Enzymes of the Cellulase Complex

2 Enzymes of the Cellulase Complex K. W . K I N G

Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch002

Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, Blacksburg, Va. 24061 M A H M O O D I. VESSAL Pahlavi University, Shirz, Iran The shortcomings of cellulose itself, and of cellulose deriva tives as substrates for studying the mechanism of cellulase action are pointed out. The oligosacchardies—prepared

with

a radioactive, or chemical label—are useful in determining the bond preference of enzymes and hence whether a com ponent is of the endo-β-1 --> 4 glucanase or exo-β-1 --> 4 glucanase type.

' T p h e first part of this paper w i l l be concerned with an experimental approach to the cellulase complex which involves (1) the effect of supramolecular structure on activity of cellulolytic systems, (2) the effect of chemical modification of cellulose on the activity of cellulase, and (3) the use of oligosaccharides i n determination of the mode of action of cellulolytic components. A

Effect of Supramolecular Structure on Activity of Cellulolytic Systems. A n earlier review (8) dealt with the general problem of deter mining the position at which cellulases attack their substrates. A number of possible approaches were considered, and some preliminary data from application of certain of these approaches were presented. In the interim we have been able to extend/these mode of action or site-of-attack studies to some new highly purified enzyme preparations. In addition the tech niques have been used in studying the means by which Cellvibrio gilvus conserves the glucosyl bond energy of cellulosic substrates that are being used as energy sources. This discussion is largely limited to our own data, not because we are unaware of the excellent work of others i n this field, but because it is extremely difficult to correlate it with what we have found i n the organisms we have used. Present address: Research Corp., 405 Lexington Ave., New York, N. Y. 10017. 7 In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.


8

CELLULASES A N D THEIR APPLICATIONS

For purposes of discussion we can divide the several alternative approaches into those employing long chain substrates as distinct from those using relatively short oligosaccharides. The difference is really more than a matter of size alone for invariably the long chain substrates are also characterized by heterogeneiety in molecular size. In addition, they involve either molecular and supramolecular architectural features that are of enzymatic significance as in the case of the native celluloses, or steric and electrostatic features that modify enzymatic action as i n the case of the water soluble derivatives. These two characteristics of the high D P substrates introduce elements of uncertainty into their use to elucidate the site of action of cellulases which are avoided by use of the lower D P water soluble oligosaccharides. It is, therefore, our inclination to use the more precise, analytically quantitative procedures which the oligosaccharides make possible. That such features of cellulose as the crystal lattice form are significant determinants of cellulase action has only recently been established (19), although a great deal remains to be learned about the enzymatic importance of cellulose fine structure. It is clearly established, however, that each of the three water-stable crystal forms of cellulose is distinct in the rate at which it is hydrolyzed and in its properties as an inducer of cellulase. For example the Trichoderma viride cellulase from culture extracts exhibits a lower activation energy in attacking the crystal lattice form used in culture growth, than in attacking the other lattice forms (Table I ) . Table I. Activation Energy (calories/mole) in Degradation of Celluloses of Differing Crystal Lattice by Enzymes Produced on the Two Water-stable Lattices , Lattice Used to Produce Enzyme I II

Lattice Form of Substrate I

II

3270 6190

6940 5430

In addition the degradation of lattice I and II substrates follows Schutz kinetics quite closely while the kinetics of degradation of lattice I V cannot be identified (19). There is also real uncertainty about what reaction is being catalyzed by Ci-components in general. Several lines of evidence suggest that the Ci-reaction on hydrocellulose may not be a hydrolytic event at all. The fragmentation process releasing tiny micelles (11,16) may involve simply a cleavage of the intermolecular hydrogen bonding system. The reaction rate is essentially unaffected by substitution of D 0 for H 0 in the reaction system (10), an observation that is highly improbable for a hydro2

2

In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.


