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Structural characterization of carbohydrate attached to the glycoprotein cellulase enzymes of Trichoderma reesei QM 9414

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Title:
Structural characterization of carbohydrate attached to the glycoprotein cellulase enzymes of Trichoderma reesei QM 9414
Creator:
Du Meé, Charles Philip Roger de Chasteigner, 1955-
Publication Date:
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English
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xlii, 176 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Acetates ( jstor )
Amino acids ( jstor )
Carbohydrates ( jstor )
Enzymes ( jstor )
Glycoproteins ( jstor )
Oligosaccharides ( jstor )
Signals ( jstor )
Sugar alcohols ( jstor )
Sugars ( jstor )
Trisaccharides ( jstor )
Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Carbohydrate Sequence ( mesh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF ( mesh )
Glycoproteins ( mesh )
Mitosporic Fungi ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 166-174.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charles Philip Roger de Chasteigner du Meé.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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STRUCTURAL CHARACTERIZATION
ATTACHED TO THE GLYCOPROTEIN
OF Trichoderma reesei


OF CARBOHYDRATE
CELLULASE ENZYMES
QM 9414


CHARLES PHILIP ROGER de CHASTEIGNER du MEE






















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1984




STRUCTURAL CHARACTERIZATION OF CARBOHYDRATE
ATTACHED TO THE GLYCOPROTEIN CELLULASE ENZYMES
OF Trichoderma reesei QM 9414
By
CHARLES PHILIP ROGER de CHASTEIGNER du MEE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984


I wish to dedicate this work first to my wife, Linda, who stood
by my side and who provided encouragement, love and emotional support
throughout and also to my two sons, Colin and Ian, who were both born
during this time.
I would also like to dedicate this work to two people who
instilled in me the drive to succeed and to enjoy science and without
whom I may not have followed the course I did. Firstly, Dr. Bob Coley,
who tauaht me the fun of organic chemistry and secondly Dr. Bob
Dekker, who introduced me to biochemical research and helped develop
my confidence and ambition.


ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Ross D. Brown, Jr., for
his suoDort and enthusiasm for my doctoral work and also for his
advice and direction during my graduate career.
I would like to sincerely thank several colleagues who were
invaluable during various phases of the project: Ms. Cindy Jackson and
Dr. John Gander for their contributions and performance of NMR
exDeriments, Mr. Mike Trehy and Mr. Jim Templeton for performing the
gas chromatography/mass spectrometry analyses, Ms. Virginia Wiley for
operating the amino acid analyzer and Dr. Mikelina Gritzali for the
preparation of the antisera. I would also like to thank Mr. William
Chirico for his innumerable helpful suagestions and hints over the
past five years and for his being a good friend and confidant.
Last, but not least, I would like to thank my advisory committee
for their helpful suggestions and recommendations and also Dr. Peter
McGuire in whose laboratory I was able to learn the art of in vitro
translation systems.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vi i i
ABBREVIATIONS xi
ABSTRACT xi i
INTRODUCTION 1
Glycoproteins 1
Funqal Glycoenzymes 4
Cellulases 6
Structure:Function Relationships for Fungal Glycoenzymes 14
Assessment 15
EXPERIMENTAL PROCEDURES 17
Materials 17
Methods 20
RESULTS AND DISCUSSION 56
Cellobiohydrolase 1(D) 56
Cellobiohydrolase II 88
Endoqlucanases 118
SUMMARY 144
APPENDICES 152
A. Electron impact mass spectra of peracetylated
alditols 152


B. Electron impact mass spectra of peracetylated
aldononitriles 154
C. Electron impact mass spectra of partially methylated
alditol acetates 156
D. Chemical ionization mass spectra of peracetylated
alditols 159
E. Chemical ionization mass spectra of peracetylated
aldononitriles 161
F. Chemical ionization mass spectra of partially
methylated alditol acetates 163
REFERENCES 166
BIOGRAPHICAL SKETCH 175


LIST OF TABLES
Table No. Page
I.Carbohydrate content of Trichoderma
cel luase enzymes 8
II.Neutral carbohydrate composition of
cellobiohydrolase 1(D) 60
III.Oligosaccharides released by preparative
reductive 6-elimination of
cellobiohydrolase 1(D) 64
IV.Composition of oligosaccharides released from
cellobiohydrolase 1(D) by reductive
6-elimination 65
V.Selected ^C-NMR signals due
carbohydrate covalently attached to
cellobiohydrolase 1(D) and due to purified
oligosaccharides released from this enzyme by
reductive 6-elimination 81
VI.Proton-NMR signal assignments of anomeric protons
of purified oligosaccharides released from
cellobiohydrolase 1(D) reductive 6-elimination ... 86
VII.Neutral carbohydrate composition of
cellobiohydrolase II 92
VIII.Oligosaccharides released by preparative
reductive 6-elimination of
cellobiohydrolase II 96
IX.Composition of oligosaccharides released from
cellobiohydrolase II by reductive
6-elimination 97
X.Selected ^C-NMR signals due
cellobiohydrolase II and due to the purified
trisaccharide released from this enzyme by
reductive 6-elimination 110
XI.Proton-NMR signal assignments of anomeric protons
of purified oligosaccharides released from
cellobiohydrolase II by reductive 6-elimination .. 116


Table No.
Page
XII.
Selected C-NMR signals due to
carbohydrate covalently attached to the
endoglucanases of T. reesei QM 9414 ...
138


LIST OF FIGURES
Fiqure No. Paae
1. Isocratic elution pattern from DEAE-Sephadex column
chromatography of the extracellular protein
preparation from reesei QM 941 arown on
cellulose 22
2. Isocratic elution pattern from SP-Sephadex column
chromatography of cellobiohydrolase II and the
endoglucanases which had been eluted from a
DEAE-Sephadex column 25
3. Gas chromatoaraphic separation of peracetylated
alditol and aldononitrile derivatives of neutral
monosaccharides released from cellobiohydrolase 1(D)
after reductive 13-elimination and subsequent acid
hydrolysis 32
4. Efficacy of sugar release from cellobiohydrolase 1(D)
by reductive 6-elimination 35
5. Quantitation of aldoses as the peracetylated
aldononitriles 38
6. Quantitation of alditols as the peracetylated
alditols 40
7. Molecular ion scanning of peracetylated alditol and
aldononitrile derivatives of monosaccharides released
from cellobiohydrolase 1(D) following reductive
6-elimination and subsequent acid hydrolysis 50
8. Polyacrylamide disc qel electroDhoresis of crude
extracellular protein prepared from T. reesei
QM 9414 and highly purified cellobiohydrolase 1(D) .. 58
9. Separation on a Biogel P-2 column of oliqosaccharides
released from cellobiohydrolase 1(D) by reductive
6-elimination 62
10.Methylation analysis of cellobiohydrolase 1(D) and
the oligosaccharides released from cellobiohydrolase
1(D) by reductive 6-elimination 68


Fiqure No.
Paae
11. ComDarison of electron impact spectra obtained from
partially methylated alditol acetates of the
tetrasaccharide from cellobiohydrolase 1(D) with
standards 71
12. HPLC separation of the products of sequential
glycosidase digestion of oligosaccharides released
from cellobiohydrolase 1(D) by reductive
13-elimination 73
13. HPLC separation of the products of acetolysis of
oligosaccharides released from cellobiohydrolase 1(D)
by reductive 6-elimination 77
13
14. Proton decoupled C-NMR spectra of
cellobiohydrolase 1(D) and the oliqosaccharides
released from cellobiohydrolase 1(D) by reductive
6-elimination 80
15. Proton-NMR spectra at 300 MHz of olioosaccharides
released from cellobiohydrolase 1(D) by reductive
6-elimination 85
16. Polyacrylamide disc oel electroDhoresis of crude
extracellular protein preoared from T. reesei
QM 9414 highly purified cellobiohydrolase II 90
17. Separation on a Biooel P-2 column of oligosaccharides
released from cellobiohydrolase II by reductive
8-elimination 95
18. Methylation analyses of cellobiohydrolase II and the
oliqosaccharides released from cellobiohydrolase II
by reductive 8-elimination 100
19. HPLC separation of the products of sequential
glycosidase digestion of oligosaccharides released
from cellobiohydrolase II by reductive
6-elimination 103
20. HPLC separation of the products of acetolysis of the
trisaccharide released from cellobiohydrolase II by
reductive 6-elimination 107
i
21. Proton decoupled C-NMR spectra of
cellobiohydrolase II and the trisaccharide released
from cellobiohydrolase II by reductive
6-elimination 109
22. Proton-NMR spectra at 300 MHz of oliqosaccharides
released from cellobiohydrolase II by reductive
8-elimination 115


Fiqure No.
Paqe
23.Polyacrylamide disc gel electrophoresis of successive
protein fractions eluted isocratically from an
SP-Sephadex column during chromatography of
T. reesei endoqlucanases 120
24. Elution pattern from Sephacryl S-200 column
chromatograDhy of endoglucanase E 123
25. Polyacrylamide disc gel electrophoresis of successive
protein fractions eluted from a SeDhacryl S-200
column during chromatography of endoglucanase E 125
26. Elution pattern from SP-Sephadex column
chromatography of endoglucanase B and endoglucanase D
using a pH gradient 128
27. Polyacrylamide disc oel electrophoresis of successive
protein fractions eluted from an SP-Sephadex column
during chromatooraphy of endoglucanase B and
endoolucanase D using a pH gradient 130
28. Elution pattern from Sephadex G-75 column
chromatography of endoglucanase B 132
29. Polyacrylamide disc gel electrophoresis of successive
protein fractions eluted from a Sephadex G-75 column
during chromatoqranhy of endoglucanase B 134
30. Polyacrylamide disc gel electroohoresis of crude
extracellular protein prepared from T. reesei
QM 9414 and purified endogl ucanases 136
13
31. Proton decoupled C-NMR spectra of the
endoolucanases purified from 1\_ reesei QM 9414
grown on cellulose 140
32. Proposed structures of the oligosaccharides
covalently attached to the glycoprotein cellulases
from T. reesei QM 9414 146


ABBREVIATIONS
CBH 1(D)
-
Cellobiohydrolase 1(D) from T. reesei QM 9414
CBH II
-
Cellobiohydrolase II from T. reesei QM 9414
GLC
-
Gas-liquid chromatography
GC/MS
-
Gas chromatography/mass spectrometry
GC/MS/DS
-
Gas chromatograph/mass spectrometer/data system
El
-
Electron impact
-
Chemical ionization
M
-
Molecular ion
NMR
-
Nuclear maqnetic resonance
HPLC
-
High pressure (performance) liquid chromatoqraDhy
PAA
-
Peracetylated alditol
PAAN
-
Peracetylated aldononitri1e
DEAE
-
Diethyl ami noethyl
SP
-
SulfoproDyl
Ser
-
Serine
Thr
-
Threonine
Asn
-
Asoaraqine
Abu
-
-aminobutyric acid
Xyl
-
Xylose
Man
-
Mannose
Glc
-
G1ucose
GlcNAc
M,M M G,
-
N-acetylglucosamine
Â¥
Oligosaccharides released from the cellobiohydrolases
(where M = Man and G = Glc)
DMSO
-
Dimethylsulfoxide
TSP
-
(Trimethyl si 1 ane)-1-propane sulfonate


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STRUCTURAL CHARACTERIZATION OF CARBOHYDRATE
ATTACHED TO THE GLYCOPROTEIN CELLULASE ENZYMES
OF Trichoderma reesei QM 9414
By
CHARLES PHILIP ROGER de CHASTEIGNER du MEE
August, 1984
Chairman: Dr. Ross D. Brown, Jr.
Major Department: Biochemistry and Molecular Bioloay
Cel 1 obi ohydrol ases 1(D) and II were purified by ion exchange
chromatography from an extracel 1 ul ar culture filtrate of Tri choderma
reesei QM 9414. Neutral sugar composition of each was determined by
gas-liquid chromatographic analysis of the peracetylated alditol and
a 1 d on on i t r i 1 e acetate derivatives of sugars released by either
reductive 13-e 1 i mination or acid hydrolysis. Mannose and glucose were
found to be the only neutral sugars and were covalently attached to
the proteins through alkali-labile mannosyl residues. Analysis of the
alkaline borohydri de-rel eased carbohydrate by high pressure liquid
chromatography (HPLC) demonstrated that each molecule of
cel lobi ohydrol ase 1(D) contained 5.9% carbohydrate comprising 0.7
tetra-, 4.2 tri-, 1.1 di- and 1.2 monosaccharides and that
cel 1 obi ohydrol ase II contained 18.9% carbohydrate comprising 14.9
tri-, 1.6 di- and 9.0 monosaccharides. The purified oligosaccharides
were shown by methylation analysis to contain (1-2) and (1-6)


glycosidic linkages and the position of 6-substituted residues was
confirmed by acetolysis. The sequence and anomeric nature of the sugar
residues in each oligosaccharide was determined by sequential
glycosidase digestion and all the residues were found to be a-linked.
1 3
Proton decoupled C-NMR analysis suggested that for
cel 1 obi ohydrol ase 1(D), each oligosaccharide was attached to a
threonyl residue on the polypeptide and for cellobiohydrolase II, each
oligosaccharide was attached to threonyl and seryl residues on the
polypeptide. These data were supDorted by amino acid analysis for
a-ami nobutyr i c acid after 8-e 1 i mi n a t i on and palladium chloride
13
treatment of each protein. Coupled C-NMR analysis confirmed that
the anomeric carbon of each suqar residue was in the a-configuration
and ^H-NMR identified the presence of mannosyl and a-alucosyl
residues in the purified o 1 i qos ac ch arides. The structures of the
oligosaccharides attached to the enzymes were determined to be
mannosyla(l-2)glucosyla(l-6 ) man nosy la( 1-2 )mannose, ql ucosyla( 1-6)
mannosyla(l-2)mannose, mannosyla(l-2)mannose and mannnose. This is the
first report of a unique glucosyla(l-6)mannose linkaae. Thus, work
with the cellobiohydrolases and the endoglucanases indicates that the
predominant cel luase enzymes secreted by T. reesei QM 9414 are each
glycosylated with similar oliaosaccharides .


