VOLUME II

PRINCIPLES OF THE ENZYMATIC DEGRADATION OF CELLULOSE

MOLECULAR MECHANISMS OF ENZYMATIC CELLULOSE DEGRADATION



109. After considering the applied aspects of biotechnology of enzymatic hydrolysis of cellulosic materials (Volume I, Sections A-F), we will give an overview of the current state of understanding of the biochemical mechanisms of enzymatic cellulose degradation. This material will likely be useful both in developing new biotechnological approaches in the field and for educational purposes.


A. BACKGROUND


110. Cellulases are the group of hydrolytic enzymes that are capable of hydrolyzing insoluble cellulose to glucose. They are produced by microorganisms, plants and animals (in the latter case by symbiotic microorganisms) usually as a cellulase system composed of several distinct enzymes. Three types of enzymes are traditionally assigned to the cellulase system: endoglucanases (endo-1,4-ß-glucanases, or 1,4-ß-D-glucan 4-glucanohydrolases, EC 3.2.1.4), cellobiohydrolases (exo-1,4-ß-glucanases, or 1,4-ß-D-glucan cellobiohydrolases, EC 3.2.1.91) and cellobiases (ß-glucosidases, or ß-D-glucoside glucohydrolases, EC 3.2.1.21). All of the principal enzymes of the cellulase system have been purified to homogeneity in numerous laboratories over the last 15-20 years.

111. Cellulolytic enzymes isolated from various sources differ in their molecular characteristics (molecular weight, amino acid composition and sequence, isoelectric point, carbohydrate content), capacity to adsorb to cellulose, catalytic activity and substrate specificity. Furthermore, cellulase components from almost any organism occur in multiple forms. All this leads to an enormous variety of cellulase systems from various sources that differ in their composition and in their catalytic characteristics for the hydrolysis of cellulose and its numerous morphological and chemical derivatives.

112. Some cellulases, particularly of bacterial origin, are known to be strongly associated with microbial cells (and apparently perform their catalytic function in vivo in an "immobilized" state), in contrast to the extensively studied extracellular fungal cellulases. Some bacterial cellulases are organized in supramolecular structures, cellulosomes. In contrast, many other multicomponent cellulase systems apparently lack spatial organization.

113. Cellulase systems often have only two or three individual components (including at least one endoglucanase), but in a number of cases thorough resolution of a cellulase system from a single source revealed 15-20 or more individual components (Hayn & Esterbauer, 1985; Knowles et al., 1987a,b; Sprey, 1988). Fungal cellobiohydrolases sometimes show a marked tendency to form aggregates with endoglucanases that are extremely difficult to break into component parts (Wood et al., 1989).

114. The appearance of multiple components of cellulase complexes can sometimes be explained by posttranslational modifications of cellulases that result from limited proteolysis during the process of recovery of cellulases from biological systems. In other cases it has been demonstrated unequivocally that the existence of several cellulases in one organism is the result of their being coded by different genes (Bhat et al., 1989a; Bhat & Wood, 1989a,b). In any case, the multicomponent nature of cellulases makes the study of their action on cellulose and elucidation of their basic mechanisms of action complicated. The fact that cellulose itself is a water insoluble polymeric material with a complex supramolecular structure that is difficult to characterize unequivocally (the physicochemical definition of size of particles, degree of polymerization, degree of crystallinity, specific surface area, etc.) makes the task even more challenging.

115. Synergism between the individual components of a cellulase system acting on insoluble cellulose adds further complexities to studying the mechanisms of action of cellulases, i.e. the action of a mixture of two or more individual cellulolytic components is greater than the sum of the action of each component. The main problem in studying this phenomenon is that the synergistic effect varies depending on which of the multiple forms of the cellulolytic components have been used in the study, the source of cellulases and the nature of the cellulose substrate (amorphous or crystalline, to mention the two extreme variants) used for the experiment. Precise quantitative parameters that correlate with these variables have not as yet been defined.

116. Glucose and cellobiose are the main products of the enzymatic hydrolysis of cellulose; the second can be considered an intermediate product of hydrolysis, but it is often present in the reaction mixture in large quantities. Some cellulolytic enzymes form cellobiose essentially as the only product of cellulose hydrolysis. The nature of glucose and cellobiose formation was historically used as one of the main criteria for the definition of cellulolytic enzymes. However, a large amount of experimental material has accumulated that suggests that the substrate specificity of cellulases relates to a broad spectrum of hydrolysis products and depends significantly on the cellulose used in the experiment, the presence in the reaction mixture of other components of the cellulase system and possibly on yet other factors that are yet unrecognized.

117. Finally, the substrate specificity of endoglucanases and cellobiohydrolases appears to overlap (Sadana & Patil, 1985; Enari & Niku-Paavola, 1987; Kyriacou et al., 1987; Biely & Markovic, 1988; Wood et al., 1988; Knowles et al., 1988; Lachke & Deshpande, 1988; Bhat et al., 1989), but the relationship of the structure and function of these components to their reaction mechanism is not known. This will only be understood when the structural and functional characteristics of their active sites are elucidated. However, published studies of that kind do not exist in spite of the hundreds of publications on cellulases that have appeared in the last few years.

118. It is now more apparent that the mode of action of fungal cellulases and bacterial cellulases differ significantly, at least in vivo. Hydrolysis of cellulose by bacterial cellulases apparently occurs at sites where contact between bacterial cells and the cellulose surface is made. This takes place at the location of structurally ordered multicomponent formations (cellulosomes), which bind to the cell wall of a bacterium and organize into catalytically active "protuberances" (Lamed & Bayer, 1986). Therefore, it may be that extracellular bacterial cellulases (detected in culture media in vastly lower quantities compared with fungal cellulases) are only "splinters" of cellulosomes and polycellulosomes. Thus, their study might only give indirect indications regarding the mechanism of the degradation of cellulose by bacterial cellulases. Cellulolytic fungi can secrete a complete cellulase system (with perhaps the exception of cellobiases, which often serve as intracellular, cell-wall bound enzymes), and the examination of such extracellular fungal cellulase systems (rather than extracellular bacterial ones) might yield more relevant data regarding the mechanisms involved in the enzymatic degradation of cellulose.

119. At first sight it might seem that a study of cellulases (a system that requires characterization both of the multicomponent cellulase complex and the cellulosic substrates) might be impractical and that this area is destined to remain an empirical collection of experimental facts, an impression reinforced by past modes of presentation of data. Indeed, many published studies appear as a mass of numbers that are difficult to interpret on a mechanistic level. However, this impression is not fully justified by the facts. A number of fundamental questions of the biochemistry and enzymology of the hydrolysis of cellulose need resolution. These questions include the following:

    (1) Why do some cellulase systems have the capacity to hydrolyze both crystalline and amorphous cellulose while others can only degrade amorphous cellulose?

    (2) Why are some cellulolytic enzymes (endoglucanases and cellobiohydrolases) synergistic with some isoenzymes but not with others, even though the substrate specificity (endoglucanases or cellobiohydrolases) may be the same for both groups of isoenzymes? Why in several cases are cellobiohydrolases synergistic with endoglucanases from some sources while not with the same type of enzyme from other sources? What is the basis of the so called "exo-exo" synergism (i.e., mutually enhanced catalytic action of two cellobiohydrolases)?

    (3) Do individual components of cellulase exist (endoglucanases or cellobiohydrolases) that are able to hydrolyze crystalline cellulose alone and, if not, why?

    (4) If the individual cellobiohydrolase indeed shows the endoglucanase type of activity towards an insoluble cellulose (there are a number of indications that this may be the case), then why does it not hydrolyze the water soluble derivative, carboxymethyl cellulose?

    (5) How can glucose be formed from cellulose under the action of an individual endoglucanase?

    (6) What are the similarities and differences in the mechanisms of cellulose hydrolysis by cellulases of bacterial and fungal origin?

    (7) Why do cellulolytic enzymes differ in their affinity for cellulose? [The range of values of the adsorption constants for different endoglucanases varies a thousand-fold or more (Rabinovich et al., 1983; Klyosov et al., 1987)]. How does this difference in the adsorption capacity reflect in the catalytic activity of the enzymes?

    (8) How do adsorbed cellulases behave on the cellulose surface? How does cellulase (endoglucanase and/or cellobiohydrolase) adsorbed to cellulose manage to cleave at multiple sites without being desorbed from the substrate's surface, or - alternatively - does a cellulolytic enzyme desorb after each scission in order to bind again at another site?

    (9) What is the basis of defibrillation (short fiber formation) of cellulose in the action of certain cellulases? What is the cause? Why do some cellulases exhibit this mechano-chemical effect on cellulose whereas others do not?

120. None of these questions is purely mechanistic. Moreover, all of them address extremely important issues whose answers promise to yield practical applications.

121. These questions have not been answered satisfactorily to date but in various forms are the subject of much discussion in numerous publications. Their resolution would be a major step forward in understanding the mechanisms not only of the enzymatic conversion of cellulose but also of the biological degradation of biopolymers and their utilization in general. Summarized below are some new ideas and experimental data that focus on answers to the questions posed above.


B. OVERVIEW


122. Many basic but unsolved problems relating to the enzymatic hydrolysis of cellulose are related to the composition of cellulase systems from various sources and to properties of their individual components. It is unclear to a large extent how the composition of cellulase systems (a set of both of their individual components and of their isoenzymes or multiple forms) determines the reactivity of the system in relation to amorphous and crystalline cellulose.

123. Still open is the question about a "minimal" composition of a cellulase system that is effective against crystalline cellulose, i.e. capable of hydrolyzing the latter rather rapidly and up to a high degree of conversion; a comment can be made, however, that in this context the term "minimal" refers not so much to the number of components but to their properties. It could be that a mixture of two cellulolytic components (e.g. an endoglucanase and a cellobiohydrolase having a certain set of properties) hydrolyzes cellulose more efficiently than a system consisting of a few dozen cellulases (also including endoglucanases and cellobiohydrolases but with different functional characteristics). That "certain set of properties" that leads to efficient hydrolysis of cellulose, and its accompanying molecular basis of action, are the principal pieces of the puzzle of what constitutes "effective" cellulases. Its elucidation will be a major step forward in understanding the biochemical mechanisms of cellulose degradation.

124. Adsorption of cellulases on cellulose provides not only physical contact between the enzyme and the substrate, but in many cases plays an important role in the efficiency of the enzymatic hydrolysis of cellulose. A recent finding that cellulolytic enzymes can be subdivided into those that are tightly and those that are loosely adsorbed (Klyosov, 1987, 1990; Klyosov et al., 1982a, 1986, 1987) seems to explain both the puzzling features of synergism in the action of cellulolytic components toward insoluble cellulose and the mechanism of the enzymatic hydrolysis of crystalline cellulose.

125. The previous hypotheses were based, however, on limited experimental evidence and cannot be considered as yet to have proven the theory. It is generally unknown, for example, why tightly adsorbed endoglucanases are significantly more reactive toward crystalline cellulose than are weakly adsorbed enzymes. Why are the differences in the efficiency of the action of tightly and weakly adsorbed endoglucanases less apparent in the hydrolysis of amorphous cellulose? These are key questions for the contemporary biochemistry and enzymology of cellulose hydrolysis. The answer to them is apparently tied to the mechano-chemical action of cellulases on crystalline cellulose and their behavior as surfactants.

126. In this report attention to these problems is focused on four areas, which essentially comprise what can be defined as molecular mechanisms of enzymatic cellulose degradation: (i) kinetics and mechanisms of enzymatic cellulose degradation, (ii) adsorption of cellulases on cellulose, (iii) behavior of adsorbed cellulases on cellulose surfaces and (iv) synergism between individual enzymatic components of the cellulase system.


C. KINETICS AND MECHANISMS OF ENZYMATIC CELLULOSE DEGRADATION


127. It was well known that cellulase systems from some biological sources can hydrolyze both amorphous and crystalline cellulose ("complete", "full-value", or "true" cellulases), whereas other cellulase systems are active only toward amorphous cellulose ("non-complete", or "low-value"). The latter are practically inactive toward crystalline cellulose (the degree of conversion does not usually exceed 2-5% and can be explained easily as reflecting the hydrolysis of the amorphous fraction of cellulose).

128. This phenomenon was a central theme of enzymology of cellulases since the early 1950s, until a quarter of a century later, when it was suggested (and proven experimentally in a number of cases) that "full-value" cellulase systems contain a cellobiohydrolase (Eriksson & Pettersson, 1975; Gow & Wood, 1988). Since then, two additional hypotheses have appeared:

    - "Full-value" cellulase systems contain an endoglucanase that adsorbs tightly on cellulose (Rabinovich et al., 1981, 1983; Klyosov, 1987, 1990; Klyosov et al., 1982a, 1986, 1987; Chernoglazov et al., 1988)
and
    - they contain an endoglucanase capable of producing glucose as one of the major products resulting from hydrolysis (Klyosov et al., 1987).

129. All three of these hypotheses are based on experimental data. However, there is no indication that these hypotheses represent alternatives. Thus, it might be that a "full-value" cellulase system of "minimal" composition could contain two enzymes only - a cellobiohydrolase with certain properties (yet to be revealed) and an endoglucanase tightly adsorbed on cellulose and producing appreciable amounts of glucose during cellulose hydrolysis. It is possible also that the biosynthesis of such cellobiohydrolase- and endoglucanase-containing "full-value" systems is coordinated somehow in certain microorganisms, which comprise a "complete" cellulase system.

130. The first hypothesis ("full-value" cellulase systems contain a cellobiohydrolase) is supported by the following experimental data: all the "complete", or "full-value" cellulase systems that have been studied so far (e.g., those from T. reesei, T. viride, T. koningii, S. pulverulentum, Penicillium pinophilum, Fusarium solani, F. lini, Sclerotium rolfsii) indeed contain cellobiohydrolases (Eriksson & Pettersson, 1975; Wood & McCrae, 1986a,b; Lachke & Deshpande, 1988; Gow & Wood, 1988; Wood et al., 1988). The cellobiohydrolases reinforce the catalytic action of endoglucanases from the same systems by interacting synergistically. This in turn leads to an increase in the overall cellulolytic activity of the system apparently rendering it "full-value".