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Cellulose Complex

9

lytic reaction. The activation energy of the C i reaction has been observed to be as low as 3000 calories/mole (19), a value more i n line with hydrogen bond cleavage than with hydrolysis, and the bonds that must be broken i n order to disaggregate the micelles of native cellulose are hydrogen bonds not glycosidic bonds. Should the Ci-reaction prove in fact to be non-hydrolytic i n nature, the whole question of site of action would be altered radically. W i t h crystalline substrates another factor contributing to uncertainty regarding the site of action can be seen from study of models. Figure 1 represents a Hirschfelder model of a glucosyl, a cellobiosyl, a celloheptaosyl, and an oligosaccharide fragment of D P = 14. A l l are i n the conformation of the dominant natural crystal lattice I form. The black atoms are carbon; the greys are hydrogen; and the white atoms are oxygen. The glycosyl bonds are easily located by the oxygen pairs lying very close together representing the glucosyl oxygens and the ring oxygens. Close study of the oligosaccharide chains shows that only alternate glucosyl bonds are exposed to attack by enzyme, the other glucosyls being heavily masked and facing the opposite direction, into the body of the micelle. Attack by enzymes onto the mass of such molecules that a crystal lattice represents would therefore be restricted to exposed glucosyl bonds, freeing fragments of D P = 2, 4, 6, 8, etc. These fragments would still be held i n the crystal lattice by hydrogen bonds, two such bonds per glucosyl. Kinetic energy sufficient to break four hydrogen bonds would be needed to free a cellobiosyl into solution. Freeing a tetrasaccharide would involve rupturing eight hydrogen bonds. Considering i n addition the increasing mass as the fragment becomes longer, it is evident that a completely non-discriminating random glucanase acting on a crystalline substrate would yield more than twice as much cellobiose as cellotetraose, and the other oligosaccharides would be freed i n even lesser amounts. Such a system could give all the appearances of an endwise acting glucanase producing essentially only cellobiose as a reaction product. For this reason the exclusive production of cellobiose from attack on such substrates cannot legitimately be interpreted as evidence of endwise attack. Inherently, then, the nature of the substrate invalidates an experimental approach to cellulase studies which is perfectly valid with the amylases and many other enzymes attacking water soluble polysaccharides. The compactness and rigid intra- and intermolecular positioning of native cellulose materials also creates problems i n interpreting data on the changes i n average chain length as expressed by D P measurements and end group analysis. Rigidly endwise glucanases removing terminal glucosyls like the exo-glucanase of T. triride (14) fail to attack crystalline hydrocelluoses to a significant degree. Furthermore, a random acting


10



cellulase presented with crystalline substrate would not necessarily find the surface array of atoms on the crystallite correct for effective enzyme attachment at all. Problems of this type are presumably the reason for the variations i n activation energy, thermal stability, and reaction rate of the various cellulase components of T. viride (19). Clearing, then, crystalline or semi-crystalline celluloses have serious drawbacks for studies of the site of attack by cellulases.

Figure 1. Fisher-Hirschfelder models of glucose, cellulose, and oligosaccharides of 7 and 14 monomers in the boat-chair conformation found in the lattice I structure Effect of Chemical Modification of Cellulose on the Activity of Cellulase. Soluble cellulose derivatives like carboxymethyl and hydroxyethyl cellulose have an advantage in being randomly coiled, but they are not without limitations. The two basic problems are those created by


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Cellulose Complex


I

TIME

Figure 2. Changes in the intrinsic viscosity of carboxymethylcellulose solutions during hydrolysis by purified /3-glucanases of Aspergillus niger (14). Numbers refer to different cellulase components steric and electrostatic interactions with enzyme. As examples, the highly purified /?-glucanases of Aspergillus niger show quite well (11, 14) the nature of the action on carboxymethylcellulose (Figure 2 ) . The action of Component 6 is of particular interest. After a very high rate of reaction, the velocity slows down to nearly zero far short of completion. Subsequent addition of Component 4 then carries the reaction to essential completion. This behavior appears to indicate that either sterically or electrostatically most of the glucosyl bonds of the carboxymethylcellulose are protected from attack by Component 6, but Component 4 can still make effective contact. The electrostatic blocking of an enzyme component from the same organism is indicated. From the p K of the carboxymethyl group one can calculate the percent dissociation of the carboxyl groups at any given p H . If then one measures the rate of hydrolysis of carboxymethylcellulose over a range of pH's covering 0.6 to 9 0 % association of the carboxyls, the data i n Figure 3 are obtained. Over a wide range of pH's the reaction velocity is directly proportional to the log of concentration of unionized carboxyls indicating that ionization of the substrate essentially protects it from enzymatic attack. This property is also reflected in the frequent observation that the p H optima for non-ionizing native celluloses differ markedly from the p H optima for attack on carboxymethylcellulose by the same enzyme (Figure 4 ) . a