INTRODUCTION
Ghose [1] has estimated the annual worldwide production of bio
degradable substances through photosynthesis at approximately 1.8 x
10 ^ tons, of which 40% is cellulose. As much as 25% of this [2]
could be made available for conversion processes, a significant amount
of which occurs as agricultural and municipal wastes. Therefore, in
the future, cellulose must be regarded as an important potential
source of fuel, food and chemical feedstocks.
Enzymatic saccharification of cellulose has proved to be both
specific and efficient, and among the organisms tested, Trichoderma
species showed the highest levels of extracellular activity [3]. Most
fungal cellulases have been shown to contain covalently attached carbo
hydrate, although the complete structure of the oligosaccharides
attached to the polypeptide of a cellulase enzyme has never been
elucidated.
Glycoproteins
Proteins which have covalently associated carbohydrate are
termed glycoproteins [4]. These are a diverse group of macromolecules
found throughout nature in plants, animals and microorganisms.
Glycoproteins are implicated in a wide variety of functions including
cell adhesion, molecular recognition, structural support, lubrication,


2
hormone control, blood clotting and enzyme catalysis. With the
increased sophistication of modern analytical methods, the structure
of the oligosaccharides attached to many glycoproteins have been
determined and are well documented [4-8]. The oligosaccharide side
chains of glycoproteins fall into two general classes, (i) those which
are attached via an N_-gl ycos i d i c linkage from an N^-acetyl gl ucos-
aminyl residue to the amide nitrogen of an asparaginyl residue on the
protein and (ii) those which are 0_-g 1 ycosidi cal ly linked from a
neutral sugar or an N-acetylgalactosaminyl residue to either a seryl,
threonyl, hydroxyprolyl or hydroxylysyl residue on the protein.
The N_-linked oligosaccharides are similar in that they all
contain a pentasaccharide core structure, Mana(l-3)[Man a(l-6)]Man-
B( 1 -4 )G 1 cNAcS (1 -4 )G 1 cNAc(3-(Asn ) attached to the protein. This
class of oligosaccharides comprises three general groups, (i) those
that have only mannosyl residues in addition to the core are termed
"simple", (ii) those that have one or more of a variety of sugars,
galactose, N_-acetyl gal actosami ne, sialic acid and/or fucose in
addition to the core, are termed "complex" and (iii) those that have a
mixture of "simple" and "complex" are termed "hybrid". The (^-linked
class of glycoproteins has a specific amino acid sequence requirement
for addition of the carbohydrate to the protein. The sequence Asn-X-
Thr(Ser) must be present, where X can be almost any amino acid except
proline [5],
Oligosaccharides of this class are transferred from a lipid-
linked precursor en bloc to the protein. This precursor is a large
branched structure containing mannose and glucose. Following transfer,
the glucosyl residues and most of the mannosyl residues are removed


3
and the core is subsequently enlarged to the simple, complex or hybrid
forms. The glycoproteins containing these types of oligosaccharides
are represented diversely in nature. They are cell surface [9,10],
secretory [11,12], plasma [13,14] and hormone polypeptides [15,16] to
mention a few.
The ^-linked oligosaccharides are not known to have such an
amino acid sequence requirement, although it has been suggested that a
conformational prerequisite, i.e. a S-turn, is necessary. Sugars are
thought to be added directly to the Drotein one by one rather than by
transfer of an oligosaccharide en bloc. This class of oliqosaccha-
rides also comorises three general groups, (i) those that have
N_-acetyl gal actosami ne attached to the hydroxyl oroup of serine or
threonine, (ii) those that have neutral sugars attached to the
hydroxyl group of hydroxylysine or hydroxyproline and (iii) those that
have neutral sugars attached to the hydroxyl group of serine or
threonine. The oligosaccharides of the first group are generally
short, one to six residues in length, although some are much larger.
They may also contain other sugars such as galactose, fucose,
N_-acetyl gl ucosami ne and/or sialic acid on the chains. This qroup is
exemplified by the mucins, but also includes antifreeze glycoproteins
[17], chorionic gonadotropin [18], blood group substances [19] and
cartilage keratan sulfate [20]. The second class is characterized by
several structural proteins, the collagens [21,22] and basement
membrane proteins [23,24] in animals, which have galactose 0-linked
to hydroxy lysine and a group of plant glycoproteins called extensins,
which have arabinose and galactose attached to hydroxyproline. For the
third group, the neutral sugar can be xylose in the case of


4
chondroitin sulfate and dermatan sulfate [25], galactose in the case
of cuticle collagen [26,27], fucose in the case of several mammalian
cell line glycoproteins [28,29] and mannose in the case of fungal
[30-38] and yeast [39,40] glycoproteins.
Fungal Glycoenzymes
With the exception of cuticle collagen from Nereis virens,
which was found by Spiro and Bhoyroo [26] to contain a unique acidic
di saccharide of 6-0_-a-D-glucuronosyl-0-D-mannose, all of the qlyco-
Droteins currently known to contain a mannosyl-0-Thr(Ser) linkage
are of fungal origin. Of these, the majority are fungal carbohydrases
which are secreted from the respective organisms.
The qlucoamylases ((1,4)(1,6)-ot-D-glucanglucohydrolases) from a
number of Aspergillus species have been analyzed for carbohydrate
content and composition [31-35]. Pazur et al. [31] removed the neutral
carbohydrate of glucoamylase I from A. n i qer wi th mild alkaline
treatment. The oligosaccharide chains released ranged in size from one
to five sugars, with 20 monosaccharides, all being mannose; 11
d i s acch ar i d es all mannobiose; and the 1 arger chains which were
combinations of glucose, galactose and mannose. Methylation and
G.C./M.S. analysis determined the disaccharides to be (1-2) linked and
the larger species to be branched and containing (1-3) and (1-6)
glycosidic bonds. The structures of these larger species, however,
were never definitively described. Manjunath and Rao [33,34] prepared
a series of g 1 ycopeptides from glucoamylase II of A. niger following
extensive pronase digestion. Monosaccharides were subsequently


5
*
released from the glycopeptides by sequential glycosidase digestion
and the products separated and identified by paper chromatography. A
series of tr i s acchari des were described with the general formula,
X-Man-Man-, where X was either glucose, galactose, mannose, N-acetyl-
glucosamine or xylose. The residues were a-linked in each case. The
procedure used, however, did not exclude the possibility of branched
structures, as substitution of individual sugars was not determined by
classical methylation analysis. Carbohydrate and amino acid analysis
following reductive 0-el imi nation identified mannose as the only
sugar attached to either threonine or serine on the polypeptide.
Comparison of g 1 ucoamylases I and II from A^ niger 1,2 and 3, Asper-
qi 1 1 us f o e t i d u s and Aspergillus cand idus showed that most of the
enzymes contained mannose, glucose, galactose, xylose and glucosamine
[35]. The Aspergillus gl ucoamyl ases are heavily glycosylated and
contain between 48 and 151 monosaccharides covalently attached to each
protein molecule [35,35]
Rosenthal and Nordin [37] have characterized a mycodextranase
( end o-1, 4 a-D-g 1 u c an a s e ) from Penici Ilium melinii. Glycosidase
digestions, Smith degradation and methylation analysis indicate that
the oligosaccharides are present as about 25 mannose, Glca(l-2)Man and
Mana(l-2)Glca(l-2)Man side chains.
Raizada and Schutzbach [38] characterized a Man
a( 1-2 ) Mana( 1-2 )Manni to 1 oligosaccaharide from a major cell envelope
glycoprotein of the fungus imperfectus, Cryptococcus laurentii. The
tri saccharide was removed from the protein by reductive 0-elimination
3
using H-NaBH^ and the rad i o 1 abe 11ed product isolated by gel


6
filtration. Characterization was performed by glycosidase digestion
and comparison of the products with standards by paper chromatography.
While not enzymatic in nature, Saccharomyces cerevisiae cel 1
wall mannan was shown by Nakajima and Ballou [39] to contain a number
of similar 0^-linked mannooligosaccharides. These were released from
the protein by mild alkaline treatment and individually purified by
gel filtration. Glycosidase digestion and gas chromatography were used
to identify a series of related oligosaccharides, the largest being
Man a( 1-3) Manad-2) Man a( 1-2) Man.
Cel lu ases
Most cellulases are of fungal origin and are glycoprotein in
nature, but relatively little is known about their structure. Unlike
their a-glucanase counterparts such as the glucoamylases and the
dextranases, the cellulases are thought to exist as a system of
enzymes.
The cellulase system of Trichoderma reesei QM 9414 comprises
three types of enzymes which act synergistical ly and can effect the
complete conversion of both amorphous and crystalline cellulose to
glucose [41,42]. The system consists of several endo-1,4-13-Dglucana-
ses (EC 3.2.1.4), two exo -1,4-S-D-cellobiohydrolases (EC 3.2.1.91)
and one or more S-D-glucosidases (EC 3.2.1.21)[43,44]. Endoglucan-
ases release soluble oligosaccharides and free chain ends from
cellulose, cellobiohydrolases release cellobiose from free chain ends
and B-glucosidases release glucose from soluble cel Tooligo
saccharides .


7
It has been shown that the biosynthesis of endoglucanases and
cel I obi ohydrol ases can be induced by addition of sophorose (0-B-D-
gl ucopyranosyl-1, 2-0_-gl ucopyranose ) to resting cells of this
organism [43,45-47]. Gritzali and Brown [43] have shown that cellobio-
hydrolase I, form D (CBH 1(D)) from cellulose-grown and sophorose-
induced Tri choderma cultures have the same electrophoretic mobility,
amino acid composition and total carbohydrate content suggesting that
they are the same protein. CBH 1(D) is so named as it has been shown
to have the same amino acid composition as three other forms of
cellobiohydrolase I, A, B and C from Trichoderma viride [43,48]. Gum
and Brown [48] proposed that these forms only differ structurally in
the amount of covalently attached carbohydrate. Cellobiohydrolase II
(CBH II) from cellulose-grown cells also exhibited the same
electroDhoretic pattern as its induced counterpart although no
comparative compositional studies on the induced enzyme were
performed. CBH 1(D) and CBH II have been shown by several criteria to
be separate proteins. Apart from electrophoretic mobility, the two
iave distinct amino acid compositions [42,49]. Fagerstam and
Pettersson [42] have also determined that the sequence of the
N-terminal 20 amino acids for each polypeptide was different, although
both N-terminii were blocked by a pyroglutamic acid residue.
Most of the cellulolytic enzymes isolated from Trichoderma are
glycoprotein in nature [30,43,48-56], although some have no covalently
bound carbohydrate [57,58]. The carbohydrate content of each type of
cel luase from this genus varies considerably and is summarized in
Table I. Gum and Brown [30] have presented the only attempt so far at
structural character i zati on of the carbohydrate of a cellulolytic


TABLE I
CARBOHYDRATE CONTENT OF TRICHODERMA CELLULASES
ENZYME3 MOL. WT. ORGANISMb CARBOHYDRATE SUGARS*
(Daltons) CONTENT (WT*) (No./Molecule) REF.
CELLOBIOHYDROLASES
CBH
CpI 3.80]
--
T. koninqii
9
N.D.
50
CBH
[pi 3.95]
--
T. koninqii
33
N.D.
50
CBH
IV
46000
T. viride
3.3
N.D.
51
CBH
42000
T. viride
9.2
Man(16)*
Glc(6)
52
CBH
A [1(A)]
53000
T. viride
1.4
Man(3)*
Glc(l)
48
CBH
B [1(B)]
53000
T. viride
5.8
Man(12)*
Glc(5)
48
CBH
C [1(C)]
53000
T. viride
10.4
Man(22)*
Glc(6)
Gal(3)
48
CBH
D [1(D)]
53000
T. viride
6.7
Man(13) *
G1 c (7)
48
CBH
C [1(C)]
48400
T. viride
Man(26.4)
Glc(4.8)
Gal(2.4)
GlcNHo(3.4)
Man(13)
Glc(4)
GlcNac(4)
30
CBH
1(D)
54000
T. reesei QM 9414
4.3
49
CBH
II
54000
T. reesei QM 9414
19.5
Man(51)
Glc(20)
49


ENDOGLUCANASES
ENDO
1
13000
T. koningii
N.D.
N.D.
53
ENDO
3a
48000
T. koninqii
N.D.
N.D.
53
ENDO
3b
48000
T. koninqii
N.D.
N.D.
53
ENDO
4
31000
T. koninqii
N.D.
N.D.
53
ENDO
51000
T. viride QM
9414
0

57
ENDO
20000
T. viride QM
9414
0
--
58
ENDO
I
12500
T. viride
21
N.D.
54
ENDO
II
50000
T. viride
12
N.D.
54
ENDO
II
37200
T. viride
4.5
Man(5)*
Gal(2)
G1 c (2)
55
ENDO
III
52000
T. viride
15
Man(33)*
Gal(5)
Glc(6)
55
ENDO
IV
49500
T. viride
15.2
Man(30)*
Gal(6)
G1 c (6)
55
ENDO
46700
T. reesei QM
9414
10.6
Man(32)
Glc(9)
49
a) Enzymes are named accordina to the original publication referenced; some enzymes with
identical primary structure may have been given different names.
b) T. yiride QM 9414 and T. reesei QM 9414 are the same organism [59].
#) Number of residues per protein molecule.
*) Number of residues calculated from weight percent data in original reference.