131. However, the presence of a cellobiohydrolase in cellulase systems might not be the only criteria necessary for making it "complete". Thus, the addition of cellobiohydrolases from T. koningii or F. solani to "low-value" cellulase preparations from Myrothecium verrucaria, Stachybotrys atra, Gliocladium roseum, Memnoniella echinata, Ruminococcus albus, R. flavefaciens or Bacteroides succinogenes does not lead to synergism and to the corresponding enhancement of the cellulolytic activity of the preparations (Wood et al., 1988). The same has been observed upon addition of cellobiohydrolase I from T. reesei to the endoglucanase from a "low-value" cellulase preparation of Aspergillus niger (Lee et al., 1988). Thus, the presence of a cellobiohydrolase in a cellulase complex does not seem to be a decisive factor in making the complex "full-value". Other enzymes (or some other properties of the enzyme) are apparently necessary. The respective recent experimental data are given in Current Status of Enzymology and Genetic Engineering of Cellulases (this volume).

132. Other literature data on "kinetics and mechanisms" that are pertinent to molecular mechanisms of the enzymatic cellulose degradation are difficult to find (if, indeed, they exist at all). Data on kinetics are typically based on empirical assumptions and aim at a formal engineering description of the process. There are seemingly no data on relationships between the specificity of cellulases (in terms of soluble products/sugars formed, as glucose and/or cellobiose vs. time) and efficiency of the degradation of soluble and insoluble celluloses. Apparently, there are no quantitative data on cellulase-catalyzed transglycosylation and its effect on the efficiency of cellulose degradation and on the pattern of soluble sugars formed as a result. It is rather obvious that considering "molecular mechanisms" of cellulose degradation in the absence of such primary biochemical information would be premature. Current Status of Enzymology and Genetic Engineering of Cellulases (this volume), however, outlines some experimental approaches in this area.


D. ADSORPTION OF CELLULASES ON CELLULOSE


133. The adsorption of cellulases on cellulose provides more than the conventional physical contact between enzyme and substrate. As first shown in the early 1980s (Rabinovich et al., 1981; Rabinovich et al., 1982; Klyosov et al., 1982a, 1983) the adsorption process in many cases plays an important role in the efficiency of the ensuing enzymatic hydrolysis of cellulose, primarily crystalline cellulose. Using mainly unresolved cellulase complexes, the rate of the enzymatic hydrolysis of crystalline cellulose is often determined by the rule: the better the adsorption, the better the catalysis (Klyosov, 1986, 1987, 1988, 1990).

134. The efficiency of cellulase adsorption on the surface of cellulose is characterized by the partition coefficient (K_p) of the enzyme between the substrate surface and the water phase. K_p is numerically equal to the ratio of the quantity of the enzyme adsorbed in units of mass (conventionally expressed as 1 g) or surface (usually 1 m²) of cellulose to the concentration of the enzyme at equilibrium in the bulk solution under linear conditions of the adsorption isotherm (Rabinovich et al., 1981, 1982, 1983; Klyosov et al., 1986, 1987).

135. Adsorption equilibrium is usually reached in 1-3 min (Klyosov et al., 1982a). It was found that the adsorption capacity of endoglucanases from various sources for crystalline cellulose varies significantly, over a range greater than 1000-fold: from the very weakly adsorbed endoglucanase from Aspergillus foetidus (K_p = 0.0035 l/g) to the tightly adsorbed endoglucanase from Clostridium thermocellum (K_p = 5.5 l/g).

136. The elucidation of the structure of binding domains for some cellulolytic enzymes (Van Tilbeurgh et al., 1986; Tomme et al., 1988; Johansson et al., 1989) in part explains the difference in the adsorption capacity of cellulases. But the question remains: why do binding domains of different cellulases differ in their affinity for cellulose? One reason might reside in the carbohydrate moiety of the domains, since they have been shown to be important for adsorption of the enzymes onto cellulose (Klyosov et al., 1987; Chernoglazov et al., 1988; Bhat et al., 1989a,b).

137. According to recent data (Tikhomirov et al., 1987) in some cases there is a certain correlation between the hydrophobicity of endoglucanases and their capacity to adsorb to cellulose. These data, however, cannot be considered systematic, and their significance in general remains to be evaluated further.


E. BEHAVIOR OF CELLULASES ADSORBED ON THE CELLULOSE SURFACE


138. Published electron microscopic data show localization of cellulase clusters on the surface of cellulose (White & Brown, 1981; Chanzy et al., 1983), but these data provide little information that would define the molecular mechanisms of enzymatic degradation. To the best of our knowledge, there are no published data on the dynamic behavior of cellulases adsorbed on cellulose, except for the preliminary data of Rabinovich et al. (1984, 1985a) (see following section).

139. Although unproven as yet, there appears to be an additional characteristic property of the cellulase/cellulose system related to the adsorption properties of cellulases. This concerns the phenomenon of defibrillation, or dispersion of cellulose during the initial steps of the hydrolytic process in the action of cellulases, which has been known since the early 1960s and was described in detail by Halliwell and King (Halliwell, 1966; King, 1966; Halliwell & Riaz, 1970).

140. More recently, the first step in the enzymatic degradation of the microfibrils of cellulose was shown to be their stratification along the longitudinal axis leading to the formation of even thinner subfibrils (White & Brown, 1981; Chanzy & Henrissat, 1983; Chanzy et al., 1983).

141. There are divided opinions about which enzyme of the cellulase system is the key to the dispersion (or defibrillation) of crystalline cellulose. White & Brown (1981) considered this enzyme to be endoglucanase, while Chanzy et al. (1983) believed it to be cellobiohydrolase I. More recent relevant data are given in the Current Status of Enzymology and Genetic Engineering of Cellulases section of this volume.


F. SYNERGISM BETWEEN INDIVIDUAL ENZYMATIC COMPONENTS OF THE CELLULASE SYSTEM


142. Synergism results in an increase of the efficiency of the joint action of two or more components of a system as compared with their individual actions. For cellulose hydrolysis synergism of the enzymes involved causes the velocity of formation of soluble products and the degree of conversion of cellulose under simultaneous action of several components of the cellulase system to exceed the sum of velocities of formation of the products when each of the components acts separately.

143. Synergism is one of the most remarkable features of cellulase systems and as a rule differentiates "complete" from "non-complete" systems. On the other hand, synergism between the individual components of a cellulase system directed toward insoluble cellulose further complicates the study of the mechanisms of action of cellulases.

144. The main challenge in the study of synergism is that variations of the synergistic effect depend on which of the multiple forms of the cellulolytic components have been used in the study, the source of cellulases and on the cellulose substrate (amorphous or crystalline, to mention the two extreme variants) used for the experiment. Up to the present, quantitative parameters defining these points have not been described.

145. Synergism in cellulase systems was first described over a quarter of a century ago (Mandels & Reese, 1964) and the possible molecular mechanisms involved have remained the subject of much discussion. Until recently two types of synergism were observed: (a) synergism among endoglucanases and cellobiohydrolases and (b) synergism upon the addition of cellobiase to a cellulase complex. Synergism of the first type has usually been described as endoglucanase cleavage of cellulose polymeric chains that creates chain ends that serve as substrates for exo-cellulases, initially cellobiohydrolases. This easily explains why the separate actions of endoglucanase and cellobiohydrolase are significantly less efficient in the formation of soluble products (mainly cellobiose) than the joint action of the two enzymes.

146. Synergism of the second type is usually explained by cellobiase rapidly converting cellobiose (formed under action of two other components of the cellulase system) to glucose, thereby eliminating the product inhibition by cellobiose - which is a more powerful inhibitor of the enzymatic hydrolysis of cellulose than glucose.

147. Although the nature of synergism in cellulose systems has seemingly been explained (albeit in simplistic terms), the molecular mechanisms that are involved remain the subject of much discussion. First, it is not known why the addition of cellobiohydrolase enhances the action of endoglucanase on insoluble, particularly crystalline, cellulose (the latter enzyme supposedly initiates the hydrolysis). Neither is it understood why among all endoglucanases and cellobiohydrolases only a limited number exhibit synergism.

148. Indeed, cross-synergism between endoglucanase from one microbial source and cellobiohydrolase from another, or that between cellobiohydrolase and different endoglucanases from the same source is rarely observed (Wood & McCrae, 1986b; Wood et al., 1988, 1989; Gow & Wood, 1988). Finally, there is no simple interpretation of synergism that can explain the "exo-exo" synergism that occurs with two cellobiohydrolases and has been described for hydrolysis of insoluble cellulose (Fagerstam & Pettersson, 1980; Henrissat et al., 1985; Wood & McCrae, 1986b; Woodward et al., 1988a).


G. HOW BACTERIAL CELLULASES ACT ON CELLULOSE. PROTUBERANCES, POLYCELLULOSOMES, CELLULOSOMES AND THEIR COMPONENTS


149. It has been mentioned in the Background (section A) that bacterial cellulases are organized differently than fungal ones. This is reflected in some instances in the quantities of cellulases in cultural liquids after culturing fungal and bacterial producers. Cellulolytic fungi secrete relatively large amounts of enzyme into the culture fluids [the best fungal mutant strains produce as much as 20 g/l of extracellular cellulase protein, which far exceeds the secretion of these proteins by any other known noncellulolytic producer (Mandels, 1985)], while the rumen fluid of ruminants (cattle, moose, for example) contains extremely low amounts (3 to 4 orders of magnitude less than that of fungal culture liquids) of free extracellular cellulases (Ernst et al., 1985). Similar low amounts of extracellular cellulases are produced by bacteria associated with the rumen cultured on synthetic media (Ernst et al., 1985). These and other data apparently show that extracellular (free) cellulases do not play any important role in the biological transformation of cellulose in the rumen.

150. However, cellulases from the rumen are extremely active in vivo. In current technological reactors proportionately larger amounts of enzymes, one hundred- to one thousand-fold more than in the rumen, are required for cellulose hydrolysis. Moreover, volume production of these reactors in terms of cellulose degradation (in g/liter/h) is proportionately much less than that of the cellulolytic apparatus of ruminants (Ernst et al., 1985).

151. In the rumen almost all cellulases are located on the surface of bacterial cells. Thus, activity of free (extracellular) rumen cellulases is equal to 0.0032±0.0001 units/ml whereas the activity of cell-bound cellulases equals 30 units/g (Ernst et al., 1985). Additionally, in cattle only 5% of the cellulase activity in the rumen is due to extracellular cellulases - the remainder is bound (Ernst et al., 1985).

152. Evolutionary processes have led to the extremely economical use of cellulases in the rumen in comparison with that of the extracellular fungal cellulases. Indeed, it is wasteful for ruminants to produce large amounts of extracellular protein in the digestive tract, since a major fraction of it would immediately and tightly adsorb to nondigestable lignin (Chernoglazov et al., 1988) and pass out of the animal. Instead, the hydrolysis of cellulose in the rumen takes place by means of direct contact between cellulose and cellulolytic microorganisms. The latter then pass into the digestive tract where they are degraded, thus supplying essential components for subsequent metabolism (Ernst et al., 1985).

153. Recent data support this. By means of scanning electron microscopy, distinct protuberant structures were observed on cells of all cellulolytic strains, which include a variety of both gram-negative and gram-positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacterial species. Those structures were absent on the surfaces of noncellulolytic bacteria (Lamed et al., 1987; Lamed & Bayer, 1988). Moreover, those characteristic protuberant structures were absent when the cellulolytic bacteria were grown on glucose and did not produce cellulases. When the same bacteria were grown on cellobiose or cellulose, cellulolytic enzymes were produced and the protuberant structures appeared on their surfaces (Lamed et al., 1987).

154. Studies of the cellulolytic bacterium Clostridium thermocellum have shown (Bayer & Lamed, 1986; Lamed & Bayer, 1986) that the protuberant surface structures appeared to be rather complex formations, either hemiellipsoidal or hemispherical in shape, and ranged from 130 to 200 nm in length and 60 to 100 nm in height. They consist of "polycellulosomes" comprised of cellulosomes - the multisubunit protein formations consisting of cellulases tightly bound to cellulose. Protuberances apparently play an important role in attachment (adhesion) of cellulolytic cells to cellulose surfaces (Lamed & Bayer, 1986, 1988; Bayer & Lamed, 1986; Lamed et al., 1987). Considering the reported size of the isolated cellulosome [about 18 nm (Lamed et al., 1983a)], several hundred may be contained in the individual protuberant structures (Lamed & Bayer, 1986).

155. The surface cellulase ensembles are rather easily destroyed during incubation of the bacterial cells in water solutions, leading to the appearance of cellulase activity in the solution (Wood et al., 1982; Ernst et al., 1985). Thus, the incubation of Cl. thermocellum cells at 50° (pH of the culture liquid 6.8) results in the nearly complete transfer of the endoglucanase activity to the solution over 50-60 min. Similar times for the transfer of the enzymes to solution were also observed for the bacterial cellulases from the rumen of a cow (Ernst et al., 1985; Bolobova et al., 1988).

156. Investigations of the temperature dependence of this transfer process have shown that the activation energy for the degradation of polycellulosomes on the surface of the bacterial cells ranges from 6.1 Kcal/mole (for bacterial cellulases from the rumen) to 7.6 Kcal/mole (for cellulases from Cl. thermocellum). This represents the cleavage of weak (noncovalent) bonds between the enzyme molecules in cellulosomes and their aggregates (Ernst et al., 1985; Bolobova et al., 1988).

157. It should be noted that after practically all endoglucanases are transferred from the bacterial cell surface into solution the cells completely lose the ability to adsorb to cellulose; for example, in a 2.6% microcrystalline cellulose solution 85% of Cl. thermocellum cells can initially adsorb to cellulose by virtue of the surface cellulase ensembles. After elimination of cellulases from the cell surface, however, only 5% of the cells adsorb to cellulose. At the same time soluble extracellular endoglucanases retain their ability to adsorb tightly to cellulose (Bolobova et al., 1988). This indicates that the binding of Cl. thermocellum cells to cellulose takes place via cellulase ensembles on the cell surface.

158. There is not yet a generally accepted and clear classification for the supramolecular protein structures composing "protuberances" on cellulolytic bacterial surfaces. The term "cellulase ensembles" has been used to describe these structures. Another is "polycellulosomes". The term "cellulosomes" was coined in the first half of the 1980s (Bayer et al., 1983; Lamed et al., 1983a,b) to describe the multisubunit complexes of bacterial cellulolytic enzymes able to hydrolyze crystalline cellulose and capable of retaining in solution certain cluster structures after the disintegration of surface cellulase ensembles.

159. It may be the case that cellulosomes contain not only endo- but also exo-glucanases (cellobiohydrolases, for example) that act synergistically during the hydrolysis of crystalline cellulose. However, this hypothesis (Lamed & Bayer, 1986) is not supported experimentally as yet. Another definition of cellulosomes is also possible - a multisubunit protein formation of minimal size that retains its ability to hydrolyze crystalline cellulose and exists both in solution and on bacterial cell surfaces.