12



These steric and ionization effects seriously limit the usefulness of substituted celluloses in site-of-action studies. Further uncertainties are introduced by the fact that the substitution reactions used to solubilize cellulose distribute the substituents randomly on the polysaccharide. As a consequence the reaction products become a very large family of variously substituted fragments rather than a single product or a few identifiable oligosaccharides.

*

REACTION VELOCITY Figure 3. Enzymatic hydrolysis of carboxymethylcellulose as a function of ionization of substrate carboxyls

3

4

5

6

7

8

PH

Figure 4. Effect of pH on enzymatic hydrolysis of charged and uncharged cellulosic substrates (12), CMC = Carboxymethylcellulose



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Cellulase Complex

13

Some progress has been possible using carboxymethylcellulose and similar soluble derivatives by relating change i n intrinsic viscosity, a parameter related to chain length, to changes in end groups during the course of enzymatic hydrolysis (23). Random cleaving endoglucanases from Myrothecium verrucaria and mineral acids catalyze a hydrolysis in which the log of the increment in end groups is proportional to change in average chain length (Figure 5). In contrast the endwise cleaving cellulases of Cellvibrio gilvus yielded data showing a linear relationship between the log of the change in chain length to the log of the change i n number of end groups (Figure 6). The use of the term "endwise-cleaving cellulases" in this sentence is for enzymes that remove cellobiose (rather than glucose units) from the chain end. This action, unreported for other cellulase systems, is analogous to that of /3-amylase, and like the latter it acts by inversion (22). A n unfortunate aspect of this approach is that a theoretical basis for these relationships has not been discovered.

CHANGE IN CHAIN LENGTH Figure 5. Relationship of change in number of end groups to change in chain length during random hydrolysis of carboxymethylcellulose by cellulase of Myrothecium verrucaria (23) The disadvantages, then, of the high molecular weight substrates in study of the mode of action of cellulases are many and derive from inherent properties of the substrate molecules themselves. But in spite of the obvious disadvantages, modified celluloses have been useful in simple assays for determining cellulase activity. Recently, fresh attempts


14



have been made to obtain molecular activity data from viscosity measurements (2, 3 ) . Values of 29 bonds broken per second were obtained. Others have shown that the presence of a substituent on the cellulose interferes with enzyme hydrolysis at adjacent linkages (13). Hydrolysis proceeds much more rapidly where three unsubstituted glucose units occur together, than where there are only two ( 3 ) .

LOG CHANGE IN END GROUPS Figure 6. Relationship of change in chain length and change in end groups during hydrolysis of carboxymethylcellulose by exoglucanases of Cellvibrio gilvus (23) Use of Oligosaccharides in Determination of the Mode of Action of Cellulolytic Components. Broadly the oligosaccharides invite three approaches to determination of the site of action: isotopic labelling, chemical labelling, and use of unlabelled substrates. Specific labelling of cellobiose can be readily achieved by use of cellobiose phosphorylase (24). glucose- C + glucose-l-P0 ?± cellobiose- C + H P 0 14

4

14

3

4

This reaction goes very well using cellobiose phosphorylase from Cellvibrio gilvus yielding cellobiose in which only the reducing glucosyl is radioactive. W i t h the cellodextrin phosphorylase from Clostridium thermocellum it should be equally easy to prepare cellotriose, cellotetraose, etc. with the reducing terminal glucosyl labelled. Because of the good yields and the purity of the products, these procedures are far superior to the earlier ones using an Acetobacter xylinum enzyme to produce labelled oligosaccharides (9, 22).


