous
by SDS electrophoresis and
,
.'
ultracentrifugqtion .. 'It is an acidic protein with MW approxit'
\
'~ately 20,000: daltons, pl ~f ~.2 and pH optimum for activity
'
0
of 5.5-6.0.
'
Other glucosirlase activities associaten with the
t)
1
t
crude cellulase preparation were not detectable in th.
pure
....
!
enzyme prèparation.
.Compctrison of amino a'cid composl tion of
the BS cellulase with the BI cellulase
(~v
,
.
of 70,000 derived
from cJIl wall rnaterial of the pea and purified by similar
\
1
'.
rneans), showed ttem to be distinct enti~ies with no possibility
<
~
of one being derived from the other.
"
Functions have yet to be assigned ao the BS (and to BI)
cellulase. , l;\ role for c.ellulase in auxin-regulated laterai
i
/
cell expqnsion i.~the pea epicotyl has been proposed (Fan' and
\
Maclachlan, 1966, 1967; Datko and Maclachlan, 1968). CelluIase activity may cause wall loosening
(Puesi~k,
,may, paradoxically, enhance c\llulose synthesis
the number of
p~imer
1969) and it
~Y
increasing
~
chain ends in the pre-existing cellulose
<\.
(Spencer 'et al, 1971).
Auxin treatment also 1eads to xylem
vess~forrnàtion ~nd adventitious roo~ developme~t in this ~
tissue (Datko and Mac1achlan, 19~8), both of which involve cell
,
. wall dissolution' and therefore possi'rrly cellulase action
(Sasson, 1965;
speci~ic
Sheldrake, 1970).
The preparation of. cellulase-
antibodies provides a means for identification and
, 77
/
localization of this enzyme during development. "They have
•
been used in a recent study (Verma et al, 1975) to idenfiny
.
BS cellulase as one of the products ',formed in vitro by a
r~boso~al protein~synthesizing
2,4-~-treated
system acting on mRNA from
..
peas.
.
,
.
1
f
"
1
, 1
"
..
'\
,,
J
,
,
t
,.
1
/
v---.
\,
.'
\
/
•
78
•
\
{
L'TERATURE CITED
•
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.
.'
"
1
.,
\.
'.
."
,.
1
Q
.'
•
,
84
1
·'
,.
.,
CONTRIBUTIONS TO ORIGINAL KNOWLEDGE
't
It has been known for many years that treatrnent'with
r
auxi~Ibrings
1
about a great increase in soluble cellulase
activity (BS cellulase) in growing regions of
p~a
epicotyls.
The present work has shown t~at this BS cellulase exists as
a single'MW entity in crude
±his-tissue.
pho~hate
" extracts of
buffer
A method of purifying this enzyme to homogeneity
has been devised.
Using this rnethod, enough enzyme can be
prepared in pure farm to allow production of monospecific
rabbit antiserum ta thi~.
A tool now exists'for further
studies of the role of ceIIulase in higher plants.
"
".
).
\
,
',.