160. Cellulosomes apparently have a well defined and repeating molecular structure (Lamed & Bayer, 1988). It is rather difficult, however, to describe let alone reconstruct this structure experimentally. This is due to interference with cellulases in solution by multiple supramolecular formations, both aggregates and partial degradation products of cellulosomes (polycellulosomes and their fragments) and individual cellulolytic enzymes (Tikhomirov et al., 1988).

161. According to HonNami and coworkers (1985, 1986) Cl. thermocellum JW20 in solution can form aggregates of cellulase complexes of molecular weight 100 million (a large complex) and 5 million (a small complex), both of which can be further degraded to smaller fragments of molecular weight 210,000. Another study (Tikhomirov et al., 1988) reported that cellulase ensembles of Cl. thermocellum degrade in solution to aggregates of molecular weight 2-4 million, 200-300,000 and 40-100,000 and eventually dissociate into two main endoglucanases of molecular weight 75,900 (which are highly electrostatically interactive) and another of molecular weight 44,700.

162. As a rule, individual endoglucanases are much less active toward microcrystalline cellulose than are supramolecular cellulase aggregates in the culture fluid of Cl. thermocellum (Ermolova et al., 1988; Tikhomirov et al., 1989). By some estimates cellulosomes of Cl. thermocellum YS consist of at least 14 protein subunits each of about 18 nm in size (Lamed et al., 1983a).

163. One can conclude from these reports that studying the action of individual protein components of bacterial cellulase complexes (separate protein fragments of cellulosomes) may not provide data that describe the actual catalytic behavior of cellulosomes in solution or even on the surface of the bacterial cell. The mode of action of bacterial cellulases apparently differs significantly from that of fungal cellulases. Mainly, this lies in the fact that bacterial cellulases act as complex surface supramolecular ensembles, whereas extracellular fungal cellulases can apparently act individually and independently from each other.

164. Recently new data have demonstrated the complexity of the spatial organization of active bacterial cellulases (Lamed & Bayer, 1986, 1988; Bayer & Lamed, 1986). When the bacterial cell comes into contact with insoluble cellulose, polycellulosomal protuberances in the contact zone are transformed yielding an amorphous or fibrous network. This serves to bridge the cell and the cellulose surface (a distance up to 400-500 nm separates the two). The fibrous material may form "contact corridors" that direct cellulose degradation products, i.e., glucose and cellobiose, toward the cell surface and then to the respective compartments of the bacterial cell for subsequent metabolic breakdown (Lamed & Bayer, 1986).





CURRENT STATUS OF ENZYMOLOGY AND GENETIC ENGINEERING OF CELLULASES



A.   ADSORPTION OF CELLULASES ON CELLULOSE. WHAT DIFFERENTIATES "FULL-VALUE" CELLULASE SYSTEMS FROM "LOW-VALUE" TYPES?


165. Fig. 1 shows how crystalline cellulose distinguishes between "low-value" and "full-value" cellulase systems.





Transition from amorphous to crystalline cellulose, with a degree of crystallinity of 60-70% or higher, leads to rapid increase of the ratio of the hydrolysis rates for a "complete" system (e.g. from Trichoderma reesei) to a "low-value" system (e.g. from Sporotrichum dimorphosporum) (Klyosov et al., 1982). Here the horizontal axis indicates the degree of crystallinity for ten different cellulose preparations and the vertical axis the ratio of the velocities of the hydrolysis of the preparations under the action of the best (T. reesei) and the poorest (S. dimorphosporum) cellulases in terms of their adsorption. One can see that for the hydrolysis of amorphous cellulose (at a degree of crystallinity up to about 40%) cellulases from both sources show a similar hydrolyzing ability. However, crystalline cellulose turns out to be much more resistant to the weakly adsorbed cellulase (from Sporotrichum) than to the tightly adsorbed one (from Trichoderma), resulting in a sharp increase in the ratio of velocities. In other words, a tightly bound cellulase is more advantageous for the hydrolysis of crystalline cellulose than a weakly bound one. It should be emphasized that the surface concentrations of the endoglucanases in Fig. 1 were equal (Klyosov et al., 1982).

166. Considering the hydrolytic mechanism, it should be emphasized that the higher the crystallinity of cellulose, the slower the hydrolysis by weakly adsorbed enzymes. On the contrary, cellulose is hydrolyzed rather well by tightly adsorbed enzymes. These data are shown in Fig. 1.

167. It is remarkable that the point of intersection with the vertical axis in Fig. 1 (where the degree of crystallinity is equal to zero or, more precisely, where the term crystallinity loses its meaning) corresponds to hydrolysis of CM-cellulose, as shown experimentally. Thus, this plot accommodates data for all three types of cellulose substrates, i.e., soluble, amorphous and crystalline cellulose, and allows one to follow the change in reactivity of cellulases for all three types.

168. It seems that all known "full-value" cellulase systems studied contain at least one cellobiohydrolase (see Section C), and also possess endoglucanases that adsorb tightly to cellulose (Rabinovich et al, 1981, 1983; Klyosov et al., 1986). Moreover, the more catalytically active the cellulase preparations are in relation to the solubilization of crystalline cellulose, the more tightly the endoglucanases they contain are adsorbed (Fig. 2).





169. These and other similar data (see Table below) are the basis for the proposition: the better the adsorption, the better the catalysis (Klyosov et al., 1986). This is discussed in detail below in Section 2, which describes the adsorption of cellulases to cellulose.



B. ADSORPTION OF CELLULASES TO THE CELLULOSE SURFACE: PHENOMENOLOGY, TIGHTNESS AND ROLE OF ADSORPTION IN CATALYSIS


170. The proposition: the better the adsorption, the better the catalysis (Klyosov et al., 1986; Klyosov, 1988) was verified by measuring the adsorption capacity of endoglucanases for 26 cellulase preparations, 10 of which were highly purified (Klyosov, 1988). Tightly bound endoglucanases are produced by some fungi (Trichoderma, Myceliophthora, Aspergillus), bacteria (Clostridium) and actinomyces (Actinomyces diastaticus). Experimental data indeed demonstrate that the adsorption capacity of endoglucanases is the principal factor in defining the hydrolysis of amorphous and crystalline cellulose.

171. It is worthwhile noting that the adsorption capacity of endoglucanases does not depend on the degree of crystallinity of cellulose (Fig. 3).



Here, values of the partition coefficients (Kp) are shown for the adsorption of two endoglucanases, from T. reesei and Sporotrichum dimorphosporum, on ten samples of cellulose (from amorphous up to nearly completely crystalline). It can be seen that the cellulases are adsorbed equally to all cellulose samples in spite of their different degrees of crystallinity (Klyosov et al., 1982). Fig. 3 also shows that endoglucanases from T. reesei adsorb to all celluloses an order of magnitude more efficiently than endoglucanases from S. dimorphosporum.

172. Fig. 4 shows the principal differences in the hydrolysis of amorphous and crystalline cellulose by cellulases having different adsorption capacities.



The data show that for the efficient hydrolysis of amorphous cellulose only quantity (that is activity) of a cellulase preparation is important. On the contrary, for the hydrolysis of crystalline cellulose not only quantity of cellulases is important but mainly their quality, that is, the ability of endoglucanases to be adsorbed strongly onto the surfaces (Rabinovich et al., 1981, 1983; Klyosov et al., 1982a, 1983, 1986; Klyosov, 1988).

173. Another illustration of the importance of adsorption on the hydrolysis of crystalline cellulose is given in Fig. 5. The hydrolysis of soluble, amorphous and crystalline cellulose by twelve cellulase preparations from various microbial sources is shown with regard to their respective activities as a function of their partition coefficients (K_p).





174. Fig. 5 demonstrates that a ~20-fold increase in the adsorption capacity of endoglucanases (K_p from 0.02 up to 0.37 l/g) does not change the rate of hydrolysis of CM-cellulose. The rate of hydrolysis of amorphous cellulose is increased about two-fold, whereas the rate of hydrolysis of crystalline cellulose is increased ~50-fold (Klyosov et al., 1982). This increase in the hydrolysis rate is proportional to the adsorption capacity of the cellulases. If a cellulase binds fairly tightly, crystalline cellulose is hydrolyzed at a level nearly 30% of that for amorphous cellulose (Klyosov et al., 1982).
175. All these examples involve the action of endoglucanases in a mixture of cellulase complexes that contains many enzymes. In order to demonstrate the validity of the proposition "The better the adsorption, the better the catalysis" for the individual endoglucanases, experiments were performed with six highly purified multiple forms of endoglucanases from T. reesei (Fig. 6). Indeed, the increase in adsorption capacity of the purified enzymes parallels their reactivity toward crystalline cellulose (Klyosov et al., 1987).




176. Although important, adsorption capacity is not always the defining variable. Other properties are also critical, as described below (see Fig. 7, section C) for some individual fungal endoglucanases. As explained, endoglucanases from various sources can differ significantly not only in adsorption capacity but also in a number of other important characteristics, e.g., the ability to catalyze transglycosylation reactions to form glucose. This latter property may be just as significant as the role of adsorption capacity of the enzymes.


C. TRANSGLYCOSYLATION AS A POSSIBLE MECHANISM OF GLUCOSE FORMATION FROM POLYMERIC CELLULOSE


177. It was regarded as an established principle that endoglucanases do not cleave short derivatives of glucose, e.g. cellobiose or p-nitrophenyl-ß-glucoside (p-NPG). Endoglucanases indeed are inactive with these compounds, indicating that they are enzymes of the endo-wise type.

178. It has turned out, however, that in the presence of polymeric cellulose (CM-cellulose, as well as amorphous or crystalline cellulose) a highly purified endoglucanase from T. viride rapidly cleaves p-nitrophenyl glucoside (with rapid accumulation of p-nitrophenol). The introduction of insoluble cellulose (microcrystalline or amorphous) to the reaction mixture results in a 200- to 400-fold increase in the rate of p-NPG breakdown by endoglucanase. The introduction of soluble CM-cellulose causes an even greater acceleration of the reaction rate. This results in a rate of conversion of p-NPG by the endoglucanase comparable to that of hydrolysis of specific polysaccharide substrates by this enzyme (Kraeva et al., 1986).

179. At first, such an unexpected change in the established specificity of the endoglucanase seems to be inexplicable. However, analysis of the composition of the residual insoluble substrate leads to clarification: the p-nitrophenyl residues are incorporated into cellulose in the course of the enzymatic reaction (Kraeva et al., 1986). The simplest explanation for this phenomenon may be that the breakdown of p-NPG by endoglucanase occurs via a transglucosylation mechanism with intermediate formation of insoluble p-nitrophenyl-cellooligosaccharides (covalently bound to the insoluble cellulose matrix), from which the enzyme cleaves p-nitrophenol.

180. Developing this hypothesis further, it might be that in the presence of cellulose some endoglucanases can convert cellobiose to glucose via transglucosylation. If this is the case, it would explain why some "full-value" cellulase systems, having little or no cellobiase, produce appreciable glucose (a typical case - cellulases from T. reesei).

181. Finally, this hypothesis might explain two more puzzling phenomena (a) why the ratio between concentrations of the two main products of the enzymatic hydrolysis of cellulose, i.e. cellobiose and glucose, is shifted toward glucose when the amount of cellulose is increased in the reaction system (Morozov et al., 1987) and (b) why during the enzymatic hydrolysis of an insoluble cellulose the addition of cellobiose to the reaction mixture leads to a decrease of the initial rate of cellobiose formation, but not that of glucose, and, on the contrary, the addition of glucose inhibits the formation of glucose but not that of cellobiose (Gusakov et al., 1985).

182. Apparently, the formation of cellobiose from cellulose by endoglucanases is a one substrate reaction (not counting water as a substrate because it is in great excess in the system), and the formation of glucose by endoglucanase is actually a two substrate reaction (cellobiose and cellulose are the substrates). Since both reactions are characterized by their separate Michaelis constants and other kinetic parameters, the differences in the substrate concentration and addition of the product to the reaction mixture should affect each process differently. In other words, to explain the two above-mentioned phenomena it is enough to assume that the transglucosylation reaction has a higher Michaelis constant than that for the direct hydrolysis of cellulose by endoglucanase, and that the transglucosylation reaction is inhibited by glucose but not by cellobiose, which is its substrate. If one can measure these parameters experimentally, it would be possible to screen cellulases systematically and choose the enzymes with the highest capacity for producing glucose (and not the less-fermentable cellobiose). This would be very advantageous from an industrial point of view.

183. It is clear that for the definite verification of the proposition described in this section additional experiments are needed, for example, the examination of the hydrolysis of labeled cellobiose by purified endoglucanase in the presence of cellulose by measuring the incorporation of radioactivity into the cellulose matrix. This should also be done at different cellulose concentrations and with the addition of unlabeled glucose to the reaction system. To the best of our knowledge, these experiments have not been described in the literature.

184. The property of cellulases to form predominantly glucose seems to be extremely important. Recent experimental data on the reactivity of purified endoglucanases from a number of cellulase systems have led to a rather unexpected conclusion: the capacity of individual endoglucanases to split crystalline cellulose turns out to be directly related to their capacity to form glucose as a soluble product of hydrolysis (Klyosov, 1990) - the more random the endoglucanase action, the less active they are in relation to the hydrolysis of crystalline cellulose, and the less glucose (compared with that of cellobiose) they form as the products of hydrolysis of crystalline cellulose (Fig. 7).




185. From these data two conclusions follow: (i) the capacity to form glucose might be more important for the activity of an individual endoglucanase toward crystalline cellulose than the capacity to adsorb tightly to cellulose, (ii) the proposition "the better the adsorption, the better the catalysis" is probably not absolute. It may pertain more specifically to unfractionated cellulase systems, or to individual endoglucanases from the same microorganism (see Fig. 6).

186. Thus, if reactivity of an endoglucanase toward crystalline cellulose is low, it will yield cellobiose almost exclusively. A more reactive endoglucanase, in contrast, produces more glucose. The latter is apparently formed as a result of transglucosylation catalyzed by the endoglucanase (see below). Transglucosylation, therefore, might be an important reaction in "full-value" cellulase systems. Indeed, a "non-cellobiase" pathway for glucose formation is known to play an important role in the action of "full-value" cellulase systems (Klyosov & Rabinovich, 1980).


D. MECHANO-CHEMICAL ACTION OF CELLULASE ON CELLULOSE: DISORDERING, DEFIBRILLATION AND DISPERSION


187. Recent data indicate convincingly that both endoglucanase and cellobiohydrolase can disperse cellulose and that the dispersion can result from hydrolytic and mechanical action on cellulose.