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CeUulase Complex

Table II. Products of Hydrolysis of End Labelled Cellohexaose- C in Relation to Bond Cleaved 14

Bond Cleaved

Products

1 2 3

cellopentaose- C + glucose cellotetraose- C + cellobiose cellotriose- C + cellobiose cellobiose- C -f- cellotetraose glucose- C + cellopentaose

4


5

14

14

14

14

14

W i t h these substrates the approach is direct. Numbering the bonds of cellohexaose 1 through 5 starting from the non-reducing end, the results i n Table II are predicted. The advantage of the labelled over the unlabelled substrates is immediately apparent. Cleavage at bonds 1 and 5 or 2 and 4 yield identical products and without the labelled groups no distinction can be made. The isotopically labelled substrates then permit reliable identification of the site of attack, and where more than one bond is attacked as is usually (4, 9, 22, 23) but not always (4,11,14) the case, the relative frequencies of attack at each bond can be quantitatively desired.

FRACTION Figure 7. Analysis of normal and reduced members of the cellulose oligosaccharide series by charcoal chromatography. Components from left to right: Glucose (Gl); reduced cellobiose (G2H borohydride reduction); cellobiose (G2); reduced cellotriose (G3H); cellotriose (G3); reduced cellotetraose (G4H); cellotetraose (G4), and reduced cellopentaose (G5H)


16


Chemical labelling of oligosaccharides in our experience has been considerably simpler, less expensive, just as definitive and to date free of steric effects. Reduction of the reducing glucosyl to sorbitol gives a series of non-reducing oligosaccharides in quantitative yield (4, 9, 22) which can be separated chromatographically and analyzed quantitatively very conveniently (Figure 7). In this approach, attack on the various bonds of cellulose is indicated as follows (Table III).


Table III. Relation of Products of Hydrolysis of Reduced Cellohexaose to Bond Cleaved Bond Attacked

Products

1 2 3 4 5

glucose + tetraglucosylsorbitol cellobiose + triglucosylsorbitol cellotriose + diglucosylsorbitol cellotetraose + glucosylsorbitol cellopentaose + sorbitol

Table IV.

Relative Frequencies of Bond Attack in Hydrolysis of Cellohexaose by Acid and Enzymes Relative Frequency of Attack Bond Number

Enzyme

1

2

C. gilvus I C. gilvus II C. gilvus III C. gilvus IV A. niger I HC1 Control

0 0 0 0 1

1 1 2 3 0 1

3 2

1

4 0

1

1 1 0

0 0 1

5

0 0

1

0 0 0 0 0 1

This approach to the problem establishes the site of attack just as reliably as does the use of isotopically labelled substrates and at considerably less expense and inconvenience. The data (Table I V ) indicate that the A . niger enzyme is a typical exo-/M,4 glucanase—i.e., an enzyme which removes single glucose units from the end of the cellulose molecule. A Consideration of Enzymes Involved in the Breakdown of Cellulose The Tricboderma viride Cellulase Complex. M a n y cellulolytic organisms secrete cellulase as a multiple component enzyme system. Separation of physically distinguishable components by electrophoretic or chromatographic techniques has been repeatedly demonstrated. It is more difficult to prove that these components are distinct enzymes with different functions in terms of substrate specificity and mode—or mecha-



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Cellulase Complex

nism—of action. However, in recent years such distinctions have in fact been shown for a number of cellulase systems. Many of these cell free systems are unable to catalyze hydrolysis of crystalline cellulose to any appreciable degree and are therefore useful only as model systems. The Trichoderma viride complex is a true cellulase in the most rigid sense, being able to convert crystalline, amorphous, and chemically derived celluloses quantitatively to glucose. It has been established that (a) the system is multi-enzymatic, ( b ) that at least three enzyme components are both physically and enzymatically distinct, and (c) that all three components play essential roles in the overall process of converting cellulose to glucose. Table V . Amino Acid Composition of the Endo and Exoglucanases of Trichoderma viride Moles/mole of Protein Amino Acid Lysine Histidine Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia

Endoglucanase

b

12 9 15 74 44 40 46 21 62 46 0-1 30 2 30 36 17 18 93

Exoglw 14 7 16 65 38 46 35 34 55 50 3 25 5 21 32 18 19 130

°A molecular weight of 76,600, determined by sedimentation velocity-diffusion coefficient measurements, was used in calculation. A molecular weight of 49,000, estimated from sedimentation velocity data, was used. The presence of cystine is questionable. 6