10
glycoenzyme. All the neutral carbohydrate was found to be released
from cel 1 obi ohydrol ase C from Trichoderma reesei QM 9414, with
alkaline borohydride (reductive 13-elimination), whereas the amino
sugars remained attached to the polypeptide. It was calculated that
the neutral carbohydrate was attached to serinyl and threoninyl
residues at an average of 16.7 sites per enzyme molecule. A range of
small oligosaccharides were released, with the mono- and
tri saccharides being the most abundant. Periodate oxidation and
G.C./M.S. of partially methylated residues determined that (i) the
oligosaccharides were unbranched, (ii) most of the linkages between
the neutral sugars in the chains are (1-6) and (iii) mannose was the
sugar linking the oligosaccharides to the hydroxy amino acids.
Glycosidase experiments suggested that a-mannose was present at the
non-reducing ends of some chains and that the disaccharides were
mannobiose.
More recently, Fagerstam et al. [60] have sequenced most of CBH
1(D) and found that all of the 0^-1 inked carbohydrate was attached
within a short region, 20 amino acids in length, located 32 residues
from the C-terminus of the polypeptide. Due to the presence of the
carbohydrate, this short region was never sequenced, but was found to
contain seven threonyl and three seryl residues. Shoemaker et al. [61]
have now sequenced the gene encoding CBH I obtained from
Trichoderma reesei strain L27. The nucleic acid sequence was shown
to contain two introns and, by comparison to the previously available
amino acid sequence, the region of carbohydrate attachment was shown
to contain eight threonyl and three seryl residues.


11
The 13-gl ucos i dases produced by Trichoderma have been found to
contain little or no carbohydrate. Berghem and Pettersson [62]
reported no carbohydrate attached to an extracellular 13-glucosidase
from T. reesei using an orcinol-sulfuric acid method. Chirico [44]
found that a 13-gl ucosidase from the same organism would not bind to
Concanavalin A-Sepharose, indicating the lack of avai1 able a-glucosyl
or a-mannosyl residues. However, four residues of neutral sugar and
two residues of N_-acetylglucosamine were estimated per enzyme
molecule based on phen o 1-s u 1 f ur i c acid and amino sugar analysis,
respectively. The identity of the neutral sugar was not determined.
This was quite different from the enzyme isolated by Emert [63] from
T v i r i d e which was found to contain 10.3% carbohydrate. Gong et
al [56] also determined that a cellobiase (13-glucosidase) activity
from another T. v i r i d e preparation did not bind to Concanavalin
A-Sepharose. Wood and McCrae [64] purified two 13-glucosidases, BG1
and BG2, from Trichoderma koningii. Neither enzyme bound to Concan
avalin A-Sepharose although BG2 was shown to contain 2% neutral carbo
hydrate by a phenol-sulfuric acid determination. By the same method,
no carbohydrate was found associated with BG1.
Glycosylated cellulases are also produced by organisms other
than Tr i choderma, although no detailed carbohydrate analyses have
been performed on them. Eriksson and Pettersson [65] have purified
five endogl ucanases from the white-rot fungus, Sporotrichum pulveru-
1 entum, of which four are glycosylated. The four glycoenzymes, T^,
T 2 b "^3a anc* were found to contain 10.5%, 7.8%, 4.7% and
2.2% carbohydrate, respectively, as determined by qas-liquid chromato
graphy as the alditol acetates. T^ was found to have 19 mannosyl and


12
2 glucosyl residues and T was found to have 5 mannosyl, 7
galactosyl, 1 glucosyl and 1 arabinosyl residue per enzyme molecule.
Kanda et al., using the phenol-sulfuric acid method, showed that both
an endocel 1 ul ase [66] and an exocellulase [67] from the fungus
Irpex lacteus were glycosylated, containing 12.2% and 2.4% carbo
hydrate, respectively. Beguin and Eisen [68] purified three endocellu-
lases, I, CA and CB, from Cel 1 ulomonas. While enzymes CA and CB were
found to stain with periodic acid-fuchsin and also bound to Concana-
valin A-Sepharose, typical of glycoproteins, enzyme I did neither and
was proposed to be unglycosylated. The amount and nature of the carbo
hydrate were not determined. Tong et al. [69] determined that three
cel lu ases, I, II and III, and a (3-gl ucosidase from the thermophilic
fungus Thermoascus aurantiacus, were all glycosylated based on the
results of anthrone-sulfuric acid analysis. They were found to contain
33%, 5.5%, 2.6% and 1.8% carbohydrate, respectively. Ait et al. [70]
found that el ectrophoret i c gels of a cellulase from Clostridium
thermocel 1 urn stained with periodic acid-fuchsin suggesting that it
also is glycosylated.
Wood and McCrae [71] separated four cellobiohydrolase forms, A,
B, C and D, from Fusariurn solani, by isoelectric focussing. Phenol-
sulfuric acid analysis indicated that they contained 21%, 10%, 12% and
1% carbohydrate, respectively. These same workers later compared the
carbohydrate composition of this cellobiohydrolase activity with that
of cellobiohydrolases from Penici 11ium funiculosum and T,
k o n i n g i i [72]. Of the carbohydrate attached to F. solani cello-
biohydrolase (all four species), 83% was mannose, 7% was xylose, 4%
was glucose and 1% was galactose. The carbohydrate component of the


13
cel 1 obiohydrolase activity of T. koningii (two species) was found
to be composed of 73% mannose, 27% glucose and a trace amount of
xylose. The structure of the carbohydrate of any of these enzymes has
not been reported.
Whereas the B-glucosidases of Trichoderma are either not
glycosylated or are so at a low level [44,56,62-64], enzymes from
other species are reported to be glycosylated to a greater extent.
Shewale and Sadana [73] purified four B-alucosidases from the fungus
Sc 1 eroti urn ro 1 f si i and suggested that they were glycoproteins due
to their affinity for Concanavalin A-Sepharose and to a positive
reaction to periodic acid-fuchsin staining. Rudick and Elbein [74]
determined that a 6-g1ucosidase from Aspergillus fumigatus
contained glucosamine and mannose based on the results of paper and
gas-liquid chromatogr aphy. Aporoximately 15 moles of mannose and 2
moles of glucosamine were found per mole protein. That either sugar
could only be released by strong alkaline treatment with IN NaOH at
100C for 6 hours suggested that the carbohydrate was associated
with the protein via a glucosaminyl-peptide linkage. Hirayama et al.
[75] purified a glycoprotein B-glucosidase from the phytopathogenic
fungus, Pyricularia oryzae. Although the enzyme contained 101
mannosyl and 13 glucosyl residues per protein molecule, as determined
by gas-liquid chromatography of the trif1uoroacetyl derivatives, only
5% of the carbohydrate could be selectively removed using an
cc-mannosidase.


14
Structure:Function Relationships for Fungal Glycoenzymes
While little is known about the structure of the carbohydrate
attached to fungal cellulases, even less is known about its function.
Hayashida and Yoshioka [76] were able to partially remove the carbo
hydrate attached to an exocellulase (Avicelase) and an endocelluase
(CMCase) from the thermophilic fungus Humicola insolens YH-8 by
either chemical or enzymatic treatment. Treatment of either with a
mi xed-g 1 ycos i d ase preparation was shown to release about 65% of the
carbohydrate. The removal of the carbohydrate by this method did not
appear to affect the ability of either enzyme to hydrolyze crystalline
cellulose (Avicel), although the thermal and oH stability of both were
decreased. Treatment with periodate oxidation and Smith degradation
was shown to release about 90% of the carbohydrate from each enzyme.
After this chemical degradation, activity of each on Avicel was
decreased about 30% and pH and thermal stability of each was also
further reduced. There was no apparent effect of covalently bound
carbohydrate on substrate specificity since cellobiose was the
predominant product of enzymatic hydrolysis of cellulose by either the
native, 65%-carbohydrate depleted or 90%-carbohydrate depleted exo- or
endocel 1 u 1 ase. However, in neither treatment was all the carbohydrate
removed and in the case of the chemical treatment, the effect on the
polypeptide was not determined. These results suggest that the
carbohydrate is important primarily for stability of the enzymes.
Extensive periodate oxidation of glucoamylase I from Asper
g' 11 u s n i ger was shown to cause a marked loss in the stability of
the enzyme [77]. Glucoamylase I, normally heavily glycosylated at


15
about 45 sites on the polypeptide, was demonstrated to precipitate out
of solution when two-thirds of the carbohydrate was removed by this
method. Thus, for this enzyme, solubility and perhaps transfer to the
aqueous phase of the culture medium may be affected significantly by
the glycosylation.
To understand the importance of carbohydrate covalently-bound to
glycoproteins it is necessary to know the role of the protein to which
it is attached. Whereas the role of some glycoproteins is not known,
glycoenzymes offer a dynamic function for which one can study binding
and catalysis. Thus, definitive information can be achieved either by
preventing glycosylation or by removing the carbohydrate from the
post-transl ationally modified polypeptide. In spite of our knowledge
concerning the enzymic properties of fungal glycoenzymes, and
cellulases in particular, there is little known regarding the three-
dimensional structure of these enzymes. If the carbohydrate can be
removed from these glycoenzymes, the polypeptide may be more amenable
to crystallization and X-ray diffraction studies.
Assessment
Physico-chemical studies have revealed the molecular weight,
isoelectric point, amino acid composition and mora recently, the amino
acid sequence of CBH 1(D) and the genetic sequence encoding the
information for synthesis of CBH 1(D) messenger RNA. Peptide isolation
has shown that the (^-glycosylated residues are confined to a short
sequence on the protein. With the availability of sophisticated
instrumentation such as high resolution nuclear magnetic resonance


16
(NMR ) and gas chromatography/mass spectrometry (GC/MS), the stucture
of oligosaccharides attached to glycoproteins can be accurately
elucidated. The role the carbohydrate plays in cellulase function
cannot be fully understood unless the structure is known; this
information will enable further exploration of the molecular basis for
cellulase biosynthesis, secretion, stability, activity and microhetero
geneity. Successful analyses, of course, depend on pure preparations
of proteins and oligosaccharides to ensure a definitive structural
description.


EXPERIMENTAL PROCEDURES
Materials
Enzymes--Crude extracellular preparation from
Trichoderma reesei QM 9414, grown on Avicel (microcrystalline
cellulose), was a gift from Gulf Oil Chemicals Company, Merriam, KS.
I3-G1 ucos i dase from Trichoderma reesei QM 9414; prepared by
William Chirico.
a-Gl u cos i d as e (maltase) from Yeast (Lot #202900); Calbiochem,
Los Angeles, CA.
a-Gl ucosidase (Type I) from Yeast (Lot #39B-5370), a-glucosidase
(Type VI) from Yeast (Lot #21F-8106), pullulanase from
Enterobacter aerogenes (Lot #99C-0273l), amyl oglucosidase from
Aspergillus niger (Lot #72F-0560 ); Sigma Chemical Company, St.
Louis, MO.
a-Mannosidase from Jack Bean (Lot #7064101); Boehringer Mannheim
Corporation, New York, NY.
Chromatographic Suppl ies--DEAE-Sephadex A-50, SP-Sephadex
C-50; Pharmacia Fine Chemicals, Piscataway, NJ.
Dowex 50W (200-400 mesh; H+ form), Biogel P-2 (-400 mesh);
BioRad Laboratories, Richmond, CA.


18
Amberlite MB-3 (mixed bed resin, Amberlite IR-120 and Amberlite
IRA-410, fully regenerated); Mal 1inckrodt, Inc., Paris, KY.
0 V 2 2 5 ( cyanopropyl methyl phenyl-methyl silicone), Chromosorb
G-HP (80-100 mesh); Varian Associates, Sunnyvale, CA.
Neopentyl glycol succinate; Alltech Associates, Deerfield, IL.
Gas Chrom Q (100-120 mesh); Applied Science, State College, PA.
Whatman Partisil PXS 10/25 PAC column (bonded cyano-amino type,
polar phase); Whatman Chemical Separations Division, Clifton, NJ.
Substrates--CM-Cellulose 7HP; Hercules Powder Company,
Wilmington, DE.
Avicel PH 101 (microcrystalline cellulose); American Viscose
Division, FMC Corporation, Newark, DE.
Walseth cellulose (Dhosphoric acid-swollen cellulose); prepared
from Avicel PH 101 by the method of Wood [78].
d_-Ni troDhenyl -a-D-mannopyr anoside (Lot #701993); Calbiochem,
Los Angeles, CA.
2_-N i t r opheny 1-a-D-g 1 u copyr anos i d e (Lot #09053-3); Pierce
Chemical Company, Rockford, IL.
p_-Nitrophenyl-B-D-mannopyranoside (Lot #62C-1270),
£_-ni trophenyl-B-D-gl ucopyranoside (Lot #88C-5039); Sigma Chemical
Company, St. Louis, M0.
Carbohydrate Standards--Dextrose; National Bureau of
Standards, Washington, DC.
D-Xylose, D-xylitol, D-galactose, gentiobiose, Yeast mannan;
Sigma Chemical Company, St. Louis, M0.