188. The hydrolytic dispersion takes the form of enzymatic cleavage of cellulose macromolecules. Mechanical dispersion is induced by the adsorption of (cellulolytic) enzymes to cellulose defects (disturbances of the crystalline structure of cellulose, e.g., microcracks) followed by their penetration into the interfibrillar space (Rabinovich et al., 1982). This in turn concentrates the enzyme in the defects (as a result of a larger contact of binding sites of the enzyme with the cellulose surface), resulting in an increase in the mechanical pressure on the walls of pores, cavities and microcracks of cellulose (Klyosov & Rabinovich, 1980).

189. Water in the defects in turn penetrates still further inside the capillary spaces, breaking hydrogen bonds between the cellulose molecules and forcing apart and solvating the microcrystallites. In turn, the adsorbed cellulases prevent the newly formed surfaces from sticking back together, a tentative mechanism for the enzymatic dispersion not only of microcrystalline cellulose but also of native cellulose with a high degree of polymerization, e.g., cotton.

190. This process can be visualized easily by means of a simple experiment in which microcrystalline cellulose (Avicel) is placed as an aqueous suspension in an ultrasonic field for a few minutes and then left to precipitate spontaneously (coagulate). The results are recorded spectrophotometrically.

191. During the first 3-4 min after sonication, the suspension is relatively stable, and its optical density does not change. That is, the coagulation of small non-aggregated particles of cellulose proceeds slowly. Then a period of rapid coagulation and precipitation of flakes occurs, with a concomitant sharp decrease in the optical density of the suspension (Fig. 8a, curve 1). However, if tightly adsorbed cellulases are added to the suspension right after sonication, the suspension is stabilized and the velocity at which the particles precipitate is significantly decreased - even with a very small amount of added enzymes (0.02-0.04 mg/ml) (Fig. 8a, curves 3-7, Fig. 8b, curves 7,8). Weakly adsorbed cellulases do not affect the sedimentation rate appreciably (Fig. 8a, curve 2), nor do other non-cellulase proteins that have been added to the suspension (bovine serum albumin, ovalbumin, human gamma-globulin, trypsin, a-chymotrypsin, lysozyme, horse radish peroxidase, ß-glucosidase, glucose oxidase, hexokinase, horse liver alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glutamate dehydrogenase, malate dehydrogenase; some of these are shown in Fig. 8b, curves 2-5).




192. Shaking the cellulose suspension repeatedly in the presence of tightly adsorbed enzymes stabilizes it further, the maximal effect of the stabilization occurs over 30-40 min. Moreover, the optical density of the suspension after the addition of small amounts of the tightly adsorbed enzyme followed by shaking, is higher than the initial O.D. value (of the control) as a result of smaller particle formation (Fig. 8a,b). This is brought about by mechanical dispersion of cellulose under the action of cellulases since the time of contact of cellulose with the enzymes is too short to expect any significant hydrolytic dispersion.

193. Thus tightly adsorbed cellulases, both endoglucanases and cellobiohydrolases, but not loosely adsorbed cellulases and other proteins, cause mechanical dispersion of cellulose particles and prevent their adhesion. These effects no doubt play an important role in the enzymatic degradation of insoluble (native and microcrystalline) cellulose.

194. Apparently, for amorphous cellulose, the entire surface is easily accessible to enzymatic attack; therefore, the penetration of enzymes into inner areas of the substrate does not necessarily require the mechanical dispersion of cellulose by tightly adsorbed cellulases that prevent stabilized microfibrils from self-association. Since there is a high degree of hydration and mobility of cellulose macromolecules, the hydrolysis of amorphous cellulose by weakly adsorbed cellulases is quite possible. However, as presented above, the presence of tightly adsorbed cellulases is necessary for hydrolysis of crystalline cellulose.

195. It should be emphasized that for the efficient hydrolysis of cotton, fibers of which contain many amorphous connective areas bound to crystalline regions, the presence of both tightly and loosely adsorbed cellulases is important. For the hydrolysis of microcrystalline cellulose, though, in which the amorphous connective areas are practically absent, the presence of tightly adsorbed enzymes (endoglucanases or cellobiohydrolases) is enough for efficient hydrolysis. These principles of the enzymatic hydrolysis of cellulose are confirmed by experimental data. Indeed, both the individual endoglucanases and the individual cellobiohydrolases that can tightly adsorb onto cellulose are able to hydrolyze microcrystalline cellulose extensively. This is particularly efficient in the presence of cellobiase, which eliminates the inhibiting effect of cellobiose (Klyosov et al., 1987). On the contrary, the individual components of cellulase systems assayed separately are not capable of degrading native, highly-ordered cellulose such as cotton (Wood & McCrae, 1986b).


E. BEHAVIOR OF CELLULASES ADSORBED ON THE CELLULOSE SURFACE


196. What is the consequence of events that occur on the cellulose surface after successful attack by the adsorbed enzyme on the glucosidic bond of an insoluble cellulosic substrate? Two major schemes can be visualized: first, the attack is followed by desorption of the enzyme into the bulk solution with subsequent readsorption to another part of the substrate, or, second, the enzyme moves along the cellulose surface performing a series of successive attacks without desorbing from the surface. In the latter case the above question might be posed as follows: how many catalytic acts (on average) does the adsorbed enzyme make before it desorbs from the cellulose surface?

197. A theoretical and experimental approach has been developed (Rabinovich et al., 1984, 1985a,b) to answer this question. Data can be obtained on the adsorption capacity of cellulosic materials in relation to cellulases and on the velocity of desorption of cellulases from cellulosic materials into the bulk solution. It appears that cellulolytic enzymes perform hundreds to thousands of catalytic acts without leaving the cellulose surface. At the same time the transfer of the enzymes between cellulose particles occurs much faster than the complete degradation of these particles. Finally, data on the character and velocity of the "erosion" (or, alternatively, the "smoothing") of the cellulose surface in the course of enzymatic attack can be obtained (Rabinovich et al., 1985a).

198. The approach is based on the use of cellulose-containing adsorbed enzymes and a standard dyed insoluble cellulose added to the reaction system. At a characteristic time after both preparations of cellulose are mixed, the dye appears in solution as the result of redistribution of the enzyme between the surfaces of the non-dyed (under study) and dyed (standard) cellulose.

199. The kinetic analysis of the formation of the dyed product in the system shows (Rabinovich et al., 1985b) that the time-lag on average corresponds to the period in which the enzyme remains bound to the substrate particle upon which it was originally adsorbed; and the stationary phase of the curve corresponds to the quantity of the enzyme transferred from the non-dyed cellulose to the dyed species before a new adsorption equilibrium is attained in the system.

200. Experiments with various cellulases and various cellulose samples show that the average life-span of cellulases on the surface of microcrystalline cellulose is 25-35 min, and on amorphous cellulose (regenerated from phosphoric acid) - 60-75 min. Since the turnover number (or the catalytic constant) for endoglucanases from various microbial sources towards crystalline cellulose usually varies from 0.01 to 0.7 sec^-1, and from 1.0 to 5.6 sec^-1 for amorphous cellulose, it is readily apparent from the ratio of retention time to turnover number that cellulases perform dozens and hundreds of catalytic acts (hydrolyzing crystalline cellulose) or even thousands of acts (hydrolyzing amorphous cellulose) without leaving the surface of the cellulose substrate.


F. SYNERGISM BETWEEN COMPONENTS OF THE CELLULASE SYSTEM: MOLECULAR MECHANISMS FOR ENZYMATIC HYDROLYSIS


201. Apart from the well-known synergism between endoglucanases and cellobiohydrolases, another kind of synergism was detected. This occurs between two endoglucanases during the hydrolysis of crystalline cellulose (Rabinovich et al., 1986a), and can not be explained in the framework of the simple "consecutive" action of endo- and exo-enzymes.

202. This new type of synergism might be the key to explaining the puzzling phenomena of synergism in general. Synergism between two endoglucanases is observed for enzymes of which one is adsorbed tightly, while the other is adsorbed weakly (Rabinovich et al., 1986a). The adsorption constants, i.e. the partition constants for these endoglucanases (from T. viride) between the cellulose surface and the water phase differ ten-fold (1.3±0.1 l/g to 0.15±0.02 l/g). The joint action of the two enzymes leads to greater hydrolysis of microcrystalline cellulose than can be obtained by each enzyme separately. Moreover, for the degree of conversion of the substrate (10, 20, 30, 35 and 40 per cent), the extent of synergism (measured in relation to the velocity of glucose formation in the presence of an excess of cellobiase) is equal to 1.3, 1.5, 2.0, 2.3 and 2.5, respectively (Rabinovich et al., 1986a).

203. When a mixture of the tightly and loosely adsorbed endoglucanases is employed the degree of conversion of microcrystalline cellulose was greater than 75%. In contrast, a loosely adsorbed endoglucanase loses its activity after a degree of conversion of only 30-40%, even when more enzyme is added to the reaction.

204. This same study (Rabinovich et al., 1986a) shows that the sites for the attack of the substrate by loosely and tightly adsorbed endoglucanases are different, and the addition of the loosely adsorbed endoglucanase to cellulose previously hydrolyzed with the tightly adsorbed enzyme leads to a significant increase in velocity of hydrolysis as well as in the degree of conversion of microcrystalline cellulose (more than 60%). Moreover, the experiments have shown that the rate of hydrolysis by a combination of the loosely and tightly adsorbed cellulases were 6 to 20 times higher than that of the tightly adsorbed cellulases alone (Rabinovich et al., 1986a).

205. Thus, the following proposition can be advanced: the efficiency of degradation of crystalline cellulose is enhanced when the enzyme system contains cellulases with both high and low affinity to cellulose, that is, they exhibit synergism. The molecular mechanisms of the exo-endo and endo-endo types of synergism differ in principle. It appears that tightly bound enzyme (with K_p 1-5 l/g) binds to cellulose at sites where the crystalline structure of the substrate is disturbed, and induces a dispersion (defibrillation) of the crystallites as a result of the above-mentioned mechano-chemical effects. These tightly adsorbed enzymes can then penetrate intercrystalline regions and open new sites for the action of weakly adsorbed enzymes (with K_p 0.02-0.04 l/g). The latter act rapidly only on disordered (amorphous) regions of cellulose, situated at peripheral parts of microcrystallites. These regions could also become accessible as a result of the dispersion of cellulose crystallites. This apparently explains the mechanism of synergism between tightly and weakly adsorbed cellulases - not only between endoglucanases themselves but also between cellobiohydrolases as well as their combinations.

206. The above can explain most (if not all) of the puzzling features of "endo-exo", "exo-exo", and "endo-endo" synergisms described in this section. It is now clearer why "cross-synergism" between endoglucanases and cellobiohydrolases from different sources (Gow & Wood, 1988) is found only for the enzymes from complete (full-value) cellulase systems, but is not observed for any combinations of cellobiohydrolases from complete cellulase systems and endoglucanases from non-complete (low-value) cellulases (of fungi Myrothecium verrucaria, Stachybotrys atra and Memnoniella echinata, or bacteria Ruminococcus albus, R. flavefaciens and Bacteroides succinogenes).

207. The apparent reason is that all (to the best of our knowledge) known complete cellulase systems contain tightly adsorbed endoglucanases (see above), and that all known endoglucanases from the above listed fungal and bacterial sources of non-complete cellulase systems contain enzymes that are weakly adsorbed to cellulose (Klyosov, 1988). Gow and Wood (1988) came closest to the explanation of the phenomenon when indicating that synergism between fungal cellobiohydrolase and endoglucanase from different microorganisms occurs only in those situations where the endoglucanase has been isolated from a cellulase preparation that also contained a cellobiohydrolase, i.e., from a "true" cellulase preparation.

208. In terms of molecular mechanisms, it seems that synergism occurs only in those situations where the endoglucanase is able to adsorb tightly to cellulose. This occurs for all major forms of endoglucanases from "true" cellulase systems (Klyosov, 1988). This conclusion is consistent with recent work (Lee et al., 1988), which demonstrated that no synergism was found when the tightly adsorbed cellobiohydrolase I from T. reesei was incubated with either of two weakly adsorbed endoglucanases from Aspergillus niger.

209. Recent work by Wood and co-workers (Bhat et al., 1989a,b; Wood et al., 1989) demonstrated that a mixture of cellobiohydrolases I and II from Penicillium pinophilum exhibits significant synergism only with tightly adsorbed endoglucanases III and V from the same source, and not with weakly adsorbed endoglucanases I, II and IV. This serves as convincing evidence that endoglucanases must exhibit tight adsorption in order for synergism with cellobiohydrolases to occur.

210. In the light of this concept it is not surprising that the tightly adsorbed endoglucanase (Klyosov, 1988) from bacterium Cl. thermocellum demonstrates cross-synergism with the cellobiohydrolase from T. koningii (Gow & Wood, 1988). However, the existence of the cross-synergism does not necessarily imply that a cellobiohydrolase exists in the cellulase complex of Cl. thermocellum, as was suggested (Gow & Wood, 1988). It might also be expected that endoglucanases with different adsorption capacities may be present in the cellulase complex of Cl. thermocellum. Indeed, endoglucanases have been found in various strains of Cl. thermocellum whose adsorption capacities differ by 30 to 300 (Klyosov, 1988).

211. The existence of the synergism between two cellobiohydrolases can now be explained. If one is capable of being adsorbed tightly to cellulose, it may lead to the mechano-chemical dispersion of crystalline cellulose. This will in turn allow a second cellobiohydrolase to attack newly formed surfaces and/or exposed amorphous portions of the substrate.

212. Cellobiohydrolase I from T. reesei indeed binds more tightly to crystalline cellulose (as well as to cellophane and viscose) than does cellobiohydrolase II, thus experimentally verifying this suggestion. This was shown by means of "affel" (affinity + electrophoresis), a new method that combines the affinity binding of cellulases onto cellulose with the subsequent isoelectric focusing of proteins tightly adsorbed to the carrier (Rabinovich et al., 1985c). The cellulose particles together with the adsorbed cellulases (directly from the culture liquid of T. reesei) were introduced into the polyacrylamide gel after thorough rinsing with water. The proteins were then subjected to electrodesorption and isoelectric focusing. Cellobiohydrolase I was transferred completely from the culture liquid onto the cellulose surface, whereas the adsorption of cellobiohydrolase II was much less.