The C i component is required for attack on crystalline cellulose. It has been considered by some workers to be a hydrolase attacking crystalline cellulose and producing cellobiose as a product (10, 11) and by others to be an enzyme of undefined nature converting crystalline cellulose to a form that can be acted on by the glucanases. The glucanase ( C ) components have the capacity to attack amorphous cellulose or x


18


cellulose derivatives, and include endo-/M-»4 glucanase(s) and exo-01 - » 4 glucanase(s). Physically they differ markedly i n adsorption characteristics, amino acid composition (Table V ) , molecular weight, and thermal stability. Enzymologically they differ only slightly in activation energies for hydrolysis of both cellulodextrins and carboxymethylcellulose, but are conspicuously different i n mode of action, K values for cellulodextrin series (Table V I ) , optimum substrate chain length, and p H optimum. One, the endoglucanase, is essentially random i n its action; the other, the exoglucanase, is endwise. This exo-£-l -> 4 glucanase is typical of other exo-glucanases (a) i n removing single glucose units from the non-reducing end of the cellulose chain, ( b ) i n its relative affinity for oligosaccharides, and (c) i n that it acts by inversion—i.e., it produces o-glucose (Figure 8 ) . The presence of both types of glucanase i n filtrates from a single organism was also observed with A. niger.


m

Table VI. Michaelis Constants for Cellulose Polymer Series: Endo and Exoglucanases of Trichoderma viride Michaelis Constants Substrate

Endoglucanase

Exoglucanase

Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose

190 X 10" Af 31 X 10" M 28 X 10" M 7.0 X 10" M 1.0 X 10" M°

220 X 10" Af ° 18 X 10~ M 6.5 X 10" M° 6.0 X 10" M 16.0 X lO^Af

4

a

4

4

4

4

5

5

B

5

5

0

° These Lineweaver-Burk plots showed apparent substrate inhibition at concentrations greater than 0.05M. To develop a clear understanding of the role of the three components in degradation, the attack on both crystalline and amorphous substrates with ultimately complete conversion to glucose must be investigated. Attack on the crystalline regions may be attributed to the C i , the primary product (97%) in our work being cellobiose, at the present stage of purification of that enzyme (11-fold over the crude). Because the endoenzyme has a greater apparent affinity for substrates of long chain length than the exoglucanase, the endo-glucanase must be assigned the dominant role of initiating the attack on "amorphous" substrate. Here the major products are cellulodextrins and a trace of glucose. The exo-glucanase, then, completes the hydrolysis of the products to glucose. The relation of the roles of the endoglucanase and exoglucanase can be looked at as representing generation of new chain ends by the random acting endoglucanase, thus increasing the effective concentration of substrate for the endwise-acting glucogenic exoglucanase.



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Cellulose Complex

20

30 40 MINUTES

19

50

Figure 8. Changes in optical rotation during hydrolysis of reduced cellotetraose by the exoglucanase of T. viride, and after basecatalyzed mutarotation (14) The system as a whole is therefore not merely multi-enzymic b y happenstance, but necessarily so for both maximum rate of attack on native cellulose and for maximum completeness of conversion to glucose. Metabolism of Cellulose Oligosaccharides by Cellvibrio gilvus. In contrast to the cellulase complex of T. viride which forms glucose as the principal product of hydrolysis, the hydrolysis of cellulose i n Cellvibrio gilvus results i n the formation of chiefly cellobiose and cellotriose (Table I V ) . F r o m our studies on the metabolism of these oligosaccharides the following picture has emerged. C . gilvus grows well on cellulose and on oligosaccharides but poorly on glucose, a phenomenon quite common among polysaccharide-using bacteria and never explained metabolically. Analysis of fermentation products (6, 7) and metabolic load studies indicated that glucose and cellobiose were differently metabolized, and phosphorolysis (7) of cellobiose was demonstrated. Different metabolic routes for the two halves of cellobiose were predicted (7) and later demonstrated (24). The extracellular cellulase system was found to consist of at least 4 C components of primarily endwise action (4, 22, 23) and a C i component, the major extracellular products being cellobiose and cellotriose. Later it was found that the cells took up all members of the cellulose oligosaccharide family x