19
D-Mannose; Calbiochem, Los Angeles, CA.
Cellobiose; Eastman Kodak Company, Rochester, NY.
Kojibiose, nigerose; gifts from Dr. Seiya Chiba, Department of
Agricultural Chemistry, Hokkaido University, Sapporo, Japan.
Isomaltose; Applied Science Laboratories, Inc., State College,
PA.
Dextran T-10; Pharmacia Fine Chemicals, Piscataway, NJ.
Chemi_cal_s--Acetic Anhydride; Mal 1 inckrodt, Inc., Paris, KY.
Acetonitrile (HPLC grade), dimethyl sulfoxide (kept dry over 3A
molecular seives, methyl iodide, palladium (II) Chloride,
hydroxylamine hydrochloride; Fisher Scientific Company, Fair Lawn, NJ.
Acrylamide (>99.9%); BioRad Laboratories, Richmond, CA.
Ampholytes ( Amphol i ne pH 3.5-9.5); LKB Produkter AB, Broinma,
Sweden.
Basic Fuchsin (91% dye); Eastman Kodak Company, Rochester, NY.
Bromophenol Blue; Canalco, Rockville, MD.
Coomassie Brilliant Blue R250, Column Coat,
N N-methylene-bis-acrylamide, ammonium persulfate; Miles Laboratories,
Inc., Elkhart, IN.
Deuterium oxide ( 99.96 atom % D); Aldrich Chemical Company,
Inc., Milwaukee, WI.
Disodium succinate hexahydrate (A grade); Calbiochem, Los
Angeles, CA.
Periodic acid; G. Frederick Smith Chemical Company, Columbus,
OH.


20
I I
Pyridine (silylation grade), N N N ,N -tetramethylethylene
diamine (TEMED); Pierce Chemical Company, Rockford, IL.
Riboflavin, L-a-amino-n-butyric acid, Freunds Complete Adjuvent;
Sigma Chemical Company, St. Louis, MO.
Sodium borohydride, sodium hydride (50% in oil dispersion); Alfa
Products, Danvers, MA.
All other chemicals were reagent grade.
Methods
Purification of Cellobiohydrolase I(D)--Cenobiohydrolase 1(D)
(CBH 1(D)) was purified from an extracel 1 ular culture filtrate of
Tr i chod erma by a modification of the method described by Gritzali
[49]. A crude precipitate produced by adding ammonium sulfate (65%
saturation) to a solution of the extracellular material from
Tr i chod erma r e e s e i QM 9414 grown on Avicel was used as the source
of the enzyme. This precipitate was dissolved in distilled water,
dialyzed and 1 yophi11ized The resultina pale yellow powder (5.0-7.5
g) was dissolved in 150 ml of 50 mM sodium succinate, 3 mM sodium
azide, pH 6.0. The mixture was then filtered slowly through glass
fibre paper (Whatman, grade 934 AH) and the filtrate applied to a
DEAE-Sephadex A-50 column (14 x 22 cm) equilibrated with the same
buffer. C e 1 1 o b i o h y d r o 1 a s e II, ar y 1-13-D-g 1 ucos i d ase and
endo-1,4-S-D-gl ucanase activity were eluted isocratical ly at a flow
rate of 400 ml/h (Fig. 1). CBH 1(D) was obtained in high purity when
the pH of the elution buffer was lowered in a stepwise fashion to 3.6.
The previous method [49] involved elution of CBH 1(D) in the presence


FIGURE 1
Isocratic elution pattern from DEAE-Sephadex column
chromatography of the extracellular protein
preparation from T_^ reesei QM 9414 grown on
cel 1ulose.
Conditions of elution were described in Experimental
Procedures. The volume of each fraction was 25 ml.


FRACTION
ENDOGLUCANASE ACTIVITY [A0S /min/mj] ( D-O)
-i -* w ro
o I
22
250


23
of 0.5M NaCI, and while this was adequate for that cellulase
preparation, with this batch under the same conditions, several
impurities eluted with the enzyme. In the absence of the salt, CBH
1(D) eluted at pH 3.6 with approximately one bed volume of buffer and
was separated from minor contaminants.
Purification of Ce 11 ob i ohyd ro 1 as e I I--Ce1 lobiohydrolase II
(CBH II) was also purified by the method of Gritzali [49]. The
proteins eluting isocratically at pH 6.0 from the DEAE-Sephadex column
used to separate CBH 1(D) (fractions 45-105 ) were pooled. This
material, containing CBH II, endogl ucanase and 13 g 1 ucos idase
activity, was dialyzed against 2mM sodium succinate containing 3mM
sodium azide at pH 4.5, concentrated by ultrafiltration to 30 ml and
applied to an SP-Sephadex C-50 column (4.4 x 55 cm). All endoglucanase
activity eluted isocratically (Fig. 2) and CBH II was obtained in high
purity when eluted in a batchwise manner with an elutant of 8mM sodium
succinate containing 3mM sodium azide at pH 4.5 (flow rate = 70 ml/h).
The previous method [49] eluted these enzymes in the same buffer at pH
5.0, but elution at the lower pH was conducted in this case to better
separate the endogl ucanases for further purification. (3-Glucosidase
activity was subsequently eluted from the column with 50mM sodium
succinate, 3mM sodium azide, pH 6.0.
Dialysis and Ultrafi1tration--Protein concentration and
dialysis were performed on either an Amicon Model 2000 High
Performance Ultrafiltration Cell (volumes up to 2 1) or an Amicon
Model 202 Ultrafilration Cell (volumes up to 200 ml)(Amicon Scientific


FIGURE 2
Isocratic elution pattern from SP-Sephadex column
chromatography of cellobiohydrolase II and the
endoglucanases which had been eluted from a
DEAE-Sephadex column.
Conditions of elution are described in "Experimental
Procedures." The volume of each fraction was 21.0 ml.


240
rm
E
c 160

a
w
^~
>-
>
t-
O
<
LU
co
<
z
<
o
3
_l
o
o
a
z
UJ
120
80
40
0
-L
1
X
80 100
FRACTION
6.0
8mM
5.0
4.0
3.0
2.0
l
E
c
o
oo
CM
UJ
o
z
<
co
cc
O
CO
CO
<
120
140
ro
cn


26
Systems, Lexington, MA). Pressure was supplied with nitrogen gas at 40
psi and 30 psi, respectively. A Diaflo PM-10 Ultrafilter, with a
molecular exclusion limit of 10,000 daltons, was used with each cell.
Protein Determi n at i on--Protein concentrations were estimated
during purification by spectrophotometric determination at 280 nm,
1%
using an approximate extinction coefficient (E at 280 nm) of 10.0.
For all quantitative analyses, protein concentration was determined
using experimentally derived extinction coefficients. Samples of
protein were dialyzed extensively against water, lyophillized and then
stored over phosphorous pentoxide for at least 10 days. Protein was
weighed and then made up into solutions of known concentrations of
between 0.3-0.8 mg/ml. The absorbance of each solution was determined
both at 260 nm and 280 nm on a Beckman DU-8 UV-Visible
Spectrophotometer (Beckman Instruments, Inc., Palo Alto, CA).
Enzyme Assays--Endo-l,4-B-D-qlucanase activity was measured
by following the decrease in viscosity of a carboxymethylcellulose
solution as described by Shoemaker and Brown [79]. Specific activity
was expressed as the change in specific fluidity/min/mg protein.
Ary 1-g 1 ycos idase activity was followed by monitoring the release of
2_-n i tropheno 1 from £_-n i tropheny 1 -gl ycosides A solution of 10 mM
substrate (0.5 ml) and an appropriate buffer (2.0 ml) were
pre incubated in small test tubes for 10 min at 40C. For
a-man n os i d as e digestions were performed with 50mM sodium citrate
containing 3mM sodium azide at pH 4.5 and for a-glucosidase reactions
were carried out in solutions of 65mM potassium dihydrogen phosphate


27
with 3mM sodium azide at pH 6.0. Aliquots of enzyme solution (2-25
pi) were then added, mixed and incubated at 40C for 20 min. The
tubes were placed in a boiling water bath for 10 min, and when cool, 1
ml sodium borate (0.5 M, pH 9.8) was added. The absorbance at 400 nm
was measured, compared to a £-ni trophenol standard curve and
specific activity expressed as pmoles £-nitrophenol released/min/mg
protein. The solution was adjusted with the sodium borate buffer to pH
9.1, a value greater than one pH unit above the pKa of
£-nitrophenol, assuring complete dissociation.
Polyacrylamide Disc Gel Electrophoresis --Disc gel
electrophoresis was performed as a routine analysis of column
fractions during purification and was used as a criterion of
homogeneity. Electrophoresis was conducted using the discontinuous
buffer system No. 1 described by Maurer [80]. The pH of the stacking
gel and separating gels were 8.3 and 9.5, respectively, and the
polyacrylamide concentration was 7.5 percent (W/V). Power was supplied
at 2 mA/tube until the bromophenol blue tracking dye had entered the
separating gel, at which point it was increased to 3 mA/tube until the
dye band was within 0.5 cm of the bottom of the tube. Separations were
performed in a Canal co electrophoresis chamber (Miles Laboratories,
Inc., Elkhart, IN) with a Hoefer PS 1200 DC power supply (Hoefer
Scientific Instruments, San Fransisco, CA).
After e 1 ectrophoresis, protein bands were fixed by immersion in
12 percent (W/V) trichloroacetic acid for 30 min after which the gels
were washed with water (3x5 min). Protein was stained with 0.1%
Coomassie Brilliant Blue R250 in methanol-water-acetic acid (45:45:10)


28
for 1 h and subsequently destained with 7% acetic acid at 40C with
frequent changes of destaining solution. Carbohydrates in the gels
were stained by the periodic acid-Schiff (PAS) method described by
Lang [81]. After fixing and washing, the gels were immersed in fresh
periodic acid (0.5% w/v) for 1 h in the dark. The acid was removed by
successive washes with 7% acetic acid (3 x 10 min). Gels were then
stained for 1 h with 1% Basic Fuchsin in 0.15N HC1 containing 1.9%
sodium metabi sulfite. Excess dye was removed by destaining with 0.1%
sodium metabi sulfi te at 40C, with repeated changes of destaining
solution.
Isoelectric Focusinq--Isoelectric points for homogeneous
proteins were determined using a BioRad Model 1415 Horizontal
Electrophoresis Cell (BioRad Laboratories Richmond, CA).
Determinations were performed with cold water (4C) passing through
the qel bed using a Haake Model FE Constant Temperature Circulator
(Haake Instruments, Inc., Saddle Brook, N. J.). Polyacryl amide gel
slabs ( 100 x 125 x 0.8 mm) were used as described by BioRad [82],
without initial prefocusing and with the modification that the
ampholytes were replaced with 0.5 ml Ampholine, pH 3.5-9.5 (LKB
Produkter AB, Bromma, Sweden), as these produced more linear pH
gradients. E1 ec t r o f ocu s i n g was performed for 2.5 h at 4.5 amps
constant current. Best results were obtained using lOpg samples of
protein applied to small strips of glass fiber paper (0.3 x 0.5 cm)
placed at the center of each lane on the gel slab. The strips were
removed 30 min after the current was turned on. After electrofocusing,
sections were cut (0.5 x 0.5 cm) down each side of the gel and placed


29
in tubes containing 1 ml 0.1M KC1 (made with degassed, deionized
water). The pH of each section was determined after 1 hour at room
temperature and the gradient calculated. Fixing, staining and
destaining were performed as described by Winter et al. [83]. Proteins
were fixed (17.3 g sulfosal icylic acid and 57.5 g trichloroacetic acid
in 500 ml water) for 1 h at room temperature, stained with Coomassie
Brilliant Blue R250 (0.46 g in 400 ml destaining solution) for 1 h and
then destained (water-ethanol-acetic acid, 67:25:8) until the
background was clear.
Carbohydrate Compos ition--Tota 1 neutral carbohydrate was
determined by the phenol-sulfuric acid method of Dubois et al. [84].
Carbohydrate components of the enzymes were determined after either
hydrolysis or B-e 1 i mi n a t i on and individual monosaccharides were
identified after separation by gas-liquid chromatography. Aldoses were
determined as the peracetyl ated aldononi tri les (PAANs) and alditols
were determined as the peracetylated alditols (PAAs).
For pheno 1 -su 1 furic acid analysis, 0.1-1.0 mg of glycoprotein
(containing 2-15 pg neutral carbohydrate) was dissolved in 0.2 ml
water in a small test tube. Phenol (0.2 ml,3%) was added followed by
1.0 ml concentrated H^SO^ with vigorous mixing using a vortex
mixer. The samples were cooled to 40C and the absorbance determined
at 400 nm, using mannose as the standard, as this is the principal
carbohydrate constituent of these glycoproteins.
Lyophi 1 1 i zed glycoproteins (1-2 mg) were hydrolyzed in 1 N HC1
(2 ml) for 2 h at 100C in sealed 5 ml Reacti-Vials (Pierce Chemical
Company, Rockford, IL). When cool, xylose and/or xylitol were added


30
as internal standards and the mixtures immediately deionized using
small columns (1.4 x 6 cm) of Amberlite MB-3 mixed bed resin. The
eluants were taken to dryness in a rotary evaporator, transferred to 5
ml React i-V i a 1 s and lyophillized. Monosaccharides were then either
converted to the peracetylated aldononitri1 es (PAAN) by a modification
of the method of Varma et al [85] or subsequently reduced and
converted to the peracetylated alditol by the method of Sawardeker et
al [86]. It was essential that these derivatives be separable and a
profile of that separation by gas-liquid chromatography is shown (see
Fig. 3).
The PAAN preparative procedure involved adding 3-5 mg
hydroxyl ami ne hydrochloride and 0.25 ml pyridine to the lyophi 11 ized
monosaccharide mixture (0.01-10mg), sealing the Reacti-Vials with
screw top teflon caps and heating at 90C for 45 min. The resultant
oximes were then peracetylated by addition of 0.25 ml acetic anhydride
and heating at 90C for 45 min. The products were taken to dryness
under nitrogen at 60C and then evaporated with toluene (3 x 1 ml) to
remove residual acetic anhydride using an N-Evap Model 106 Analytical
Evaporator (Or g an omat i on Associates, Inc., Shrewsbury, MA). The
products were subsequently partitioned between 3M HC1 (1 ml) and
CHClgd ml), the aqueous layer removed and the organic layer washed
with water (2 x 1 ml). Washing involved vigorously mixing the two
phases in the Reacti-Vial and removing the aqueous layer with a
pipette after partitioning was complete. The CHCl^ layers were then
washed with 0.5M sodium bicarbonate (1 x 1 ml) and then again with
water (1 x 1 ml). A spatula tip of MgSO^ powder was then added to
each to remove any excess water. The organic layers were then filtered


FIGURE 3
Gas chromatographic separation of peracetylated
alditol and aldononitrile derivatives of neutral
monosaccharides released from cellobiohydrolase 1(D)
after reductive B-elimination and subsequent acid
hydrolysis.
Peaks identified are per acetyl ated 1. xylononi tri le; 2.
xylitol; 3. mannononi tri 1 e; 4. gl ucononi tri le; 5. mannitol.
Conditions of separation are described in "Experimental
Procedures". The peaks eluting before the xylononi tri le
acetate peak were always present in PAAN preparations but
not in PAA preparations and are thought to be byproducts of
the reaction [88].