213. Recently published data on the molecular structure of cellobiohydrolase I from T. reesei support a possible role for this enzyme in the mechanochemical dispersion of crystalline cellulose. Small angle X-ray scattering showed that the enzyme molecule contains a long tail, the length and diameter of which is 18 nm and 4.4 nm, respectively (Schmuck et al., 1986). The tail was heavily glycosylated and played an important role in the adsorption to cellulose, and in fact, represented the adsorption domain (Van Tilbeurgh et al., 1986; Knowles et al., 1987a,b). It might be that this glycosylated tail, able to adsorb strongly to cellulose, contributes substantially to the mechanochemical effects and the subsequent dispersion of crystalline cellulose by cellobiohydrolase I.

214. On the other hand, cellobiohydrolase II from the same cellulase complex of T. reesei, which is not able to adsorb strongly, degrades amorphous portions of cellulose and renders the crystallites susceptible to subsequent dispersion by cellobiohydrolase I. Such joint behavior by cellobiohydrolases I and II might describe the nature of "exo-exo" synergism, that is, the synergism between two cellobiohydrolases.

215. It should be noted that cellulolytic enzymes are remarkably structurally organized. Recently it has been shown that four cellulases from T. reesei, an exoglucanase from the fungus Sporotrichum pulverulentum (Johansson et al., 1989) and two endoglucanases from the bacterium Cellulomonas fimi (Langsford et al., 1987; Greenwood et al., 1989) all share a two-domain structure. Moreover, a chemical modification of the carboxyl group in the active site of cellobiohydrolase I of T. reesei causes an 80% decrease in the activity of the enzyme toward microcrystalline cellulose but only a 12% decrease in its adsorption to the same substrate (Tomme & Claeyssens, 1989). This indicates that the binding and catalytic domains of cellulases are separate functionally.

216. Finally, based on the modes of action of tightly- and weakly-adsorbed cellulases, it becomes clearer why not all cellobiohydrolases and endoglucanases are synergistic when present together. Thus, according to (Wood et al., 1988) only one of two cellobiohydrolases from P. pinophilum is synergistic with the cellobiohydrolase of T. koningii or the cellobiohydrolase of F. solani. Additionally, only two of the four endoglucanases of T. koningii are synergistic with cellobiohydrolase (Wood & McCrae, 1978). Wood and McCrae suggested that this could be explained either by specific associations between endoglucanase and cellobiohydrolase on the surface of cellulose (it is not clear though why some pairs of cellulases do form associations while others do not), or by the existence of two different stereospecific cellobiohydrolases that each hydrolyze one of the two different configurations of non-reducing end groups that exist in the cellulose crystallite (Wood & McCrae, 1986b; Wood et al., 1988, 1989).

217. It appears that this suggestion can not be easily proven experimentally and thus, it will not be considered further. The explanation, rather, may turn out to be much simpler and, more importantly, more easily verified experimentally - if the synergism is due to one of the components being adsorbed tightly to cellulose. If both the components are adsorbed weakly on cellulose, synergism would not be observed, or would be observed to a much lesser extent. This might possibly explain recent observations (Wood et al., 1989) that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or between the individual cellobiohydrolases and the endoglucanases of P. pinophilum.

218. Certainly, a "kinetic" synergism of the "endo-exo" type (that is a bienzymatic consecutive reaction) can also take place in cellulolytic systems, and both types of synergism, i.e. "kinetic" and "adsorptive", can take place in the same reaction system. It should be noted, though, that these reactions may strongly depend on enzymatic hydrolysis conditions (that is, on cellulose concentration, enzyme concentration, ratio between the two, etc.). This is illustrated by the report by Sinitsyn and coworkers (1987a) that showed that the extent of synergism between endoglucanase and cellobiohydrolase from T. viride depends significantly on cellulose concentration, on the ratio of the concentration of cellobiohydrolase to that of endoglucanase and on the degree of polymerization of the substrate, among others. A computer simulation of these considerations has been published (Sinitsyn et al., 1987a; Klyosov et al., 1986b).

219. Synergism is determined by the rate limiting step in consecutive hydrolytic pathways (Klyosov & Grigorash, 1981a,b, 1982; Klyosov, 1982; Klyosov et al., 1986a,b; Mitkevich et al., 1985; Sinitsyn et al., 1987b). It is clear that if in a consecutive cellulolytic process (for example, in which endoglucanase acts first followed by cellobiohydrolase and cellobiase) the first step is rate-limiting, "kinetic" synergism will not be observed. It could be that in the example described above, when only two out of four endoglucanases were synergistic with cellobiohydrolase (Wood & McCrae, 1978), the two other endoglucanases possessed significantly lower molecular activities (that is, turnover number). This might change the rate limiting step of the enzymatic hydrolysis, in comparison with that for the first two enzymes, resulting in the loss of synergism. In a recent study (Woodward et al., 1988a,b) it was demonstrated that synergism between cellulase enzymes was dependent on their individual concentrations, and the magnitude of the synergistic effect was critically dependent on whether or not the cellulose was saturated with enzymes. Because these and many other parameters of the enzymatic hydrolysis are difficult (perhaps impossible) to control in the course of experiments, comparisons of synergisms with different cellulase systems have to be performed with care.

220. In conclusion, considerations of the adsorption characteristics of cellulolytic enzymes are needed to explain the complex features of synergism in the action of cellulolytic components toward insoluble cellulose and the mechanism of the enzymatic hydrolysis of crystalline cellulose.


G. HOW DOES ENDOGLUCANASE HYDROLYZE SOLUBLE CARBOXYMETHYL CELLULOSE?


221. It is known that the presence of the carboxymethyl substituents in CM-cellulose retards the hydrolytic action of cellulases (Klop & Kooiman, 1965). In connection with this there is a rather general belief that it is difficult, if not impossible, to compare directly on a quantitative basis activities of endoglucanases determined with CM-celluloses differing in degree of substitution.

222. It appears now, however, that these comparisons can be made. This was shown recently with a new viscometric approach to measuring endoglucanase activity in which the dynamics of the viscosity change of CM-cellulose solutions are measured at the initial periods of substrate depolymerization (Rabinovich et al., 1977; Klyosov & Rabinovich, 1980). This new approach has led to almost identical values for T. viride and Clostridium thermocellum endoglucanase activity measured with several preparations of CM-cellulose with degrees of substitution varying from 0.45 to 0.99. For all seven samples of CM-cellulose used in the study the endoglucanase activity of a commercial cellulase preparation Meicellase (Japan) was equal to 8.5 ± 1.5 micromole/min/g, and the endoglucanase activity in the culture liquid of Cl. thermocellum was equal to 57 ± 10 micromole/min/l (Bolobova et al., 1985).

223. These data lead to the conclusion that in the very initial period of the enzymatic reaction, when rather long, unsubstituted multiglucose "blocks" of CM-cellulose (as long as 5-6 and more joined glucose residues) are subjected to endoglucanase attack (Klyosov & Rabinovich, 1980), the cleavages of the polymeric substrate take place mainly at certain distances from the carboxymethyl substituents and the degree of substitution of CM-cellulose does not appreciably affect the initial velocities. This relates, however, only to the initial period of enzymatic hydrolysis. Thereafter, the most enzymatically accessible long unsubstituted sites are being depleted and the enzyme gradually turns to attacking short unsubstituted portions of the substrate. The degradation of the polymer therefore decreases.


H. WHY DO CELLOBIOHYDROLASES NOT HYDROLYZE SOLUBLE CARBOXYMETHYL CELLULOSE?


224. As might be judged from the literature, the answer to the above question is quite clear: because cellobiohydrolase must by definition cleave a polymeric substrate from its ends (from the nonreducing end, to be precise) successively cleaving cellobiose. In the case of CM-cellulose, however, the carboxymethyl substituents interfer with cleavage. Recently, though, this conjecture has been brought into question by the finding that certain cellobiohydrolases can degrade insoluble cellulose, attacking it in sites remote from the ends of cellulose chains (Enari & Niku-Paavola, 1987; Wood et al., 1988). Moreover, cellobiohydrolases can, in a number of cases, possess the specific activity of endoglucanases (Enari & Niku-Paavola, 1987; Biely & Markovic, 1988).

225. Thus, the question posed as the title of this section should be rephrased as follows: why can certain cellobiohydrolases attack resistant, insoluble cellulose (crystalline cellulose, in particular), but not the seemingly easily accessible chains in soluble CM-cellulose? This is an important point for our understanding of the mechanisms involved in cellulose hydrolysis.

226. The apparent answer turns out to be rather unexpected. It was shown recently that CM-cellulose is a very strong inhibitor of T. reesei cellobiohydrolase I, with an inhibition constant of (1.0±0.2)x10^-7 M (Rabinovich et al., 1986b). In other words, the carboxymethyl substituents not only do not interfere with the binding of cellobiohydrolase to CM-cellulose, but, on the contrary, this binding is very strong. It also becomes apparent why endoglucanases and cellobiohydrolases usually do not act synergistically in CM-cellulose hydrolysis, but do so in the hydrolysis of an insoluble cellulose: CM-cellulose as a strong inhibitor of cellobiohydrolase eliminates it "kinetically" from the reaction system.

227. Why, however, is CM-cellulose such a powerful inhibitor of cellobiohydrolase, while also serving as the specific substrate for endoglucanase? It may be that the electrostatic interactions of the negatively charged CM-cellulose with the active center of cellobiohydrolase play a key role. If this is the case, differences in the structure of the active center should be reflected in the two enzymes. Cellobiohydrolase may bind to CM-cellulose through interactions with unsubstituted regions between the neighboring carboxymethyl substituents distributed rather randomly along the polymer backbone. To the best of our knowledge, experiments to clarify these questions have not been described in the literature and await investigation.

228. It is of interest that cellobiose is also a rather powerful inhibitor of cellobiohydrolase, with an inhibition constant of 6x10^-6 M (Rabinovich et al., 1986b). In weight units this inhibition constant equals 0.0021 g/l, meaning that at a cellobiose concentration of 2.1 mg/l (about 0.0002%), cellobiohydrolase activity would be suppressed by half (if an insoluble cellulose does not adsorb cellobiohydrolase on its surface). Thus in some cases during hydrolysis of an insoluble cellulose, cellobiohydrolase activity can be completely suppressed by its product, that is cellobiose, very early in the reaction (Klyosov, 1987). This may account for the ability of some individual cellobiohydrolases to hydrolyze insoluble cellulose. In the literature this is usually called "contradictory data" (see, for example, Enari & Niku-Paavola, 1987). However, it might not be a contradiction, but rather a reflection of different properties of cellobiohydrolases from various biological sources. This hypothesis awaits experimental verification.


I. TITRATION OF THE ACTIVE CENTERS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI


229. The observation that cellobiose is a powerful inhibitor of cellobiohydrolase I from T. reesei led to its use in a novel approach for the titration of the enzyme active centers (that is, the determination of the molar concentration of enzyme active centers in solution) as well as for the determination of absolute molecular activity (the catalytic constant or turnover number) (Rabinovich et al., 1986b).

230. The approach is based on the determination of the initial velocities of the hydrolysis of the nonspecific chromogenic reference substrate (p-nitrophenyl-b-D-lactoside) by cellobiohydrolase (either purified or as a component of a cellulase system) in the presence of various concentrations of the specific inhibitor (cellobiose or CM-cellulose). A plot of the data yields a straight line that intersects with the horizontal axes at the value of the inhibition constant (for cellobiose or CM-cellulose), and the slope of which equals the reciprocal of the initial concentration of the enzyme active centers. If the inhibitor concentration is expressed in moles per liter, the enzyme concentration is also calculated in this unit (Rabinovich et al., 1986b; Klyosov et al., 1987).

231. This approach for the titration of enzyme active centers may be helpful in searching for cellobiohydrolases with increased molecular activity and produced in higher molar concentrations. This, in turn, should aid in identifying better producers of cellobiohydrolases (and other enzymes) that can subsequently be cloned genetically.


J. A BACTERIAL CELLOBIOHYDROLASE: A PROTOTYPE


232. The fact that cellulases from Cl. thermocellum are able to hydrolyze crystalline cellulose may indicate that the respective cellulase complex consists not only of endoglucanases but may also contain a cellobiohydrolase(s) (Wu & Demain, 1988; Lamed & Bayer, 1988; Gow & Wood, 1988). However, efforts to isolate a bacterial cellobiohydrolase undertaken during the last decade have been unsuccessful; in all cases the identifiable end products were endoglucanase or derivatives thereof (see Wu & Demain, 1988; Ohmiya et al., 1987; Coughlan & Ljungdahl, 1988; Lamed & Bayer, 1988). Thus, the molecular mechanism of action of the cellulase system from Cl. thermocellum (and from bacterial cellulases in general) remains to be determined.

233. There are indirect data, however, that indicate that Cl. thermocellum cellulases contain additional components that enhance the action of endoglucanases on insoluble (particularly crystalline) cellulose. It is known, for example, that the individual endoglucanases of Cl. thermocellum or their mixture can not efficiently hydrolyze crystalline cellulose, whereas the crude culture liquid of the strain is effective (Ermolova et al., 1988; Tikhomirov et al., 1989). According to other data (Wu & Demain, 1988) the degradation of crystalline cellulose does not occur when separate protein subunits of the cellulase complex of Cl. thermocellum are employed, only when a mixture of a few (unidentified) protein fractions is used. Moreover, the activity of an unfractionated cellulase system of Cl. thermocellum is increased in the presence of dithiothreitol and calcium ions whereas the individual endoglucanases are not activated with those agents (Lamed et al., 1985; Johnson & Demain, 1984).

234. It was shown that the degree of "orderliness" of the enzymatic hydrolysis of soluble cellulose by a purified endoglucanase of Cl. thermocellum, on the one hand, and the initial culture fluid, on the other, differed significantly (Tikhomirov et al., 1989). Thus, the individual homogeneous endoglucanases of molecular weight 75,900 and 44,700 elicited a typical random hydrolytic pattern on CM-cellulose (the degree of "orderliness", or the ratio of the saccharifying activity to the liquefying one was equal to 1.2±0.2); at the same time the initial culture liquid showed a more ordered activity (the degree of "orderliness" was equal to 3.3).

235. Based on an analogy with fungal cellulases and other data it was suggested that cellobiohydrolase might be the additional component(s) in the cellulase complex of Cl. thermocellum. As support for this suggestion cellulase (endoglucanase) of Cl. thermocellum was shown to act synergistically with the individual cellobiohydrolase of T. koningii to solubilize crystalline cellulose (Gow & Wood, 1988).