20



by an active transport mechanism at rates such that the cells were supplied with about 40 million glucosyls/cell/minute (20) and that the metabolic efficiency with which the oligosaccharides were used increased with chain length at a rate suggesting conservation of glucosyl bond energy. The obvious first hypothesis for explaining this phenomenon is that the oligosaccharides are cleaved phosphorolytically as appears to be true for Clostridium thermocellum (21). This suggestion, however, is ruled out by the data (Table V I I ) showing that there is intracellular hydrolysis of cellotriose and phosphorolysis only of cellobiose (25). Table VII. Fractionation of Phosphorylase and Hydrolase Activities in Cell-free Extracts of Cellvibrio gilvus Hydrolysis (fimoles/hr.) (NHJ S0 g

4

Precipitate

Crude Enzyme 0-20 % Satn. 20-40 % Satn. 40-50 % Satn. 50-60 % Satn. 60-65 % Satn. 65-75 % Satn.

Phosphorolysis (^moles/hr.)

Cellobiose

Cellotriose

Cellobiose

Cellotriose

0.66 0 0.16 0 0.22 0 0

1.44 0 0 0 0.36 0.20 0

3.44 0 1.36 1.66 0 0 0

1.80 0 0 0 0 0 0

Thus, the "apparent" phosphorolysis of cellotriose i n the crude cell extracts is i n reality the result of hydrolysis of the trisaccharide followed by phosphorolysis of the resulting cellobiose. H o w then is the energy of the glucosyl bonds i n oligosaccharides to be conserved? There is an active "glucotransferase" located somewhere in the wall of the cell which transfers non-reducing glucosyls from any of the oligosaccharides to any other but apparently not to water so that starting with any one oligosaccharide a l l of the others are produced without appreciable reduction in the total number of glucosyl bonds through hydrolysis. F o r example, with cellotriose or cellotetraose as substrates prolonged incubation shows slight if any hydrolysis (Table VIII). A t the same time chemical analyses of this same experimental system shows active transglucosylation (Figure 9 ) . Glucose is the preferential acceptor, the moles of cellobiose far exceeding the value anticipated from purely random transglucosylation. The transfer reactions follow second order kinetics as expected and when physiological concentrations of oligosaccharides—i.e., those used in growth experiments—are used the transfer product has been characterized as cellobiose. A t very low concentrations of oligosaccharide laminaribiose is produced (perhaps indicating non-specific transfer).


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Cellulase Complex

Table VIII. Retention of Glucosyl Bonds During Action of Glucotransferase of C . gilvus on Cellotriose and Cellotetraose


Total Glucosyl Bonds Incubations, hrs.

Cellotriose (^moles)

Cellotetraose (fimoles)

0 1 2 3 4

5.40 5.19 5.10 5.10 4.93

4.47 4.43 4.33 4.27 4.37

Table IX.

Characteristics of Cellulose Oligosaccharides as Donors to Glucose

Donor

M X I0"

Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose

1.16 1.18 1.19 0.36 —

V fig/ml./min.

Relative Rate as Donor

m

f

167 38 21 8 —

5 25 28 30 36

Table X . Agreement of Experimental Data with Those Predicted by Proposed Sequence of Reactions /A moles of Cellobiose Incubations, hrs.

Theoretical

Experimental

1 2 3 4

0.40 0.66 0.80 0.95

0.34 0.62 0.81 0.94

Some further characteristics of the oligosaccharides as donors to glucose are summarized (Table I X ) . Although the maximum velocity falls rapidly as the chain length increases, the relative rates of donation at physiological concentrations are of the same order to magnitude. When one assumes the occurrence of the following reactions: G + G ?± G x

x

x + 1

G + H 0-*G _ x

2

x

1

+ G _! x

+ G

G + Gj ^ G _ j + G x

x

G + P, ^ G-l-P + G 2

1

2

x



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C E L L U L A S E S A N D THEIR APPLICATIONS