ELUTION TIME (MIN)
RELATIVE INTENSITY


33
through glass wool, concentrated to about 20-50 pi and 1 pi aliquots
were analyzed by gas-liquid chromatography.
In the procedure for peracetylated alditols, monosaccharides
were reduced overnight with aqueous sodium borohydride (10 mg in 1 ml
water) at 4C. The reactions were stopped by dropwise addition of 2N
acetic acid until the effervescence stopped, using 3 drops of
n-octanol to suppress foaming. The mixtures were taken to dryness
under a stream of dry nitrogen at 60C and the residues evaporated
with methanol (3 x 1 ml) to remove excess borate as the volatile
methyl ester. The mixtures were then deionized by passage through
small Amberlite MB-3 columns and the eluants were taken to dryness on
a rotary evaporator. The samples were transferred to 5 ml
Reacti-Vials and lyophi11ized Pyridine (0.25 ml) and acetic anhydride
(0.25 ml) were added and the vials sealed and heated at 90C for 45
min. The mixtures were taken to dryness under a stream of dry nitrogen
at 60C and then evaDorated with toluene (3x1 ml). The residues
were dissolved in 20-50 pi CHC1 ^ and 1 pi aliquots analyzed by
gas-liquid chromatography.
0_- linked oligosaccharides were released by reductive
B-e1imination of the glycoDroteins, by a modification of the method
of Nakajima and Ballou [87]. To 1 or 2 milligrams of the glycoproteins
in a 5 ml Reacti-Vial were added 2 ml of 0.1N NaOH and 0.3M NaBH^.
The solution was incubated at 40C for 48 hours. During a 72 h
control experiment, no further sugars were released from the protein
after 3 hours of reductive B-elimination (Fig. 4). After
neutralization by dropwise addition of 2 N acetic acid, the samples
were evaporated with methanol to remove borate esters and then


FIGURE 4
Efficacy of sugar release from cellobiohydrolase 1(D)
by reductive 13-elimination.
Oligosaccharides released were subsequently hydrolyzed to
constituent sugars. The resultant monosaccharides were then
converted to the peracetylated alditols and aldononitri les
and analyzed by gas-liquid chromatography on 1% OV-225 as
described under "Experimental Procedures". A, Mannose;D,
Mannitol ;0> Glucose.


MOLES SUGAR RELEASED/MOLE PROTEIN
to o>

o o o


36
deionized by passage over Amberlite MB-3. The eluate was dried under
reduced pressure and hydrolyzed as previously described. The sugars
released were converted to either a mixture of PAANs and PAAs by the
PAAN method described previously or were further reduced with NaBH^
and all converted to the PAAs. Formation of mixed derivatives is
feasible as alditols, unlike aldoses, do not form the corresponding
nitrile in the presence of pyridine and hydroxylamine hydrochloride;
but do undergo peracetylation upon addition of acetic anhydride. This
der i vat i zati on method following 6-el imination provided information
regarding, not only the total neutral carbohydrate composition of the
0_-linked oligosaccharides, but also identification of the type of
sugar attached to the protein (leading to the formation of the
correspond'ng PAA) and an estimation of the number of sites at which
carbohydrate had been attached to the polypeptide.
The area of peaks resulting from gas-liquid chromatography (see
below) of the sugar derivatives were subjected to integration and were
compared to internal standards. Positive identification of derivatives
was achieved by gas chromatography/mass spectrometry (see below).
Quantitative determination of the peracetyl ated aldononi tri les of
mannose and glucose were derived from the linear relationship of their
GLC peak areas with respect to that of the xylose internal standard as
were those of the peracetylated alditols of mannitol and glucitol with
respect to the peracetylated xylitol internal standard (Figs. 5 and
6).
While alditol acetate formation proceeds essentially to
completion, Furneaux [88] has shown that formation of some
per acetyl ated al donon i tr i 1 es occurs with the formation of several


FIGURE 5
Quantitation of aldoses as the peracetylated
aldononitriles.
Aldoses (mannose (O), qlucose (#) and xylose (internal
standard)) were converted to the PAANs and the derivatives
were separated by gas-liquid chromatography on 1% OV-225 at
190C as described under "Experimental Procedures". The
ratio of peak areas of sample/standard was plotted against
the molar ratio of the sample/standard. Correction factors
necessary for calculation of unknowns were determined to be:
Man/Xyl, 0.85; Glc/Xyl, 0.94.


MOLAR RATIO
PEAK AREA
ALDONONITRILE ACETATE
XYLONONITRILE ACETATE
-i N>
b b


FIGURE 6
Quantitation of alditols as the peracetylated
alditols.
Alditols (mannitoKD), glucitol () and xylitol (internal
standard)) were converted to the PAAs and the derivatives
were separated by gas-liquid chromatograohy on 1% OV-225 at
190C as described under "Experimental Procedures". The
ratio of peak areas of sample/standard was plotted against
the molar ratio of the sample/standard. Correction factors
necessary for the calculation of unknowns were determined to
be: Mannitol/Xylitol, 0.87; Glucitol/Xy1 ito1, 0.94.


MOLAR RATIO
PEAK AREA
ALDITOL ACETATE
XYLITOL ACETATE
-* ro
b b


41
furanose and pyranose byproducts. The PAANs are more labile than the
PAAs; the former partially decompose in the injector of the gas-liquid
chromatograph^ Observations in this laboratory support these
conclusions as we have found that the yield of PAANs is lower than
that of the corresponding peracetylated alditols from derivatization
of equal amounts of the respective aldoses and alditols. Several small
unidentified peaks also eluted after these derivatives and by elution
at higher temperatures on 1% OV-225 as was seen by Furneaux. This
discrepancy was accounted for, however, by the use of internal
standards in each sample, since it was found that the aldoses behaved
reproducibly as a group, as do the alditols (Fig. 5 and 6).
Preparation of reduced oligosaccharides -01 i gos aechar ides
0_-l inked to the protein were released by treating 50-200 mq of each
glycoprotein with 0.1N NaOH and 0.3M NaSH^ at 40C for 48 hours
[87]. The reactions were performed in sealed culture tubes with teflon
screw caps in a volume of 15 ml. Reaction products were neutralized
with 2 N acetic acid using 5 drops of n-octanol to control foaming and
taken to dryness on a rotary evaporator at 50C. The residues were
evaporated with methanol several times to remove excess borate and
then deionized through a small column (50 ml) of Amberlite MB-3. The
eluant was taken to dryness, dissolved in 400 pi of water and
injected onto a Biogel P-2 (-400 mesh) column (0.25 in x 8 ft)
attached to a Waters high performance liquid chromatograph (HPLC;see
below). Oligosaccharides were eluted with water and the eluant stream
monitored with a differential refractometer. Oligosaccharides were
^Bradbury, A.G.W. (1984) Personal Communication.


42
purified either by rechromatography of pooled fractions under the same
conditions, or by passage through a Partisil PXS 10/25 PAC polar
r e v e r s e p h a s e column, also by HPLC. Composition of each
oligosaccharide and identification of the sugar attaching the
oligosaccharide to the protein was determined following mild acid
hydrolysis of each and conversion of the product to a mixture of the
alditol and al donon i tr i 1 e acetates by the PAAN method previously
described. Derivatives were then analyzed by gas-liquid
chromatography and gas chromatography/mass spectrometry.
Preparation of Methyl sulfi nyl Carbanion--The methylsulfinyl
carbanion suspension for methylation analysis was prepared by an
adaptation of the methods of Lindberg [89] and Spiro [90]. Sodium
hydride was obtained in a 50% mineral oil dispersion as it reacts
explosively with water; all glassware was acid cleaned, baked and then
kept over phosphorous pentoxide until use. Under a dry nitrogen
stream, 4.75 g of sodium hydride was weighed into a 250 ml erlenmeyer
flask, equipped with a two-hole rubber stopper (with a CaC 1 ^ trap
and a tube for nitrogen) and a small magnetic stirrer bar. To this was
added 100 ml DMS0 (dried over 3A molecular seive) to achieve a final
concentration of 2M sodium hydride, and the mixture stirred under a
nitrogen stream for one hour. Aliquots (25 ml) of the suspension were
poured immediately into 30 ml serum bottles, sealed with rubber caps
and placed on ice. The suspensions were still generating hydrogen gas;
so the caps were punctured with two syringe needles and the bottles
flushed with nitrogen until the mixture froze solid. The
methy 1 su 1 f i ny 1 carbanion suspension was stored under nitrogen (with


43
Drierite) at -20C. Under these conditions the preparation was good
for at least four years. When ready for use, the caps were punctured
again with two syringe needles and flushed with nitrogen in a
sonicator bath for one hour. Aliguots of the suspension were removed
with an 18 gauge needle (smaller needles clogged up with the thick
slurry) and the mixture frozen on ice again under a nitrogen stream.
Methyl at ion Analysis--Glycoproteins and oligosaccharides
released by reductive 6-elimination were methylated by the method of
Hakomori [91] and samles were subsequently hydrolyzed, reduced and
acetyl ated by a method adapted from those of Bjorndal et al. [92] and
Gum [93]. Samples (oligosaccharides, 0.5-1.5 mg; glycoproteins, 10
mg) were first dried over phosphorous pentoxide overnight in 5 ml
React i- Vials, dissolved in 1 ml dry DMSO and closed with
teflon/silicone seals. The seals were punctured with two syringe
needles, 1.0 ml of 2M methylsulfonylmethylsodiurn was injected and the
needles removed after flushing the vials with nitrogen. The vials were
agitated in an sonicator bath for one hour. After another 2 hours, 1
ml methyl iodide was injected dropwise and the vials sonicated for 30
min. The products were partitioned into CHCl^ (5 ml) and water (7.5
ml) and the water layers washed again with CHCl^ (2x3 ml). The
organic layers were pooled and repeatedly washed with water (3x5
ml). Excess water was removed from the organic layers by addition of
a spatula tip of MgSO^ and the supernatant filtered through glass
wool into 5 ml Reacti-Vials and evaporated to dryness under nitrogen.
Methylated oligosaccharides were then hydrolyzed with 88% formic acid
(2 ml) for 2 hours at 100C. The products were taken to dryness


44
under nitrogen and the hydrolysis completed with 0.25 M H^SO^ (1
ml) at 100C for 12 hours. Samples were transferred to small test
tubes, 0.54 g BaCO^ (10% excess) was added to neutralize the
hydrolysates and the supernatants removed after centrifugation (5000
rpm, 10 min). The pellets were extracted washed with 1 ml water,
centrifuged again and the supernates pooled. The partially methylated
monosaccharides, then in 3 ml of water, were then reduced by addition
of 20 mg NaBH^ at room temperature for 2 hours. The supernatants
were then taken to dryness in 5 ml Reacti-Vials and evaporated with
methanol (3x2 ml). After drying overnight with P ^ 0 5, the
partially methylated alditols residues were acetylated with pyridine
(0.25 ml) and acetic anhydride (0.25 ml) at 90C for 1 hour. The
reaction mixtures were taken to dryness under nitrogen at 60C,
evaporated with toluene (3x1 ml) and dissolved in 20-50 pi CHC1 ^-
Products were analyzed by gas-liquid chromatography on 3% neopentyl
glycol succinate, and positively identified by gas chromatography/mass
spectrometry (see below).
Tetramethyl and trimethyl alditol acetate standards for GLC and
GC/MS comparison were prepared by methylation of either (i) 5 mg of
yeast mannan for the mannose series or (ii) 5 mg each of the glucose
di saccharides kojibiose (al-2), nigerose (al-3), cellobiose (31-4)
and gentiobiose (61-6). Pentamethyl alditol acetate standards were
prepared after reduction of 5 mg of each of the glucose disaccharides
described with 20 mg sodium borohydride in 2 ml of water at room
temperature overnight. The reactions were stopped with 2 N acetic
acid, in the presence of n-octanol, and each sample taken to dryness
under reduced pressure. Excess borate was removed by evaporation as


45
the volatile methyl ester by addition of aliquots of methanol (3 x 10
ml) and the samples subsequently transferred to 5 ml Reacti-Vials and
lyophillized in preparation for methylation.
Acetolysis--Reduced tri- and tetrasaccharides were subjected
to acetolysis by the method of Tai et al. [94] as modified by Li et
al. [95]. Oligosaccharides (100 pg) were dissolved in 0.5 ml pyridine
and 0.5 ml acetic anhydride in 5 ml Reacti-Vials and left at room
temperature for 70-74 h. The vials were then heated at 80C for 4 h.
Samples were taken to dryness under nitrogen and evaporated with
toluene (3x1 ml). Acetylated oligosaccharides were then acetolyzed
with 1.0 ml acetic anhydride-acetic acid-H^SO^ (10:10:1) at 40C
for 16 h. The products were then partitioned into CHC1 ^ d ml) and
water (1 ml). The aqueous layer was removed and the organic layer
washed repeatedly with water to remove the color (3x3 ml). The
aqueous washes were then extracted with CHC 1 ^ (3 x 1 ml) and all the
CHC1 ^ fractions pooled and dried. The residue was then deacetylated
with 1.0 ml 0.1% sodium methanolate for 30 min at room temperature.
The reaction was neutralized with 2.0 ml ethyl acetate and the
products evaporated to dryness under nitrogen. The residue was then
dissolved in 10-30 pi of water and the products separated by polar
reverse-phase HPLC on a Whatman Partisil PXS 10/25 PAC column. The
compositions of purified oligosaccharides were then determined by
gas-liquid chromatography as the per acetylated alditol and/or
aldononitriles (see below).