236. The individual cellobiohydrolase of Cl. thermocellum has been isolated only recently, by cloning its gene into Escherichia coli. Purification and assay of the recombinant protein showed that it is indeed, as expected, cellobiohydrolase (Melnick et al., 1989a,b). The recombinant enzyme produces mainly cellobiose (93-97%) as the hydrolysis product for both crystalline and amorphous cellulose. The molecular weight of the recombinant cellobiohydrolase is 56,200, while its isoelectric point is 4.15. While active against soluble CM-cellulose, no effect on its viscosity is observed and only small quantities of reducing sugars are produced; the liquefying activity of the enzyme is at least 10,000 times less than that for saccharifying.

237. The recombinant enzyme has a characteristic hydrolysis pattern on trifluoromethyl umbelliferyl cyclooligosaccharides (MUF-G_n, where n=14). MUF-G itself is not a substrate, and no dyed fluorophore is formed from MUF-G₃. MUF-G₂ and MUF-G₄, however, are cleaved by the recombinant enzyme whereas the stationary formation of the fluorophore from the latter substrate occurs after a lag period. The study of the dynamics of hydrolysis indicated that the active center of the recombinant enzyme accommodates two glucose chains, with the site of catalysis at the glucosidic bond following the second glucose residue. The enzyme binds cellobiose 2,000 times tighter than glucose. Mapping of the active center has also shown that the recombinant bacterial enzyme is indeed cellobiohydrolase (Melnick et al., 1989a,b).


K. A NOVEL APPROACH TO THE DESIGN OF CELLULASE COMPLEXES: INTERGENERIC FUSION OF FUNGAL PROTOPLASTS


238. As previously mentioned, cellulase systems from various microbial sources are very diverse both in composition and catalytic properties. There is an occasional need, however, to "correct" the composition of a cellulase system for a predetermined change in its functional characteristics. This may be accomplished by cloning certain cellulase genes, an approach that has been used widely in the last decade for studying cellulases and obtaining new mutant strains (for recent works see Aho, 1991; Aminov et al., 1990; Baird et al., 1990; Bingle et al., 1993; Knowles et al., 1988; Beguin et al., 1988; Hazlewood et al., 1988, 1990, 1992; Chippaux, 1988; Miller et al., 1988; Yablonsky et al., 1988; Melnick et al., 1989a,b). Nuzubidze and coworkers (1988) have developed a different approach dealing with fungal protoplast fusion. In contrast to the well known intrageneric protoplast fusion they developed intergeneric fusion of fungal protoplasts.

239. In this approach, the protoplasts of two taxonomically distant fungi belonging to the different genera Trichoderma and Neurospora were fused. A successful intergeneric protoplast fusion leading to a stable fusant strain is achieved when a steroid glycoside, digitonin, is added to the recombinant system (Nuzubidze et al., 1988). Digitonin, like some other steroid saponins, may interact specifically with cell membrane components and make the protoplast fusion easier. Eventually a new producer of cellulases was obtained that secretes the enzymes of the two parent strains, that is T. viride and Neurospora crassa.

240. Unexpectedly, the new strain secreted appreciably more endoglucanase and cellobiase than either of the two parent strains. The morphology of the fusant strain was also significantly different from that of the parents. These and other characteristics of the new strain show that it is indeed a hybrid producer. The fusant strain turned out to be essentially indefinitely stable during cell culture.

241. In a subsequent work (Kirimura et al., 1989) an intergeneric hybridization of protoplasts of the fungi A. niger and T. viride was performed in a polyethylene glycol solution, but cellulolytic activities of the intergeneric fusant were not measured (see also Furlaneto & Pizzirani-Kleiner, 1992).

242. Intergeneric fusion of fungal protoplasts could become a widely accepted tool for designing new strains with predetermined composition and catalytic properties of the enzymes.


L. CELLULASES OF THE THIRD GENERATION


243. This section demonstrates how knowledge of some properties of cellulolytic enzymes, such as specific activity, adsorption capacity, thermostability, product inhibition, etc. can be translated into concrete requirements for cellulases in potential industrial applications. A new strategy for screening cellulases is discussed here: technological parameters required for maximal enzymatic hydrolysis of cellulose drive the selection of molecular characteristics. The primary focus of such screening is a search for cellulases having not greater total activity, but rather more appropriate molecular properties of the enzymes. This section shows the first results of such molecular screening for cellulases from fungi, bacteria and marine organisms.

244. During the first half of this century the enzymatic hydrolysis of cellulose was studied with a microbial biomass as the primary source of cellulolytic enzymes without separation of these enzymes (cellulases of the "first generation"), under the premise that the enzymatic hydrolysis of native cellulose can not be realized in an extracellular system.

245. The finding that extracellular cellulolytic systems can degrade native cellulose to glucose, and the introduction of the multienzyme cellulase complexes under laboratory conditions, and later on an industrial scale, gave rise to new work with cellulases, i.e. to cellulases of the "second generation". These cellulases are generally of fungal origin. The microscopic fungi Trichoderma, Aspergillus, Sporotrichum, Penicillium, Fusarium as the best-known sources.

246. These cellulases have been selected generally by screening techniques common in microbiology, that is, by selecting for high "overall" cellulolytic activity as indicated by initial velocity of hydrolysis at moderate temperatures ("model" conditions). The same criteria have been applied to selection of new mutants of cellulolytic microorganisms.

247. This approach has resulted in an extraordinarily high concentration of extracellular protein in culture media as well as in biosynthetic production of cellulases in large quantities. For T. reesei, for example, which is used by most research groups, advances in strain improvement and fermentation development permit production of titers greater than 20 g of cellulase protein/1iter as well as production of over 200 mg of extracellular enzyme protein/1iter/h, an amount that is probably much higher than that in any other industrial enzyme production process (Mandels, 1985).

248. Such an exceptionally high excretion by the strain apparently has its own "biotechnological" reasons. Obviously, in order to reach a certain "overall" enzymatic activity in culture media, nature can take either of two extreme alternatives - produce either a small amount of enzyme with a high catalytic activity (a high turnover number) or a large amount of protein with low activity. Unfortunately, it seems that in the case of Trichoderma, nature proceeded in the latter manner, resulting in the organism excreting an enormous amount of protein of relatively low catalytic activity in relation to insoluble cellulose.

249. The following data demonstrate the low extent of that activity. In a routine experiment on the hydrolysis of cellulose by Trichoderma cellulases, even when one essentially covers all cellulose surface with the enzymes, hydrolysis proceeds very slowly so that hours and even days are necessary for completion.

250. The specific activity of Trichoderma cellulase is very low even on filter paper, i.e. almost ideally pretreated cellulose, and is equal only to 0.6-0.7 IU/mg of protein (Mandels, 1985) (IU, an international unit, corresponds to the formation of 1 micromole of product per min). This indicates that the catalytic constant (kcat value, i.e., maximum velocity per mole of enzyme) for this reaction is equal to 0.5-0.6 sec^-1. Glucoamylase, for example, has a specific activity 100 times greater, i.e. 69 IU/mg of protein with starch as substrate (Mandels, 1985), which corresponds to a kcat value of 58 sec^-1. The same value for catalytic constant is shown, however, by the endoglucanase of the T. reesei cellulase complex for hydrolysis of soluble carboxymethyl cellulose (specific activity 70 IU/mg of protein, or kcat 58 sec^-1). This demonstrates that the catalytic activity of cellulases is low not for all substrates, but mainly for insoluble cellulose.

251. On the other hand, the hydrolytic activity of cellulases toward even soluble cellulose is not high compared with that of many other hydrolases in relation to their specific substrates, where the k_cat values sometimes reach hundreds and thousands of reciprocal seconds, i.e. one to two orders higher than that for T. reesei cellulases.

252. Unfortunately, industrial application of cellulases is restricted not only by a relatively low catalytic activity but also by other unfavorable properties. Recently, it became obvious that those cellulases that are the most widely used in laboratories cannot be considered to be "technological" candidates, as they cannot provide an appropriately high velocity and degree of cellulose conversion under operational conditions.

253. Thus, aside from the relatively low turnover number, T. reesei cellulases have the following principal disadvantages:
(i) low thermostability; the half-life at 65°C for endoglucanase, which is the most stable component of the cellulase complex, is only 20-40 min; this is the temperature required for pasteurization, which is a very desirable lower temperature limit for industrial production since it can obviate the problem of microbial contamination of the glucose syrup that is produced.
(ii) a high degree of inhibition by products, i.e., glucose and cellobiose; at product concentrations of 3-5%, the velocity slows, and at 8-10% hydrolysis is almost nil.
(iii) endoglucanases that comprise approximately 30% of the loosely adsorbed multiple forms of the enzymes are adsorbed on cellulose 100 times less than that of other endoglucanases (see below) from the same source and cannot remain, for example, in a flow-through reactor for continuous cellulose hydrolysis.

254. In general, considering problems that hamper industrial application of enzyme engineering development, we stress one nonobvious difficulty: sometimes the most suitable enzyme, based on microbial screening, turns out to be quite unsatisfactory for its industrial application. Actually, a search for a suitable enzyme preparation and for subsequent scale-up application by screening the corresponding strains is directed at determination of enzyme activity, i.e., to the amount of the enzyme in a system under study (a culture fluid, a cell lysate and so on). On the other hand, in enzyme engineering processes, other advantageous features of the enzyme, i.e. thermal stability, insensitivity to product inhibition, catalytic constant, the ability to adsorb to a solid support, etc., are often more important.

255. Thus, a highly active culture liquid does not necessarily result in a technologically suitable enzyme preparation, because classical screening and current technological requirements often differ when investigating an enzyme.

256. It has become rather obvious by now that the development of efficient technology for enzymatic hydrolysis of cellulose requires cellulases of the "third generation" (Klyosov, 1988), i.e. cellulases with predetermined molecular characteristics that provide the following "biotechnological" properties of the enzymes:
(i) half-life of as much as two weeks at 65°C,
(ii) the capacity to catalyze cellulose hydrolysis effectively in the presence of 20-30% glucose/cellobiose syrups,
(iii) a molecular activity towards insoluble cellulose of about 10-50 sec^-1, that is 20-100 times higher than that of T. reesei endoglucanase,
(iv) an adsorption constant (the partition coefficient between the cellulose surface and water) of 2-5 l/g, that is, the upper range for known cellulases that vary from 0.001 to 8 l/g).

257. In order to uncover sources for such "third generation" cellulases, a new strategy for screening is needed, that is, screening not only with respect to "overall" activity, but primarily with respect to the molecular properties of the enzymes. Development of this new strategy requires a re-evaluation of the approach to the determination of "biotechnological" properties of cellulases and design of new methodology for the necessary assays. The overall activity of such cellulases can be increased up to the required level for the next step of the work by means of molecular engineering and other similar novel approaches.

258. Some preliminary results of the "molecular" screening for cellulases from some fungi, bacteria and marine organisms are given below. Such screening has required development of new experimental approaches, in particular, for the determination of adsorption constants for cellulases, their catalytic rate constants and product inhibition constants (Klyosov, 1989).

259. The next table shows the values of inhibition constants for cellobiose, which is often a major product of enzymatic hydrolysis of cellulose, in relation to the activities of endoglucanases of cellulase complexes from different sources. These data indicate that the endoglucanases can be subdivided tentatively into three groups that are:
(i) relatively less sensitive to inhibition by cellobiose (K_i 100-120 g/l, or 0.29-0.35 M),
(ii) of moderate sensitivity (K_i 50-90 g/l, or 0.15-0.26 M) and
(iii) of high sensitivity (K_i 20-40 g/l, or 0.06-0.12 M).
Included in the first group, for example, are endoglucanases Actinomyces diastaticus, Aspergillus fumigatus and Clostridium thermocellum; and included in the third group is endoglucanase T. reesei.

260. In the course of cellulose hydrolysis by T. reesei cellulases, as shown in the table below, the velocity of hydrolysis should decrease two-fold at cellobiose concentration of about 3-7%. A similar decrease should be observed at cellobiose concentrations of 9-12% for cellulases from Act. diastaticus or Cl. thermocellum. These data would have been appropriate if the efficiency of the hydrolysis depended on endoglucanase only. It turns out, however, that another important component of the cellulase complex, cellobiohydrolase, is much more strongly inhibited by cellobiose, and its inhibition constant (toward a soluble substrate at low concentrations of the latter) equals (6±1) x 10^-6 M, or 0.0021 g/l (Rabinovich et al., 1986b).

261. If the last value is applicable to the hydrolysis of an insoluble cellulose, then at any reasonable value of Michaelis constants (2-50 g/l) for the enzymatic hydrolysis of cellulose and, at a wide range of cellulose concentrations (10-400 g/l), cellobiohydrolase is almost completely inhibited by reaction products even during the initial period of hydrolysis (at cellulose concentrations of 0.01 to 0.1%).




262. These data illustrate the necessity for a well-oriented search for cellobiohydrolases from various sources having cellobiose inhibition constants of 1-10 g/l (0.003-0.03 M) or higher, i.e., three orders of magnitude less sensitive to product inhibition than T. reesei cellobiohydrolase. Work in this direction has not yet begun.

263. The following table shows data on adsorption of endoglucanases from various sources on cellulose and indicates that the respective coefficients vary significantly, more than 1000-fold, from 0.0035 l/g (Asp. foetidus) up to 8.5 l/g (T. reesei). Endoglucanases from Cl. thermocellum, Act. diastaticus and Asp. versicolor are attractive in that the major fraction consists of tightly binding enzymes. As previously demonstrated (Klyosov et al., 1982, 1986; Klyosov, 1990), hydrolysis of highly ordered cellulose by cellulase complexes from various sources is described by the following proposition: the better the binding of endoglucanases, the better the catalysis. It follows from this that the cellulase complexes mentioned above could be promising for the design of efficient catalysts for cellulose hydrolysis.

264. Thermal stability of cellulases, as well as many other enzymes, are not easily comparable since the corresponding kinetic curves for enzyme inactivation are not usually described by simple kinetic regularities. A substrate such as cellulose when in an incubation mixture might produce a stabilizing effect, but this effect could in turn depend on substrate concentration, source of a cellulase complex, reaction conditions, etc. In addition, the thermal stability of cellulases changes with temperature to a different extent for different enzymes as a result of different activation energies for thermal denaturation, and this effect, in particular, makes a comparison between cellulases difficult.

265. To avoid this ambiguity as much as possible, the next table compares thermal stability of cellulases in the absence of substrate, and at one temperature only, i.e. 65°C, since this temperature is in the pasteurization range, where microbial contamination of the reaction system is less likely. To simplify the expression of thermostability of cellulases even further, and to avoid the ambiguous analysis of complex kinetic curves of thermal inactivation of enzymes, the table shows the half-life for endoglucanases only.