> TIME Figure 9. Course of transglucosylation of cellotriose by cell-free extracts of Cellvibrio gilvus (See legend to Figure 7) it is possible to predict the amount of each product to be anticipated during the initial phases of the reaction and to check these experimentally. W h e n such an experiment is performed with cellotriose as substrate the prediction of the cellobiose (or other oligosaccharide) yields is good (Table X ) . The combination of the oligosaccharide glucotransferase with the hydrolase and the cellobiose phosphorylase, then accounts fully for the conservation of the glucosyl bond energy predicted by the growth experiments. Phosphorolysis of Cellulose Oligosaccharides. Although it has not been shown that Clostridium thermocellum can freely take cellulose oligosaccharides into the cell, there is reason to believe that this organism, like C. gilvus, metabolizes these oligosaccharides intracellularly. A n enzyme, cellodextrin phosphorylase, which catalyzes the phosphorolysis of cellulose oligosaccharides, has been found in Clostridium thermocellum (21). This enzyme catalyzes the phosphorolysis of cellohexaose, cellopentaose, cellotetraose, and cellotriose, resulting i n the formation of cellobiose and glucose 1-P. Cellobiose can be broken down into glucose and glucose 1-P by cellobiose phosphorylase. A somewhat similar situation is found in protozoa where enzymes have been found that catalyze


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Cellulose Complex

The Cellulase Complex


As currently understood, the cellulase complex contains the following components (listed in the order i n which their action on cellulose occurs): 1. C i is an enzyme whose action is unspecified. It is required for the hydrolysis of highly oriented solid cellulose (Cotton, Avicel, etc.) by / M -> 4 glucanases. 2. p-1 - » 4 Glucanases ( = C ) are the hydrolytic enzymes. In his book, Eriksson (5) uses cellulase as synonymous with /M—» 4 glucanase. It is preferable to reserve the term cellulase for the C i - C complex. C is an earlier synonym which can be discarded. /3-1 —> 4 glucanase is usually measured by action on soluble cellulose derivatives, usually carboxymethylcellulose. The "x" in C emphasizes the multi-component nature of this fraction. The —» 4 glucanases are clearly of two types: (a) exo-^-1 - » 4 glucanase, successively removing single glucose units from the non-reducing end of the cellulose chain; (b) endo-/?-l-» 4 glucanases with action of a random nature, the terminal linkages generally being less susceptible to hydrolysis than internal linkages. x

x

x

x

3. /?-Glucosidases vary in their specificities. Those that act primarily on aryl-0-glucosides are not involved in cellulase action. The 0-glucosidases that are involved in cellulose breakdown are those highly active on the /?-dimers of glucose, including cellobiose. "Cellobiase" is the most descriptive designation for this group (to cellulase workers), but a more accurate name might be "0-glucodimerase," indicating the ability to act on all of the 0-dimers of glucose. /?-Glucosidases and exo-/?-l - » 4 glucanases have substrates in common, cellobiose to cellohexaose. /?-Glucosidases hydrolyze the smaller oligomers most rapidly; exo-glucanases, the larger ones. /?-Glucosidases act by retention of configuration; exo-glucanases by inversion. /?-Glucosidases are strongly inhibited by gluconolactone, exo-glucanases much less so. Finally, there is a difference in linkage specificity, the exo-enzyme generally being more specific; the 0-glucosidase less specific. Enzyme Commission numbers have been assigned to some of the cellulase components. Endo-/J-l -> 4 glucanase is E . C . 3.2.1.4. 0-glucosidase is E . C . 3.2.1.21, a designation which does not differentiate between aryl-glucosidase and dimerase (cellobiase). The e x o - / M - » 4 glucanase has sometimes been placed in E . C . 3.2.1.21, but a new number should be assigned to it. The Commission has not yet begun to think about such odd-balls as C i .