46
Glycosidase diqestion--01iqosaccharides released from the
glycoproteins by reductive 6-elimination were subjected to sequential
glycosidase digestion which provided information as to the anomeric
nature of each sugar residue and also the sequence of sugars in each
oligosaccharide. Samples of each oligosaccharide (100 pg) were placed
in 250 pi Microfuge tubes (Beckman Instruments, Inc., Palo Alto, CA)
and lyophi 11 i zed Incubation with yeast a-glucosidase (maltase) was
performed with one unit of enzyme in 0.05 ml of 0.05 M potassium
phosphate, pH 6.8, for 12 hours at 40C with 10 pi of toluene added
to ensure sterility. One unit of enzyme was then added every 12 hours
for the next 36 hours until four units of enzyme had been added.
Incubation with jack bean a-mannosidase was performed with one unit of
enzyme in 0.05 ml of 0.05 M sodium citrate, pH 4.5 for 48 hours at
40C with 10 pi toluene added. Products were separated directly by
HPLC on a Partsil PXS 10/25 PAC polar reverse-phase column, equipped
with an Bondapak AX/Corasil anion exchange precolumn. Oligosaccharides
were subsequently incubated with a different glycosidase enzyme and
the resulting monosaccharides were analyzed as the alditol and/or
aldononitrile acetates by gas-liquid chromatography.
Gas-Liquid Chromatoqraphy--Monosaccharide components of
oligosaccharides and glycoproteins were identified and quantified
after separation of acetylated derivatives by gas-liquid
chromatography and as such it was necessary to achieve a separation of
alditol and a 1donon itri 1e acetates of component sugars of the
glycoproteins (see Fig. 3). Although separation of PAANs [85] and PAAs
[86] have been reported, these methods were extensively modified to


47
permit simultaneous separation of the two classes of sugar
derivatives. All gas-liquid chromatography was performed on a Varian
Model 2700 gas chromatograph (Varian Associates, Sunnyvale, CA)
equipped with a flame ionization detector and attached to an Autolab
System 1 computing integrator (Spectra-Physics, Santa Clara, CA).
Alditol and al donon i tri le acetates were separated on a 1% OV-225 on
Chromosorb G H/P (80-100 mesh) glass column (2 mm X 8 ft.),
isothermally at 180C. Parti al ly methylated alditol acetates were
separated on a 3% neopentyl glycol succinate on Gas Chrom Q (100-120
mesh) glass column (2 mm x 20 ft.), with isothermal elution at 190C.
The injector and detector temperatures were 230C and 260C,
respectively, for both columns. Trimethyl alditol acetates were found
to be less well resolved on packed columns of 1% 0V-225, whether
separated isothermally or under a variety of temperature programmed
conditions.
Gas Chromatoqr aphy/Mass Spectrometry--Whi le separations of
the derivatized sugars can be performed by gas-liquid chromatography,
definitive identification was achieved by this technique in
conjunction with mass spectrometry. Two types of fragmentation were
used in this work, electron impact (El) and chemical ionization (Cl),
and each yields different information regarding the molecules being
identified. Mass spectrometry is not normally used for quantitation of
samples as a relatively low fraction of the sample is ionized (about
1% for El) and so all quantitation of derivatives was performed on the
gas chromatograph. El fragmentation is severe as the sample is exposed
directly to an electron beam in the ionizing chamber; hence El spectra


48
represent an array of structural fragments (fingerprints) of the
molecules. It should be mentioned that peracetylated alditol (Appendix
A) and peracetylated aldononitrile (Appendix B) derivatives of mannose
and glucose fragment identically by El; hence the need for prior
separation by gas-liquid chromatography. This is more important when
comparing the partially methylated alditol acetates, where
coordinately methylated mannose and glucose derivatives fragment
identically. Differentially methylated residues, while having many
similar fragments, give rise to unique spectra (see Appendix C).
Cl fragmentation is less severe as the ionizing chamber is
filled with an excess of reagent gas, i.e. isobutane, and thus it is
the gas molecules that are ionized directly. These ionized gas
molecules then collide with samle molecules causing a milder
fragmentation than that which occurs in El, as less energy is
transferred. Cl spectra thus provide very accurate molecular weight
information by a monitoring of the molecular ion (M+), generated by
loss of an electron from the molecule during collision. As
gal actononitrile acetate and mannitol acetate were found to coelute on
a packed 1% OV-225 column, mass fragment scanning was useful in ruling
out the presence of galactose in glycoprotein samples following
reductive B-e1imination and hydrolysis. This is shown in Fig. 7,
where the mass ion for galactononitri le acetate is 356 and none was
shown to be present under the mannitol acetate peak (M+=375). Cl
spectra for peracetylated alditol (Appendix D) and peracetylated
aldononitrile (Appendix E) standards are shown. By providing primarily
molecular weight information, Cl spectra cannot distinguish between
the various trimethyl alditol acetates prepared as standards but of


FIGURE 7
Molecular ion scanning of peracetylated alditol and
aldononitrile derivatives of monosaccharides released
from cellobiohydrolase 1(D) following reductive
B-elimination and subsequent acid hydrolysis.
Molecular ions were selected out of the profile following
chemical ionizatiorji gas chromatography/mass spectrometry.
Scanned were the M ions for: pentitol acetates (303),
hexitol acetates ( 375), pentononi tri le acetates (256) and
hexonon i tr i 1 e acetates (328). The bottom panel represents
the reconstituted ion current (RIC), or the total material
in the sample. Elution times are shown for peracetylated 1.
xylononitrile; 2. xylitol; 3. mannononitrile; 4.
glucononitrile; 5. mannitol.


ELUTION TIME (MIN:SEC)
RELATIVE INTENSITY
G> -ft
0> to o M
w ro o> u
MOLECULAR ION MONITORED
100.0


51
course can distinguish between the tri- and tetramethyl alditol
acetates (see Appendix F).
Gas chromatography/mass spectrometry was performed either on a
Finnegan 4021 GC/MS/DS or on a Finneqan TSQ GC/MS/MS/DS instrument.
All chromatographic conditions were the same as those for gas-liquid
chromatography except the 3% neopentyl glycol succinate column was
only 8 ft long. Electron impact spectra were recorded at an ionizing
potential of 70 eV with an ion source temperature of 300C. Chemical
ionization spectra were recorded at an ionizing potential of 55eV and
a filament current of 0.3A, using isobutane as the reagent gas at 5 x
10 ^ torr.
High Performance Liquid Chromatoqraphy--HPLC separations were
performed with a Waters Model 6000 pump, Model U6K injector and Model
R401 differential refractometer (Waters Associates, Inc., Milford,
MA). Analytical separations were performed by an adaptation of the
method of Gum and Brown [96], on a Partisil PXS 10/25 PAC polar
reverse-phase column (Whatman Inc., Clifton, NJ) equipped with a
Bondapak AX/Corasil anion exchange precolumn (Waters Associates).
Elution was performed at a flow rate of 1.5 ml/min at room
temperature, with either 75% or 77% acetonitrile in water and at a
pressure of 1400 psi. Preparative separations were performed on a
water-jacketed Biogel P-2 (-400 mesh) gel filtration column (0.25 in x
8 ft), with no precolumn, using a Haake Constant Temperature
Circulator. Oligosaccharides were eluted with water at 60C at a flow
rate of 0.7 ml/min and at a pressure of 700 psi.


52
N M R Analyses - N M R analyses were performed on both
glycoprotei ns and on oligosaccharides isolated from them after
preparative reductive 6-elimination and the spectra generated
provided information about the entire molecule being analyzed. For
^H-NMR, analysis of the glycoprotein is not practical due to the
excessive number of signals generated. For the oligosaccharides, the
^H-NMR provided information as to the number of anomeric residues
present and also, in the case of glucose, the nature of the anomeric
linkage. For mannose, the splitting of the anomeric proton for both a-
and 6-anomers is less than 2 Hz, which is below the resolution of the
instrument at 300 MHz.
Characterization of oligosaccharides attached to the
gl ycoprotei ns was possible by proton coupled and proton decoupled
13
C-NMR as these signals (between 60-110 ppm) were far removed from
those of amino acid residues. The decoupled spectra generated signals
for each carbon nucleus in the sample, although due to the closeness
of many of the signals, it was not possible to assian all of them. The
signals generated by the anomeric carbons, located between 98-106 ppm,
were distinct and were far downfield from the other carbohydrate
signals. These can be assigned, on the basis of literature values and
by analysis of the compounds by chemical methods. The number of
hydroxymethyl carbons can also be determined as they generate signals
at the upfield end of the carbohydrate region between 62 and 66 ppm.
But like the ^H-NMR, decoupled ^C-NMR spectra are a
characteristic fingerprint of the molecule, even though it is not
13 1
possible to assign all the signals. Coupled C-NMR, like H-NMR,
provided information as to the anomeric nature of the sugar residues


53
in the oligosaccharides. The coupling constants generated from the
anomeric carbons were characteristic of either a-linkages (170 Hz) or
(3-linkages (160 Hz), although the resolution of signals in coupled
spectra is much poorer than those acquired in the decoupled mode.
CBH 1(D) was prepared by dialyzing against water, lyophillizing
and dissolving the protein into D^O (98.5%). CBH II was less soluble
and counterions were needed, so the protein was dialyzed against 50 mM
NaCl and made to at least 60% D^O. The endoglucanases were prepared
in the same way, except that 20 mM NaCl was used. The reduced
oligosaccharides were lyophi 11 ized and picked up in 100% D^O several
times for all the NMR analyses.
13
C-NMR spectra were obtained on a Nicolet NMR Spectrometer
with a 70.5 kG field and a broadband tunable probe operating in the
Fourier Transform mode at 75.45 MHz. D^O was used as solvent in all
samples and the field was locked on the deuterium signal. Spectra were
collected with broad band proton decoupling either on or off, as
indicated in the particular experiment. Pulse widths and individual
recycle times used were 29.00 psec and 3.00 msec, respectively, and
in all experiments, the post acquisition delay time was greater than
5T^. The spectra were filtered to improve the signal to noise ratio
and this treatment broadened the lines by 1 Hz. Chemical shifts are
reported in parts per million (ppm) from internal
(trimethylsi 1 ane)-l-propane sulfonate (TSP). Under these conditions,
the anomeric carbon of 13-D-glucopyranose resonates at 98.76 ppm.
^H-NMR spectra were obtained on the same instrument with a
fixed frequency ( 300 MHz) probe and spectra were acquired at both


54
25C and 70C to eliminate the possibility of signals "hidden" under
the large HOD signal (4.77 ppm at 25C).
Amino Acid Analysis--One milligram samples of the
glycoproteins were B-eliminated with 0.1M NaOH and 0.3M NaBH^ for
48 hours at 40C. The resultant unsaturated amino acids were
subsequently reduced with palladium chloride and polypeptides
hydrolyzed to the free amino acids as described by Downs et al. [97].
Reactions were performed in sealed screw-caD culture tubes in a volume
of 2.0 ml. After alkaline treatment, 5 drops of n-octanol and a small
stirrer bar were added. One milliliter 0.8M HC1 was added followed by
0.1 ml 0.08M PdCl? and the solutions mixed. Two milliliters NaBH.
(0.66M in 0.1M NaOH) and 2.0 ml PdCl^ (0.016M in 0.8M HC1) were then
added simultaneously in a dropwise fashion, while mixing rapidly. The
total volume was now 7.1 ml and to this was added an equal volume of
concentrated HC1. The tubes were resealed with teflon liners and
heated at 110C for 24 h. Each sample was cooled, transferred
quantitatively to a 100 ml round bottom flask and taken to dryness on
a rotary evaporator at 50C. Residual HC1 was removed by repeated
evaporation with water (3 x 10 ml). The final volume was carefully
dissolved in 1 ml lithium citrate buffer (0.2 M, pH 2.2) and
transferred to a 5 ml volumetric flask. The round bottom flask was
then rinsed repeatedly (4 x 1 ml) and the washings added to the 5 ml
volumetric flask and mixed. Samples containing PdCl^ contained a
small amount of black particulate precipitate, which was allowed to
settle before analysis. Aliquots (0.1 ml) were injected onto a Beckman
Model 119CL amino acid analyzer (Beckman Instruments, Inc., Palo Alto,
CA) in the physiological column mode. a-Aminobutyric acid (product of


55
threonine 13-elimination and reduction) was detected with ninhydrin
[98] and eluted between 76-77 minutes. The peak was integrated and
compared to an internal standard, a-aminoguanidinopropionic acid.
Controls were performed on (i) samples which had undergone PdC 1 ^
treatment without previous (5-elimination, to determine if any
background a-ami nobutyric acid was produced and (i) on samples which
had undergone neither PdCl^ treatment nor 13 e 1 i mi nation to
determine any possible effects of these treatments on any amino acids
released by hydrolysis.
Antibody Preparation and Immunochemical Analysis--Antisera to
CBH 1(D), CBH II and the endoglucanases were prepared in New Zealand
White rabbits by a method adapted from that of Hurn and Chantler [99].
Samples of protein (2.5-3.0 mg) which had been dialyzed to water and
lyophi 1 1 i zed were dissolved in 1 ml water and emulsified with 3.5 ml
Freunds Complete Adjuvent. Each rabbit, 1-2 Kg in size, was injected
with a total of 2 ml of one of the proteins, 1 ml into each thigh
muscle. Booster injections were qiven three weeks later under the same
conditions. Animals were test bled periodically and each antisera
titer determined by immunodiffusion on agar against all the
cel 1 obi ohydrol ases and endoglucanases to check for cross-reactivity.
Rabbits were then sacrificed after seven weeks, 70-100 ml of whole
blood collected from each and serum prepared after removal of whole
cells by centrifugation.