Kp, l/g



266. The next table shows that the thermal stability of endoglucanases from different cellulolytic sources varies over an extraordinarily wide range, at least ten to fifty thousand times. Cellulases in this table can be subdivided tentatively into four groups:

(i) those that are very unstable at the given temperature (half-life less than 1 min),
(ii) rather unstable (half-life from several minutes to one hour),
(iii) relatively stable (half-life of several hours), and
(iv) very stable (half-life of several days or even weeks).

According to this subdivision, endoglucanases from T. reesei drop to the category of rather unstable cellulases, together with typical "low-value" cellulase complexes from Asp. niger and Asp. foetidus, which are marked also by their strong product inhibition and low adsorption to cellulose (see tables in paragraphs 261 and 265). On the other hand, endoglucanases from thermophilic Aspergillus as well as from Actinomyces and Clostridia appear to be relatively thermostable, but the most stable (among the purified preparations) turns out to be an endoglucanase from fungus Myceliophthora thermophila with a half-life 20-100 times higher than for the nearest endoglucanases from the previous group (see also Durrant et al., 1992).






267. With regard to molecular activities of cellulases and their catalytic constants, the data are too limited to draw any certain conclusions. Thus far, supportive data can be obtained only with highly purified individual cellulolytic components by measuring their specific activities referred either to 1 mg of protein or to 1 mole of enzyme. This approach was used for the determination of the catalytic constants of the endoglucanases T. reesei and M. thermophila shown in the next table. It turns out that M. thermophila endoglucanase is three times more active than that of T. reesei in CM-cellulose hydrolysis, but only if the activity is determined viscometrically. The activities are equal when determined by reducing sugars. This effect possibly reflects a difference in mode of action of the two endoglucanases (for example, the "randomness" of their action on soluble polymer). Moreover, the data support the possibility that more active cellulases than those of T. reesei can be found.



a - In an excess of cellobiase from Asp. foetidus, degree of conversion of crystalline cellulose 10%
b - Tightly binding endoglucanase, highly purified.
c - Loosely binding endoglucanase, highly purified.
d - Recombinant microorganism carrying structural Cel genes of Cl. thermocellum (strain F-7).
e - 60° C


268. Only one value for the catalytic constant for the cellobiohydrolase-catalyzed hydrolysis of the "model" synthetic substrate, p-nitrophenyl-ß-D-lactoside, has been reported (Rabinovich et al., 1986b). Detection in nature of more active and more "biotechnological" cellobiohydrolases, as mentioned above, would be very important and would make those "cellulases of the third generation" the basis of efficient bacterial strains that could improve the practical bioconversion of cellulose into glucose and other valuable products.


M. PROTEIN ENGINEERING. CELLULASES OF THE FOURTH GENERATION


269. In the long term, protein engineering may be most productive for obtaining cellulases with optimized properties (high catalytic efficiency, adsorption to cellulose substrate, thermostability, coupled with resistance to product inhibition). Over the past decade this approach, involving the modification of protein primary structure, usually by recombinant DNA technology to mutate specific sites or exchange domains, has been widely exploited for the successful determination of protein structure-function relationships, alteration of properties, and improvement of catalysis, binding, or stability. For example, the detailed mechanisms of certain amino acid tRNA synthetases have been established in this manner, which include the identities of the various amino acid residues forming interactions with substrate, product, and reaction intermediates. In the process, it was possible to design mutagenic changes that improved in vitro enzymatic activity.

270. Subtilisin, a protease of enormous industrial importance (600 tons per annum are produced for use in soap powder and for the food and leather industries) has also been successfully studied and its application-related properties improved. For example, the role of residues in the catalytic triad of this serine protease have been deciphered, specificity has been altered, resistance to oxidation increased, the pH activity profile tailored, and thermostability increased (Fersht & Winter, 1982).

271. Extensive use has been made of domain transfers. For example, enzymes have been joined to the antigen-binding domains of antibodies to make enzymes that can target blood clots or tumors. In addition, adhesion molecules have been joined to the F_c region of antibodies to block infection. This approach is in fact favored by nature to add new functions to proteins, since many proteins contain several different functional domains on the same polypeptide chain (e.g., the serine proteases and associated activator proteins in the blood clotting system) (Fersht & Winter, 1982).

272. Several requirements are necessary before this technology can be applied effectively to any specific system (e.g., cellulases): (i) knowledge of the three-dimensional structure, either directly by x-ray crystallography or indirectly through homology considerations; (ii) knowledge about the mechanism of action, and (iii) access to the cloned cDNA and an expression system. In the case of cellulases, items i and ii are satisfied in a number of instances. As part of a successful protein engineering effort it may be necessary to study additional cellulases at this level. Concerning item iii, several cellulases have been cloned and sequenced, and numerous systems are available for overexpressing these proteins in bacteria or yeast (Beguin, 1990). Thus the cellulases should be amenable to study and improvement by recombinant DNA approaches.

273. The cellulases identified by screening procedures under "cellulases of the third generation" (section L) will provide a convenient starting point for mutagenesis. It is expected that any individual cellulase may have appropriate characteristics with respect to one or even two of the four critical parameters listed above, but most likely not in all four. Three general methods can then be applied for combining or imparting desirable characteristics: random mutagenesis, site-directed mutagenesis, and domain combination.

274. As discussed, cellulases are composed of several different kinds of enzymes. No known organism can efficiently degrade native cellulose with a single enzyme species. Most bacteria have at least three types of cellulases consisting of 1) endoglucanases that cleave cellulose at random locations, 2) exoglucanases that act on the non-reducing ends releasing glucose or cellobiose, and 3) beta-glucosidase that hydrolyzes cellobiose to glucose. Fungal organisms, in general, have endoglucanase and beta-glucosidase along with cellobiohydrolase which liberates cellobiose from the non-reducing ends. These enzymes act synergistically, and since all members are essential (Beguin, 1990), all classes of cellulases will probably require modification of their properties.

275. In the following sections it is assumed that the genetic engineering discussed will pertain to all members of the cellulase family and, unless otherwise indicated, they will be considered equivalent. This concept is valid since over 50 fungal and bacterial cellulases have been sequenced and the structure of many of these enzymes is composed of domains that are more or less conserved. Interestingly, these domains are integrated in different orders in various proteins. The catalytic domains are usually the longest (between 250-300 residues). These domains, also called catalytic cores, behave as independent entities endowed with catalytic activity and defined specificities toward soluble model substrates. Although the cores exhibit considerable diversity, they can be ordered into six families based on hydrophobic cluster analysis. The noncatalytic domains, which are involved in substrate binding, are connected by a 10-30 amino acid segment that is rich in proline and hydroxy amino acids. In at least one case this stretch is heavily O-glycosylated (Beguin, 1990).


1. Increased Stability - General Considerations


276. The following discussion pertains to results that are relevant to all proteins, with subsequent recommendations that are more directly related to the problems associated with cellulases.

277. Two types of protein stability are of interest in this regard - thermodynamic and long-term. Thermodynamic stability is associated with the process of a peptide transforming from its folded, native state to an unfolded, denatured form. This process is considered reversible. The free energy of denaturation can be determined empirically, and in general the higher the free energy of activation the more stable the protein. By comparing the parameters for different enzymes and their mutants and for chemically modified forms of the enzyme, it is sometimes possible to determine the molecular basis of thermodynamic stability, thus suggesting ways for its improvement. Stability can also be studied by considering a two-step process of irreversible inactivation. The native protein can be denatured into a reversibly unfolded form as described above. This form can then undergo a further step resulting in an irreversible, inactivated form of the enzyme. There are several mechanisms of inactivation including 1) aggregation (e.g., formation of intermolecular disulfide bonds); 2) changes in primary structure (e.g., oxidation of Cys, Trp or Met or hydrolysis of peptide bonds by H⁺ or proteases; 3) loss of coenzyme from the active site; 4) dissociation of oligomeric proteins into monomers; 5) absorption on the surface of the vessel; and 6) incorrect refolding. In any case stabilization can be considered as a problem of suppression of initial unfolding and/or deceleration of irreversible folding (Mozhaev, 1993).

278. Two successful processes that inhibit unfolding have been used. Conformational motions in proteins that lead to unfolding can be constrained by immobilization or through the introduction of new stabilizing interactions by protein engineering. Among the immobilization methods tested, the most successful are those that provide multi-point attachment of the protein to a support, since stabilization often correlates with the number of bonds involved. Site-directed mutagenesis can also prevent unfolding. In general, there is a marginal preference for the native conformation over the unfolded, as a consequence of the delicate balance of stabilizing and destabilizing forces. Unfavorable amino acid changes that perturb the packing within the protein core can often be compensated for by structural adjustments of other regions of the protein molecule. The net result is the preservation of the main backbone conformation with minimal energetic loss. Stabilizing strategies include introduction of electrostatic and polar interactions (the remarkable thermostability of B. licheniformis alpha-amylase was found to be due to additional electrostatic interactions in this enzyme involving a few specific lysines; Quax et al., 1991), intensification of hydrophobic interactions, and decrease in conformational entropy of unfolding and protein unfolding. Uncompensated polar or charged residues rarely occur inside protein molecules. However, if encountered, they can be satisfied by the presence of polar or oppositely charged residues in the vicinity that have been introduced by amino acid replacements to form a stabilizing internal hydrogen bond or a salt bridge. External electrostatic interactions add overall stability (<0.5 kcal/mol). Greater stabilization (1 kcal/mol) can be achieved if a charged residue is introduced in the vicinity of alpha helices to compensate for their unsatisfied dipole, or between pairs of aromatic residues or between an aromatic residue and histidine (Mozhaev, 1993).

279. Polar residues inside protein molecules can be replaced by non-polar ones, or if cavities are present in a protein, they can be filled by a non-polar group. The energetic gain from such a replacement should be proportional to the increase in the number of hydrophobic contacts. The addition of new hydrophobic contacts into the non-polar protein core can significantly increase stability. However, the hydrophobic cores of proteins are usually densely packed and normally there is insufficient free volume for creating new non-polar contacts without concomitant formation of undesirable cavities (Mozhaev, 1993).

280. A decrease in entropy of unfolding can be accomplished by replacing amino acid residues with high conformational flexibility (e.g. Gly, Ser, Ala) with more "rigid" residues (e.g. Thr, Val, Pro). Such substitutions can stabilize the protein by about 1 kcal/residue changed (Mozhaev, 1993).

281. The most effective amino acid replacements determined from comparison of amino acid sequences going from mesophiles to thermophiles are 1) Lys to Arg; 2) Ser to Ala; 3) Gly to Ala; 4) Ser to Thr; 5) Ile to Val; 6) Lys to Ala; 7) Thr to Ala; 8) Lys to Glu; 9) Glu to Arg; 10) and Asp to Arg. These substitutions among thermophiles tend to increase hydrophobicity and decrease flexibility. This is highly significant in helices but not in beta sheets. In particular, the replacement of Gly with Ala in alpha helices is very efficient for improving thermal stability. However, different proteins have evolved different structures to achieve the same result. For example, in LDH more Ser to Ala and Lys to Arg interchanges are most prevalent whereas for G3PDH Gly to Ala and Ser to Ala changes predominate. In summary, the results of evolutionary mutations appear to have increased rigidity and hydrophobicity in alpha helical segments. The helices became more rigid and tightly packed as a result of increased hydrophobic contacts. The introduction of alanine and removal of glycine favor this trend of closely packed helices. The empirical rules proposed in order to increase thermostability include 1) location of helices in the tertiary structure; and 2) concentration of these helices by decreasing flexibility and increasing hydrophobicity (Goodenough & Jenkins, 1991).

282. Introduction of disulfide bonds by replacing pairs of residues located close to each other with cysteine often results in increased stability. One potential drawback to introducing disulfide bonds is that they can accelerate inactivation due to the chemical instability of the -SH groups following reduction. An alternative is to crosslink proteins chemically with bifunctional reagents (Pace, 1990; Goodenough & Jenkins, 1991; Mozhaev, 1993).

283. In order to increase stability, some of the irreversible processes are amenable to manipulation. Aggregation and other bimolecular processes are blocked by eliminating protein diffusion, which can be achieved by immobilization. Enriching the protein surface with similarly charged groups can also reduce protein-protein interactions (Mozhaev, 1993).

284. Chemical decomposition can also be addressed. High temperature instability can be caused by chemical deterioration of amino acids such as Cys and Met. Therefore, replacement of any nonessential Cys or Met residues with Ala can increase the stability of some enzymes (Mozhaev, 1993).

285. Asparagine and glutamine are the two main sources of amide groups in proteins and are also two of the most commonly occurring amino acid residues. The side chains do not ionize but are relatively polar. During the deamidation reaction the side chain amide linkage is hydrolyzed to form a free carboxylic acid. Deamidation of a protein introduces a new negative charge that may influence tertiary structure and stability. Deamidation of Asn occurs most frequently at Asn-Gly or Asn-Ser bonds (the deamidation of Asn at Asn-Gly in peptides occurs more rapidly than deamidation of Asn alone). Peptides in which a Ser residue follows an Asn undergo a peptide cleavage reaction at about 10% of the rate of deamidation. Thus, it may prove beneficial to remove or minimize potential deamidation sites in cellulases, especially since the rate of deamidation increases with temperature. Hydrophobic amino acids adjacent to Asn decrease deamidation. The deamidation of Gln occurs at a frequency far less than at an Asn residue. However, engineering cellulases such that deamidation of Gln is less likely to occur may increase the stability of the enzyme (Liu, 1992).

286. Irreversible inactivation by incorrect refolding can be inhibited by stabilization of the reversibly folded state rather than the native conformation. This can be accomplished by introduction of hydrophilic functions onto the protein surface. These hydrophobic "anchors" can reduce the contact area of the exposed non-polar residues with water in the reversibly unfolded state, thereby preventing incorrect refolding (Mozhaev, 1993).

287. Another stabilization method involves addition of low molecular weight compounds to the media. A common example of this is substrates and inhibitors that bind specifically to the native conformation of an enzyme and increase stability. The stability enhancement depends on the binding constant and the concentration of the compound that binds. It is best to use a compound that can be added at moderate concentrations and whose binding does not interfere with the enzyme's function. This is probably best achieved by adding sites on the surface of the protein that bind either cations or anions. For example, the conformational stability of RNase T1 is increased by 2 kcal/mol in the presence of Ca++ ions. At the other extreme the melting temperature of alpha-lactalbumin is increased by 33.8° C in the presence of calcium (Pace, 1990; Giafridea & Scarfi, 1991; Mozhaev, 1993).