24


the phosphorolysis of laminaridextrins (17, 18). These enzymes catalyze the phosphorolysis of laminaridextrins that are formed by the intracellular hydrolysis of paramylon. The phosphorolytic reactions catalyzed b y these enzymes can be described as: (Glucose) + P i (Glucose)„_i + a-glucose 1-P. They resemble the hydrolytic exoglucanases i n that they remove one unit at a time from the nonreducing end of the glucan; and at least in the /3-series, in that they act by inversion—e.g., to a-glucose-l-P from a £-glucan. A t present, knowledge of such phosphorolytic systems are limited to a few microbial species.


n

These enzymes also resemble the exo-glucanases i n their specificity —i.e., they form, or split, a single linkage type—e.g., B - l - » 3, or B - l -> 4. Some, at least, of the phosphorylases show a preference for oligomers over dimers (20); others act more readily (or exclusively) on the dimers (like the glucosidases). Most do not act on the long polymers (laminarin, etc.) and i n this respect, they differ from exo-glucanases. Experimentally, the interesting feature of these enzymes is their synthetic action. They make it possible to attach a glucose unit to another glucose, or oligomer, by a specific linkage. The acceptor molecule may possess linkages (and even sugars) that are the same or different from that being added. A variety of products can, thus, be synthesized of predetermined linkage sequence. Such products are useful for determining specificity of hydrolytic enzymes (1, 7, 21). It should be emphasized that these phosphorolytic enzymes are intracellular. They are not found i n the extracellular cellulase complex, and they are involved i n the cellulolytic systems of relatively few microorganisms. Literature

Cited

(1) Alexander, J. K., J. Bacteriol. 81, 903 (1961). (2) Almin, K. E., Eriksson, K. E., Biochim. Biophys. Acta 139, 238 (1967). (3) Almin, K. E., Eriksson, K. E., Jansson, C., Biochim. Biophys. Acta 139, 248 (1967). (4) Cole, F . E., King, K. W., Biochim. Biophys. Acta 81, 122 (1964). (5)

Eriksson, K. E., A D V A N . C H E M . SER. 95, 83 (1969).

(6) Hulcher, F. H . , King, K. W., J. Bacteriol. 76, 565 (1958). (7) Hulcher, F . H., King, K. W., J. Bacteriol. 76, 571 (1958). (8) King, K. W . , "Advances in Enzymatic Hydrolysis of Cellulose and Related Materials," E . T . Reese, E d . , p. 159, Pergamon Press, London, England, 1963. (9) King, K. W . , Virginia Agri. Exp. Sta. Tech. Bull. (Blacksburg, Virginia) 154, 1 (1964). (10) King, K. W., J. Fermentation Technol. (Japan) 43, 79 (1965). (11) King, K. W., Biochem. Biophys. Res. Comm. 24, 295 (1966). (12) King, K. W., Smibert, R. M . , Appl. Microbiol. 11, 315 (1963). (13) Klop, W., Kooiman, P., Biochim. Biophys. Acta 99, 102 (1965).



2.

KING A N D VESSAL

Cellulose Complex

25

(14) L i ,L.H.,Flora, R. M . , King, K. W., Arch. of Biochem. Biophys. 111, 439 (1965). (15) L i ,L.H.,King, K. W., Appl. Microbiol. 11, 320 (1963). (16) Liu, T . H., King, K. W., Arch. of Biochem. Biophys. 120, 462 (1967). (17) Marechal, L. R., Biochim. Biophys. Acta 146, 417 (1967). (18) Ibid., 146, 431 (1967). (19) Rautela, G. S., King, K. W., Arch. of Biochem. Biophys. 123, 589 (1968). (20) Schafer, M . L., King, K. W., J. Bacteriol. 89, 113 (1965). (21) Sheth, K., Alexander, J. K., Biochim. Biophys. Acta 148, 808 (1967). (22) Storvick, W . O., Cole, F . E., King, K. W., Biochem. 2, 1106 (1963). (23) Storvick, W . O., King, K. W., J. of Biol. Chem. 235, 303 (1960). (24) Swisher, E . J., Storvick, W. O., King, K. W., J. Bacteriol. 88, 817 (1964). (25) Vessal, M . I., Ph.D. Dissertation, Virginia Polytechnic Institute, Blacksburg, Virginia (1967). R E C E I V E D October 14,

1968.


Enzymes of the Cellulase Complex

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