RESULTS AND DISCUSSION
Cellobiohydrolase 1(D)
Molecular Properties of CBH I(D)--CBH 1(D) has been shown to
constitute almost 60% of the extrace 11 u 1 ar protein secreted by
Tri choderma reesei QM 9414 grown on cellulose. This protein has also
been found to have a molecular weight of 54,000 daltons, as determined
by sedimentation equilibrium and amino acid analysis [49]. CBH 1(D)
was obtained in highly purified form here after elution from
DEAE-Sephadex at pH 3.6, as determined by native polyacrylamide qel
electrophoresis (Figs. 1 and 8). Horizontal isoelectric focusing of
this material gave rise to a single band which was isoelectric at pH
4.14 +_ 0.06. Antisera oroduced against CBH 1(D) in rabbits generated
a single precipitin band after immunodiffusion against the homologous
protein, but had no cross-reactivity at all to CBH II or to any of the
endogl ucanases tested. An experimentally determined extinction
coefficient of 13.80 +_ 0.20 (for a 1% solution at 280 nm) was used
in each calculation and pure CBH 1(D) was found to have an
A280^A260 ratl 1*82-1.84.
Neutral Carbohydrate Composition--The carbohydrate was removed
from CBH 1(D) either by reductive (3-elimination to release 0-linked
oligosaccharides or by mild acid hydrolysis to release all sugars as


FIGURE 8
Polyacrylamide disc qel electrophoresis of crude
extracellular protein prepared from T. reesei
QM 9414 and highly purified cellobiohydrolase 1(D).
Lanes 1 and 2 contain 100 pq each of crude extracellular
protein that was apDlied to the DEAE-Sephadex column. Lanes
3 and 4 represent 40 pq each of highly purified CBH 1(D)
(from a pool of fractions 600-620) eluted from DEAE-Sephadex
at pH 3.6 (see Fig. 1). Lanes 1 and 3 were stained with
Coomassie Blue for protein and lanes 2 and 4 were stained
with the periodic acid-Schiff reagent for carbohydrate.




59
monosaccharides. The products were converted to the respective
per acetyl ated alditols and aldononitriles and analyzed by gas-liquid
chromatography (see Table II). The protein was found to contain about
13 mannosyl and 5 glucosyl residues which account for 6.0% of the 54
Kdalton molecular weight. This composition agrees well with previous
data (Table I, [49]), even though protein was determined by the Lowry
colorimetric assay and not the extinction coefficient, as in the case
of the present work. This also agrees well with a value of 5.8 weight
percent carbohydrate obtained from phenol-sulfuric acid determination
using mannose as a standard.
Analysis of the products released by reductive 13-elimination as
both the alditol acetates (derived from sugars directly attached to
the protein) and aldononitrile acetates provided information as to the
total carbohydrate composition, the number of 0^-1 inked attachment
sites to the protein and the type of sugar attached (Table II). For
CBH 1(D), mannose (identified as peracetylated mannitol) was the only
0^-linked saccharide found to be attached to the Drotein and at an
average of 5.9 +_ 0.6 sites. The fact that the total number of
neutral sugars recovered from either reductive 6-elimination or acid
hydrolysis were approximately the same suggests that all the neutral
carbohydrate was alkal i -1 abi 1 e (i.e. 0 linked). This ruled out the
possibility of large N_-linked structures, common in many
glycoproteins, as they are stable under the mild alkaline conditions
used.
0-1 i nked 01 igosaccharides--0-linked oligosaccharides
released from CBH 1(D) by reductive 6-elimination were separated by


60
TABLE II
NEUTRAL CARBOHYDRATE COMPOSITION OF
CELLOBIOHYDROLASE 1(D)
CBH 1(D) was treated with either mild acid or alkaline borohydride
and the products analyzed either as a mixture of PAANs and PAAs or, after
reduction with sodium borohydride, as the PAAs. For further details see
"Experimental Procedures.11 Values are expressed in moles/mole protein.
Peracetylated Alditols
and Aldononitri les
Method of Carbohydrate Release
Acid Hydrolysis Alkaline Borohydride
Mannononitrile 15.2 + 1.0
Glucononitrile 5.9 + 0.5
Mannitol
6.2 + 0.6
5.2 + 0.8
5.9 + 0.6
12.8 + 0.5
5.3 + 0.5
Mannitol
Glucitol
13.8 + 0.5
4.9 + 0.3


FIGURE 9
Separation on a Biogel P-2 column of oligosaccharides
released from cellobiohydrolase 1(D) by reductive
13-elimination.
200 mo CBH 1(D) was [3-eliminated with 0.1M NaOH and 0.3 M
NaBH. at 40C for 48 hours. The reaction was stopped
with42N acetic acid and the mixture taken to dryness.
Following methanol evaporation, the reduced oligosaccharides
were deionized over Amberlite MB-3, lyophi 11 ized and then
applied to a Biogel P-2 column. For further details see
"Experimental Procedures". Peaks labelled M, M M_G
and M G correspond to Mono-, di-, tri-aird
tetrasacCharides, respectively.


62
1 1 1 1
80 100 120
ELUTION TIME (MIN)


63
gel filtration on Biogel P-2 (Fig. 9), purified by rechromatography on
the same column and weighed (Table III). Analysis of the eluate
permitted an estimate of an average of 4.2 chains of trisaccharide,
1.1-1.2 of the di- and monosaccharides and 0.7 chains of
tetrasaccharide per molecule of glycoprotein; calculations were made
on the assumption that each oligosaccharide chain has one attachment
site to the protein. These account for about 7.2 oligosaccharide
chains 0^-linked to CBH 1(D) which is in fair agreement with an
estimate of 5.9 +_ 0.6 attachment sites obtained by gas
chromatography data (Table II). The 5.9% total neutral carbohydrate
found also agrees well with previous evidence from gas chromatography
(6%) and colorimetric determination (5.8%).
Analysis of the composition of each of the oligosaccharides by
gas-liquid chromatography (see Table IV) provided the first evidence
that these may be a related series. All contain mannose (as mannitol)
at the reducing terminus, and the tri- and tetrasaccharides were also
found to contain one residue of glucose. Since the peak shapes
generated from Biogel P-2 separation were symmetrical and the
stoichiometry of the gas chromatographic analysis of the di- and
tr i s acch ar i des yielded nearly integral values for each residue
indicating that each oligomer comprises one species, and is not,
therefore, a heterogeneous population. These results further
demonstrated the usefulness of the mixed alditol/aldononitrile acetate
technique in determining not only the linking sugars, but also the
composition of each oligosaccharide.


64
TABLE III
OLIGOSACCHARIDES RELEASED BY PREPARATIVE
[3-ELIMINATION OF CELLOBIOHYDROLASE 1(D)
Oligosaccharides released by reductive [3-elimination of CBH 1(D) were
separated on Biogel P-2 (see Fig. 9), pooled and lyophi 11 ized. Samples
were then dried over P ? 0 r and weighed. Calculations were made
assuming a molecular weight for CBH 1(D) of 54,000 daltons [49].
01igosaccharide
Weight Percent
of Carbohydrate
Weight Percent
of Glycoprotein
Moles per
Mole CBH 1(D)
Tetra-
13.8
0.8
0.7
Tri-
66.9
4.0
4.2
Di-
12.1
0.7
1.1
Mono-
7.1
0.4
1.2
5.9%
7.2


65
TABLE IV
COMPOSITION OF OLIGOSACCHARIDES RELEASED FROM
CELLOBIOHYDROLASE 1(D) BY REDUCTIVE 8-ELIMINATION
Reduced oligosaccharides, purified on Biogel P-2, were hydrolyzed with
mild acid and the products analyzed by gas-liquid chromatography as a
mixture of the alditol and aldononitrile acetates. For further details,
see "Experimental Procedures".
Peracetylated
Derivative
Saccharide
Mono-
Di-
Tri-
Tetra-
Mannitola
1.0
1.0
1.0
1.0
Mannononitri1e
-
1.2
1.1
1.5
Glucononitri1e
-
-
1.2
0.9
mannitol hexaacetate has been normalized to 1.0 assuming one reducing
end per oligosaccharide chain prior to B-elimination.


66
Methylation An a 1 y s i s--The types of linkages between the sugar
residues in the oligosaccharides attached to CBH 1(D) were studied by
methylation analysis. The partially methylated alditol acetates
generated were subsequently separated and identified by gas
chromatography/mass spectrometry (see Fig. 10).
The intact glycoprotein yielded three methylated species
corresponding to the 2,3,4,6-1etramethy1, 3,4,6-tr imethyl and
2,3,^-trimethyl alditol acetates which indicated the presence of
non-reducing terminal, 2-substi tuted and 6-substi tuted hexoses,
respectively (Fig. 10,Panel A). No dimethyl hexitol acetates were
observed indicating that all oligosaccharide chains are unbranched.
The ratio of the 3,4,6-/2,3,4-trimethyl species is 0.85, which agrees
well with the ratio expected (0.83) from previously determined amounts
of tri- and disaccharide attached to CBH 1(D) (see Table III). The
disaccharide yielded two partially methylated alditol acetates
corresponding to the 1, 3,4,5,6-pentamethyl and 2,3,4,6-tetramethyl
species indicating a 1-2 linked mannobiitol (Fig. 10,Panel B).
Analysis of the reduced tr i s ac c h ar i d e also reveals three peaks
corresponding to the 1,3,4,5,6-pentamethyl, 2,3,4,6-tetramethyl and
2,3,4-tr imethyl hexitol acetates (Fig. 10,Panel C); these indicated
the presence of 2-subst i tuted (reducing end), non-reducing end and
6-substi tuted residues, respectively. The tetrasaccharide generated
four peaks corresponding to the 1, 3,4,5,6-pen t ame t hy 1 ,
2,3,4,6-tetramethyl, 3,4,6- and 2,3,4-trimethyl hexitol acetates (Fig.
10,Panel D); these would be expected to arise from 2-substi tuted
alcohol (reducing-end), non-reducing end, 2-substituted and
6-substi tuted residues, respectively. The absence of the formation of


FIGURE 10
Methylation analyses of cellobiohydrolase 1(D) and
the oligosaccharides released from cellobiohydrolase
1(D) by reductive B-elimination.
G.C./M.S. profiles of partially methylated alditol acetates
generated from (A) CBH 1(D) (B) reduced disaccharide, (C)
reduced tr i s acch ar i d e and (D) reduced tetrasaccharide.
Individual peaks were positively identified by their
electron-impact spectra compared with prepared standards for
further details, see "Experimental Procedures". Arrows
indicate elution of standards: 1. 2-0-acetyl-1,3,4,5,6-
penta-O-methyl glucitol; 2. 1, 5-di-0_-acetyl-2 ,3,4,6-
tetra-fr-methy1 mannitol; 3. 1, 2,5 tr i-0_- acetyl -3,4,6-
tri-0_-methyl mannitol; 4. 1, 5,6-tr i-0_-acety 1 -2,3,4-
tri-0-methyl mannitol.


RELATIVE INTENSITY
68


69
dimethyl species from any of the oligosaccharides suggested that none
of the chains are branched.
Electron impact spectra of all the partially methylated alditol
acetates were compared with those prepared standards (Appendix C) and
examples of those from the tetrasaccharide (Fig. 10,Panel D) are shown
(Fig. 11). Glue i to! and mannitol peracetates methylated in the same
positions coeluted on 1% 0V-225 and also gave identical
electron-impact fragmentation patterns (data not shown). The
methylation profiles, in conjunction with the evidence from
glycosidase digestions (see below), suggest a series of related
oligosaccharides corresponding to Man(l-2)mannitol and
G1 c (1-6)Man(l-2)mannitol for the di- and trisaccharides, respectively.
The data for the tetrasaccharide can be interpreted as either a
Man (1-2)Glc(l-6)Man(1-2)mannitol or a Man(l-6)Glc(l-2)Man(l-2)mannitol
oligosaccharide. Evidence from acetolysis (shown below) will support
the former structure.
Glycosidase Diqestions--Q1iqosaccharides released from CBH
1(D) by reductive B-elimination were subjected to sequential
glycosidase digestion to determine the sequence of residues and the
anomeric nature of glycosidic bonds (see Fig. 12). Each of the
glycosidases were assayed for contaminating glycosidase activity using
the appropriate £_-n i tr ophenyl glycosides. a-G1 ucosidase from yeast
was found to have minor levels of other glycosidase activities
(expressed as percent of a-qlucosidase activity) as follows:
- 5 A
B-glucosidase (7.9 x 10 %), a-mannosidase (5.6 x 10 %) and
-5
B-man nos i d as e (4.7 x 10 %). The a-mannosidase from jack bean also


FIGURE 11
Comparison of electron impact spectra obtained from
partially methylated alditol acetates of the
tetrasaccharide from cellobiohydrolase 1(D) with
standards.
Panels (A), (B), (C) and (D) are spectra taken from the four
peaks in Figure 6, Panel (D). Panel (E) is a standard
pentamethyl alditol acetate from derivatization of
kojibiitol, and represents 2-0-acetyl-1,3,4,5,6-penta-0-
methyl glucitol. Panels (F), (G) and (H) are standards taken
from the partially methylated alditol acetates of yeast
mannan, and represent 1, 5-d i-0_- ace t.yl-2,3,4,6-tetra
d-methyl mannitol, 1, 2,5 -1r i-0_-acety 1-3,4,6 tr i-
0_- methyl mannitol and l,5,6-tri-0_-acetyl-2,3,4-
tr i-0_-m ethyl mannitol, respectively. The fragmentation
pattern of the pairs (A) and (E), (B) and (F), (C) and (G),
(D) and (H) were compared for positive identification.