2. Thermostability of Cellulases


288. The most direct way to obtain enzymes that are optimally stable is to isolate them initially from thermophilic organisms. Indeed, several cellulases have been isolated from thermophilic organisms, including Trichoderma harzianum and Myceliophthora thermophila. The enzyme from the latter organism has a half-life of 3 days at 65°C. The recently-determined crystal structure of the cellulase from the thermophilic bacterium Clostridium thermocellum (Juy et al., 1992) will facilitate modelling for potential site-directed mutagenesis. Although the cellulases isolated from these thermophilic organisms exhibit temperature maxima at more than 70° C, they appear to be unstable at these elevated temperatures for extended periods of time in vitro, e.g., a half-life of 70 h at 75° C for Acidothermos cellulolyticus (Tucker et al., 1989).

289. As suggested above, one method to enhance thermal stability is to introduce disulfide bonds by substituting various amino acids with pairs of cysteine residues. Knowledge of the three-dimensional structure permits introduction of the cysteine residues such that the newly formed disulfide bond does not interfere with the active site of the enzyme. In addition, the disulfide bonds should be placed in regions that minimize disruption of the protein structure while maintaining the strain energy as low as possible. This can be done by involving flexible parts of the enzyme such that the side chain and backbone can freely adjust to allow for optimal geometry and to minimize any perturbations caused by the cysteine substitutions. Flexibility is indicated by high crystallographic thermal factors and evidence for large-scale mobility. Among these regions of proteins are the N- and C-termini. Rigid regions of the protein, such as alpha-helix or beta-sheets are not suitable domains for introducing disulfides. The resulting disulfide bridge loop should also be large. In the case of T4 lysozyme thermal stability was increased by introducing cysteines and creating disulfide bonds at positions 9-164 and 21-142 (T4 lysozyme has 164 residues). Each pair of substitutions increased the Tm by more than 6° C, but did not affect catalytic activity (Matsumura et al., 1989).

290. An exposed lysine in the glucose isomerase from Actinoplanes missouriensis is involved in dimerization and is susceptible to glycation (nonenzymatic) which may be involved in destabilizing the enzyme. Since glucose is a product of cellulases and the cellulosome is a multienzyme complex, mutations of exposed lysine residues may enhance thermal stability. Changing the lysine residues to arginines reduces the possibility of glycation while maintaining the chemistry required for dimerization. Such mutations increased the half-life of the immobilized glucose isomerase from 607 to 1550 hours at 60° C (Quax et al., 1991).

291. The crystal structure of the C. thermocellum thermophilic cellulase reveals the presence of 12 alpha helices (Juy et al., 1992). Experiments dealing with small single chain proteins indicate that substitutions of a glycine residue can stabilize the enzyme by reducing the conformational entropy of the unfolded state or by direct effects on the folded state. Glycines occur with a low frequency in alpha helices and can often act as helix breakers. Substituting glycines with serine or threonine has been shown to increase the stability of enzymes (Pace, 1990).

292. Exchange of hydrophobic residues with hydrophilic residues at solvent-exposed locations is also stabilizing. Thus, for example, any alanine residues exposed to water may be substituted with a serine to increase stability (Mozhaev, 1993).

293. Immobilization of the enzyme may also increase thermal stability. Immobilization is defined as a process whereby the protein is physically located in a certain region of space or converted from a water soluble, mobile state to a water-insoluble, immobile one. The most widely used immobilization techniques are categorized according to whether the protein becomes immobilized through chemical binding or physical retention. Methods used are 1) binding of enzyme molecules to carriers by covalent bonds; 2) binding by absorptive interactions; 3) entrapment into gels, beads or fibers; 4) crosslinking or co-crosslinking with bifunctional reagents; and 5) encapsulation in microcapsules or membranes. Cellulases have been immobilized successfully with the following methods: 1) covalently linkage to agarose beads with cyanogen bromide; 2) absorption to cellulose via the cellulose binding domain; 3) entrapment in polyvinyl alcohol; and 4) encapsulation in PVA membranes by UV irradiation. All of these methods stabilize the enzyme against thermal inactivation (Giafreda & Scarfi, 1991).

294. Immobilization also increases operational stability as defined by the ability of an enzyme system to effect catalysis under given operating conditions. Operational stability is a function of the enzyme and carrier durability and the chemical and physical properties in the experimental environment. Operational stability of cellulase is further increased by the addition of lithium and ammonium sulfate to the enzyme solution. Immobilization also enhances storage stability of cellulases (Giafrida & Scarfi, 1991). Importantly, processes involving immobilization of the enzyme may decrease the concentration of product present during the reaction and therefore alleviate product inhibition problems.

295. It was demonstrated that glycosidation of two of the beta-glycosidases from species of Bacillus enhances enzyme stability. The genes for the same enzymes were expressed in bacteria and in yeast and analyzed at 70° C. The half-life of the enzymes expressed in bacteria were 10 and 5 min. The half life of the same enzymes expressed in yeast increased to 26 and 100 min, respectively. Therefore, expressing in yeast either the naturally glycosylated cellulase from fungi or from bacteria may result in an increase in stability (Dixon, 1991).

296. Protein stabilization has also been achieved by engineering metal binding sites into the enzyme. A metal can shift a protein's folding/unfolding equilibrium by interacting with higher affinity to the native state. A metal-chelating site consisting of two histidines separated by three residues engineered into an alpha-helix has been shown to stabilize a protein by 1 kcal/mol. The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein (Kellis et al., 1991). Since cellulase has 12 alpha helices, the potential for stabilization utilizing this technique is significant.


3. Enhanced Substrate Binding


297. As previously discussed, cellulases contain a cellulose binding domain (CBD) distinct from the catalytic domain that does not participate in catalysis. Besides serving as an anchor for the catalytic domain, the CBD can play a direct role in disrupting the cellulose fiber structure. These domains are generally between 30 and 130 amino acids long and are separated by a stretch of 10-30 amino acids rich in proline and threonine/serine residues. The CBD can be at either the C-terminus or the N-terminus of cellulases derived from the same organism. Thus, the CBD is at the N-terminus of an endoglucanase and at the C-terminus of the exoglucanase from Cellulomonas fimi (Beguin et al., 1992). The exogluconase from C. fimi exhibits extremely tight binding to cellulose even in the presence of high salt. This suggests that the interaction is hydrophobic, which is consistent with the conservation of four tryptophan residues in the CBDs of cellulases. The CBD of the C. fimi exoglucanase has been used in generating fusion proteins that can be immobilized non-covalently on cellulose. Proteins that have been engineered include alkaline phosphatase and a beta-glucosidase from Agrobacterium sp. The chimeric glucosidase retained over 40% of its activity when bound to cellulose and hydrolyzed a constant amount of substrate over a period of 112 h suggesting that no bound enzyme was leaching from the column, even at a flow rate of 15 ml/hr (Ong et al., 1989). Thus, preparing a chimeric cellulase using the CBD from the C. fimi exoglucanase should result in enzyme complexes that bind tightly to their substrate. Additionally, the binding of the CBD to cellulose can be enhanced by mutations that increase the hydrophobic interactions, i.e., substitutions of tryptophan, leucine, valine or isoleucine in regions thought to interact with the substrate.


4. Decrease in product inhibition


298. Since the products of the reaction of cellulases inhibit the catalytic efficiency of the native enzymes, decreasing the inhibition constant is beneficial. However, it is teleologically difficult to design such an enzyme since the product of the cellulase reaction is a hydrolyzed monomer of a polymeric substrate. It is probably easier to maintain the product formed at low concentrations so that it will not significantly inhibit the rate of reactions. This can be accomplished either by physically removing the end products from the (immobilized) enzyme or by chemically or enzymatically altering the products of the reaction such that they are no longer potent inhibitors. Since the three-dimensional structure of a cellulase has been determined, specific mutations can be introduced into the active site that decrease the affinity for the product without affecting (or hopefully even enhancing) the affinity toward the substrate. There are no general guidelines as to what mutations may be beneficial so random mutagenesis of the active site is the more promising approach.

299. Since the crystal structure of a cellulase from a thermophile has been determined, computer modelling may prove helpful in designing site-specific mutations.


5. Laboratory protocols


300. Due to the large number of mutations that can potentially be performed in an attempt to improve cellulases with respect to thermostability, operational stability and functional properties, the following criteria have to be met. One has to generate a large number of specified and random mutants. These mutants must be expressed and rapidly isolated in order to obtain information regarding the desired optimization. Finally a rapid and quantitative assay for screening the large number of generated mutants is essential. Once the enzyme(s) with the optimized characteristics have been generated, the next step is the scaling up of the procedure for industrial applications.


6. Generation of mutants


301. The polymerase chain reaction (PCR) is currently the method of choice for generating specific (Ho et al., 1989) and random mutations (Leung et al., 1989). The PCR reaction requires a clone coding for the desired protein, a machine to generate oligonucleotides of a specified sequence, a machine to perform the PCR, and supplies for generating oligonucleotides and the PCR. With the exception of obtaining the clone, all the materials are commercially available. For both types of mutations oligonucleotides complementary to the 5¢ end and identical to the 3¢ end are prepared and used as primers for the reactions. For specific mutations, a complementary set of oligonucleotides spanning the region of mutation is generated incorporating desired changes. PCR is performed under standard conditions that will normally generate a DNA fragment containing the clone with the desired changes. For random mutagenesis the conditions of the PCR reaction are altered such that the ability of the enzyme to make perfect copies of the clone's nucleotide sequence is compromised.

302. It is necessary to sequence the individual clones to ensure that the desired change in amino acid sequence has taken place. In addition, the nucleotide sequence of the randomly mutated clones which have exhibited the desired properties needs to be sequenced. Materials for determining the nucleotide sequence of a cloned piece of DNA are commercially available and will not be discussed here.

303. Genetic engineering protocols obtain a large number of different forms of the same enzyme, each of which has to be assayed separately. Simplification of the assay and the ability to do multiple assays are essential in order to screen all of the samples for the desired properties. For example, cellulases could be assayed on large sheets of filter paper impregnated with a compound that releases a dye when cleaved by the enzyme. The sheets could be spotted with numerous enzyme solutions and then incubated under conditions to assess the effectiveness of modifications (e.g., elevated temperature).

304. Work in the protein/genetic engineering area requires a DNA synthesizing machine for generating oligonucleotides. At least 4 PCR machines, 2 orbital lab environmental shakers, several regular and mini gel apparati and 500 V power supplies typically are used for preparation and analysis of mutants. Four sequencing gel apparati and two 3000 V power supplies are required for sequencing the site-directed mutants. Also needed are a -70° C freezer, other freezers, refrigerators, temperature controlled incubators and water baths. This work also requires the use of radioisotopes so space for the handling and proper disposal of radioactive materials must be provided. Analytical tools such as acrylamide gel apparatus, high performance liquid chromatography instruments, gravity flow columns, and a walk-in cold room are also essential. In addition a speed-vac/lyophilizer, dark room and x-ray film processing must be available. All of this equipment and the materials necessary to operate them are commercially available.


7. Recombinant Insulin: An Example


305. As an example, this section gives a brief history of one of the first commercially developed recombinant proteins, human insulin. Most of the material in this discussion is from the review by Ladisch & Kohlmann (1992). A large amount of insulin is needed in the United States. The market for human insulin is 100 times higher than that for another recombinant protein, the human growth hormone, which has a current usage of several kg/yr. Human insulin is required for treatment of the majority (>70%) of the estimated 12 million diabetics in the United States.

306. Insulin is a hormone consisting of two different peptide chains: the A chain (21 amino acids) and the B chain (30 amino acids) joined by disulfide bridges. Proinsulin is the biological precursor of insulin and is a single peptide chain formed when the A and B chain are connected by a third peptide (the C chain). The first recombinant expression of human insulin in bacteria was accomplished by Eli Lilly and Genentech in 1978. Each insulin peptide was expressed as a beta-galactosidase fusion protein and obtained from separate fermentations. The chimeric proteins appeared in inclusion bodies. The method used to extract the peptides from the inclusion bodies is proprietary information. Once removed from the inclusion bodies the insulin peptides were chemically cleaved from beta-galactosidase. The peptides were then combined in the presence of a mercaptan in order to obtain active hormone with a yield of approximately 60%. Because the beta-galactosidase is a large protein (~1000 amino acids), portions of the expressed fusion proteins were incomplete. A different set of fusion proteins using the Trp E gene (only 190 amino acids) and the trp promoter instead of the beta-galactosidase system increased expression 10-fold. The isolation of the insulin A and B peptides is essentially the same as for the beta-galactosidase fusions. There is a patent on the solubilization of the peptides from the inclusion bodies that results in a protein that is 95% pure.

307. Human insulin can also be produced with recombinant organisms that make intact proinsulin instead of the A and B chains separately and is the current method of choice for insulin production. It has the major advantage that only one fermentation and purification is required instead of two (one each for the A and B chains). In this case the proinsulin chain is subjected to a folding process that allows intermolecular disulfides to form, followed by enzymatic removal of the C peptide. Work is currently underway to express and isolate human insulin from yeast in an attempt to improve yields.

308. Analysis of the insulin preparations is usually performed by reverse-phase high-performance liquid chromatography (RP-HPLC) over alkylsilane supports. This procedure can separate insulin species that differ by only one amino acid.

309. Large-scale purification of insulin requires a somewhat different methodology than that used in conventional protein purification. An exceedingly high degree of purity is required. The product must be free of contaminating solvents, nutrients, metabolites, fusion proteins and other forms of insulin (e.g., proinsulin and deaamido insulin) that might be immunogenic. It is estimated that more than half of the processing costs are in the downstream purification relative to the actual purification. One published procedure for insulin purification involved ion-exchange chromatography followed by RP-HPLC and then a size exclusion step.

310. Insulin is a therapeutic protein with a history of production using recombinant DNA technology. Improvements in genetic engineering methods have facilitated the production and recovery of insulin. While the current methods yields highly purified protein, further refinements in separation is an area of continuing research. This reflects the demand for therapeutic proteins with minimal contamination and the need to reduce the high cost of purification (Ladisch & Kohlmann, 1992).

311. It is obvious that the development of new procedures for the specific construction of industrial enzymes, including cellulases, is feasible. The molecular characteristics of the "fourth generation" enzymes will be modulated by specific changes in the primary structure of enzymes produced by genetic engineering.


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