RELATIVE INTENSITY
71
MASS ION


FIGURE 12
HPLC separation of the products of sequential
glycosidase digestion of oligosaccharides released
from cellobiohydrolase 1(D) by reductive
6-elimination.
Shown are products of reduced oligosaccharides from (A) CBH
1(D) tetrasaccharide + a-mannosidase, (B) trisaccharide from
(A) + a-glucosidase, (C) disaccharide from (B) +
a-mannosidase, (D) CBH 1(D) trisaccharide + a-glucosidase,
(E) disaccharide from (D) + a-mannosidase and (F) CBH 1(D)
di saccharide + a-mannosidase. Elution times of standards are
shown: 1. mannose (glucose); 2. mannitol; 3. CBH 1(D)
reduced di saccharide; 4. CBH 1(D) reduced trisaccharide; 5.
CBH 1(D) reduced tetrasaccharide. Separations were performed
as described in "Experimental Procedures".


73
O 10 20 30 40
ELUTION TIME (MIN)


74
was found to have minor contaminating activities of B-glucosidase
(5.8 x 10^%), B-mannos i d ase (2.2 x 10~^%) and a-glucosidase (2.8
-4
x 10 %). These levels of contaminating activities are so low that
they would not be expected to be a substantial influence, unless the
incubations were for a long period of time and/or with large amounts
of enzyme.
The products of digestion were separated by polar reverse-phase
HPLC. The monosaccharides released were converted to peracetylated
alditols and/or al donon i tr i 1 es and were identified by gas-liquid
chromatography; subsequently the oligosaccharides were degraded with
an alternate glycosidase. All monosaccharides released with
ot-gl ucos i dase (Fig. 12,Panels B and D) were identified as the
per acetyl ated g 1 u con on i t r i 1 e The trisaccharide(s) was not a good
substrate for the yeast a-glucosidase and so more units of this enzyme
were necessary than in the case of the a-mannosidase. Consequently,
small amounts of mannose and mannitol were produced by the minor
a-mannosidase contamination. It should be mentioned that digestion of
the tri saccharide with B-gl ucosidase from Trichoderma did not cause
the release of glucose, indicating that the non-reducing terminal
glucose residue is not B-linked. All non-reducing terminal mannose
residues, however, were readily cleaved by a-mannosidase (Fig.
12,Panels A,C,E and F) and were identified as the mannononitrile
acetates. Recovery of the products after each digestion and the HPLC
chromatography step was about 85% in each case.
From the HPLC profiles, it was evident that all the glycosidic
linkages are in the a-configuration. The sequence of the tetra- (Fig.
12,Panels A, B and C), tri- (Fiq. 12,Panels D and E) and disaccharides


75
(Fig. 12,Panel F) was proposed to be ManaG1caManamann i to 1,
GlcctManamannitol and Manamannitol, respectively.
Acetolysi s--The reduced tri- and tetrasaccharide from CBH 1(D)
were each subjected to acetolysis in order to confirm the position of
(1-6) glycosidic linkages within the oligosaccharide chains (see
Experimental Procedures). After acetylation, oligosaccharides have
been shown to be di f ferentially susceptible to hydrolytic cleavage
depending on the nature of the glycosidic linkages. The rate of
cleavage of acetylated a-linked mannooliaosaccharides has been shown
to be (l-6)(l-3)>(l-2) [71], thus under controlled conditions, (1-6)
glycosidic bonds may be hydrolyzed preferentially.
The products of the reaction were separated by HPLC (see Fiq.
13) and then further analyzed as the peracetylated alditols and/or
a 1 d onon i t r i 1 es by gas-liquid chromatography. The trisaccharide was
cleaved into two products, a monosaccharide coeluting with a glucose
standard and reduced di saccharide coeluting with authentic reduced
di saccharide from CBH 1(D) (Fig. 13,Panel A). The monosaccharide was
converted to the peracetylated glucononitri le and the disaccharide
into a 1:1 mixture of peracetylated mannitol and peracetylated
mannononi tri 1e. This array of products from acetolysis is consistent
with a terminal glucose attached by a (1-6) glycosidic linkage to
mannobiitol. The tetrasaccharide also was cleaved into two products,
a reduced disaccharide which coeluted during HPLC analysis into
authentic reduced disaccharide from CBH 1(D) and a (unreduced)
disaccharide (Fiq. 13,Panel B). The reduced disaccharide was
converted by hydrolysis into a 1:1 mixture of peracetylated mannitol


FIGURE 13
HPLC separation of the products of acetolysis of
oligosaccharides released from cellobiohydrolase 1(D)
by reductive 13-elimination.
(A) Reduced trisaccharide and (B) reduced tetrasaccharide
were subjected to acetolysis as described under
"Experimental Procedures". 1 and 2 refer to the elution
times of glucose and reduced disaccharide from CBH 1(D),
respectively. Separations were performed on a Whatman
Parti sil PXS 10/25 PAC column eluted with 75% acetonitrile
at a flow rate of 1.5 ml/min.


77
10 20
ELUTION TIME (MIN)


78
and peracetylated mannononitrile and the disaccharide was converted to
a 1:1 mixture of per acetyl ated mannononitrile and peracetylated
gl ucononitrile. This acetolysis cleavage pattern is consistent with a
(mannose glucose) disaccharide attached by a (1-6) glycosidic bond to
mannobiito 1.
^ ^ C N M R - Proton decoupled ^C-NMR at 75.5 MHz was
performed on CBH 1(D) and the reduced tri- and disaccharides released
from CBH 1(D) with alkaline borohydride (Fig. 14 and Table V). CBH
1(D) generated three signals in the anomeric reqion (C^) at 105.1,
102.5 and 100.8 ppm, which indicates the presence of three predominant
types of hexoses (Fig. 14,Panel A). It should be mentioned that at
this resolution, minor species were not distinguishable, so it is
assumed that these signals reflect the trisaccharide (major species)
attached to the protein. The signal at 100.8 ppm has been assigned to
the glucosyl residue (C-l") at the non-reducing end. The signal at
105.1 ppm was assigned to the central mannosyl residue (C-l1) of the
tri saccharide and, although the signal is far down field from any
known standards, it is suggested that this is due to the unique nature
of the substitution of this residue. By comparison with published
values [100], the signal at 102.5 ppm was assigned to a mannosyl
residue (C-l) a-linked to a threonyl residue on the peptide. The
strength of this signal relative to those of the other anomeric
carbons suggest that most or all of the oligosaccharides are attached
to threonyl residues, rather than to seryl residues. Allerhand et al.
[100] have demonstrated that ManaThr and ManaSer standards generate
C^ signals 1 ppm apart (at 102.8 and 101.8 ppm, respectively), so


FIGURE 14
13
Proton decoupled C-NMR spectra of
cellobiohydrolase 1(D) and the oligosaccharides
released from cellobiohydrolase 1(D) by reductive
B-elimi nation.
Spectra of (A) 400 mg CBH 1(D), (B) 10.5 mg reduced
tr i sacchar ide from ¡3-elimination of CBH 1(D) and (C) 2.0 mg
reduced disaccharide from B-elimination of CBH 1(D), were
recorded at 75.46 MHz. Chemical shifts are reported in parts
per million from internal TSP. Spectra were the result of
(A) 35712 accumulations, (B) 4304 accumulations and (C)
16328 accumulations, respectively. For further details, see
"Experimental Procedures."


80
(O


81
TABLE V
SELECTED 13C-NMR SIGNALS DUE TO CARBOHYDRATE COVALENTLY
ATTACHED TO CELLOBIOHYDROLASE 1(D) AND DUE TO PURIFIED
OLIGOSACCHARIDES RELEASED FROM THIS ENZYME BY
REDUCTIVE S-ELIMINATION
13
Proton coupled and decoupled C-NMR spectra were performed at 75.5
MHz. 200 mg CBH 1(D), 10.5 mq reduced trisaccharide and 2.0 mq reduced
di saccharide \^ere used. Chemical shifts are expressed in PPM and, in
parentheses, values are expressed in Hz. For further details,
see "Experimental Procedures".
Chemical Shifts Relative to TSP
Carbon CBH 1(D) Reduced Reduced Residue
Atom Trisaccharide Disaccharide
Anomeric Carbons
C-l
102.5 (171.1)
-
-
Man
C-l
' 105.1 (173.6)
104.6 (169.2)
104.3
Man
C-l
" 100.8 (171.5)
100.9 (170.5)
-
Glc
Hydroxymethyl
Carbons
C-l
-
66.0
66.0
Mannitol
C-6
63.9
63.9
64.0
Man
C-6
1
-
63.8
Man
C-6
" 63.4
63.4
_
Glc


82
attachment to more than one type of hydroxy amino acids would be
expected to result in separate signals, which is not the case here.
Protein folding is thought not to affect the chemical shift of
carbohydrate residues [101], as it does the chemical shift of amino
acid residues.
The above data is in agreement with sequence evidence from
Pettersson [60] which suggested that all of the 0_-linked
carbohydrate is present in a 20 amino acid region located 33 residues
from the C-terminus of CBH 1(D); the work of Shoemaker et al. [61],
who have sequenced the gene for the enzyme, suggests that the region
contains 8 threonyl and 3 seryl residues. This is also in agreement
with amino acid analysis of the polypeptide from which the
carbohydrate had been removed by 8-elimination (see below).
Comparison of the CBH 1(D) anomeric carbon signals with those of
the reduced tri saccharide shows the loss of the signal at 102.5 ppm
(Fig. 14,Panel B). This was expected as the linking sugar has now been
reduced and thus has no anomeric carbon atoms. This further confirms
that this signal is due to the mannosyl residue attached to the
protein. The signals at 105.1 ppm (C-l1 ) has been shifted to 104.6
ppm, suggesting this mannosyl residue is close to the reducing end.
The signal at 100.9 ppm is unchanged, suggesting that this glucosyl
residue is furthest removed from the reducing end. This data is in
agreement with the chemical evidence of the sequence of sugars in the
tri saccharide (see above). Spectra of the reduced disaccharide reveal
a single signal at 104.3 ppm (C-l1) due to the non-reducing terminal
mannosyl residue (Fig. 14,Panel C). Comparison of the signals in the
hydroxymethyl region (63-66 ppm), indicates that the carbohydrate of


83
intact CBH 1(D) (Fig. 14,Panel A) generates two signals. Thus the
tr i s aechar i de, as it is attached to the protein, has only two
hydroxymethyl groups and must contain one 6-substituted residue. The
reduced trisaccharide reveals three signals, and thus it must contain
one 6-substituted residue (Fig. 14,Panel B). The reduced disaccharide
also gives rise to three signals, which suggests that it cannot
contain a 6-substituted residue (Fig. 14,Panel C).
13
Coupled C-NMR spectra performed on both CBH 1(D) and the
reduced trisaccharide, provide coupling constants for anomeric carbons
and yield information on the nature of those carbons. All
^J^-values (Table V) for anomeric carbons of both CBH 1(D) and
the reduced tri saccharide were between 169.2-173.6 Hz indicative of
a-linked sugar residues (3-linkages generate J-values of about 160
Hz), in agreement with the results of the glycosidase digestion (see
above).
^H-NMR--The ^H-NMR signals of anomeric protons at 300 MHz
of the reduced oligosaccharides 3-eliminated from CBH 1(D) are shown
13
(Fig. 15). As with the C-NMR, the reducing end residues do not
contain anomeric carbons and do not generate signals in this region.
At this resolution, with a 300 MHz instrument, distinction between
signals separated by less than 2 Hz was not possible and thus it was
not possible to distinguish between a- and 3-anomers of mannose by
these SDectra. Signal assignments are shown in Table VI. The glucose
signals generated from the tri- and tetrasaccharides are split by 3.5
Hz and 3.4 Hz, respectively, due to the equatorial proton at C^ and
the axial proton at which indicates that both residues are


FIGURE 15
Proton-NMR spectra at 300 MHz of oligosaccharides
released from cellobiohydrolase 1(D) by reductive
13-el imi nation.
Spectra were generated from (A) 2.0 mg reduced disaccharide,
(B) 10.5 mg reduced tri saccharide and (C) 700pg reduced
t e t r as acc h ar i d e in D0. All spectra were recorded at
25C and were the result of 200-300 accumulations. Spectra
at 70C revealed no signals hidden" under the large HOD
peak at 4.771 ppm. For further details see Experimental
Procedures".


PPM
(C)
co
en
5.0
4.8
4.6


86
TABLE VI
^-NMR SIGNAL ASSIGNMENTS OF ANOMERIC PROTONS
OF PURIFIED OLIGOSACCHARIDES RELEASED FROM
CELLOBIOHYDROLASE 1(D) BY REDUCTIVE B-EL IMINAT ION
Oligosaccharide
Anomeric
Proton
Sugar
Chemical
Shift
(PPM)
JH-H
Anomer
Disaccharide
H'
Man
4.953
<2.0
a or B
Trisaccharide
H'
Man
4.995
<2.0
a or 3
H"
Glc
4.969/
4.957
3.5
a
Tetrasaccharide
H'
Man
4.998
<2.0
a or 3
H"
Glc
4.916/
4.906
3.4
a
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