The Cellulase System of - American Chemical Society

12 The Cellulase System of Trichoderma

Downloaded by UNIV OF AUCKLAND on December 18, 2014 | http://pubs.acs.org Publication Date: June 1, 1979 | doi: 10.1021/ba-1979-0181.ch012

Relationships Between Purified Extracellular Enzymes from Induced or Cellulose-Grown Cells M I K E L I N A GRITZALI and ROSS D . B R O W N , JR. Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, V A 24061

The use of sophorose to stimulate production of the cellulase enzyme system of Trichoderma reesei QM 9414, has clarified the nature of the individual enzyme components. The multiple forms of cellobiohydrolases, endoglucanases, and β-glucosidase observed among the extracellular enzymes produced by strains of T. viride and T. reesei have compli cated interpretation of the respective catalytic roles of these proteins and their biosynthetic origins. The rapid produc tion of the system in response to 1mM sophorose has allowed isolation of the three principal components of the nascent mixture. An endoglucanase and Cellobiohydrolases I (D) and II comprise about 15%, 60% and 25%, respec tively, of the cellulase proteins. Cellobiohydrolase I, form D, is distinctly different from the newly described Cellobio hydrolase II. Other proteins, such as β-glucosidase, can account for only about one percent of the total protein. This eliminates the possibility that there is induced with the cellu lase system a significant complement of additional enzymes concerned with the degradation of either cellulose or other components of wood.

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified β-glucosidases ( E C 3.2.1.21), endo-l,4-0-r>glucanases ( E C 3.2.1.4) and 1,4-β-Ό0-8412-0460-8/79/33-181-237$06.00/0 © 1979 American Chemical Society

In Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid Catalysis; Brown, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1979.


238

HYDROLYSIS OF CELLULOSE

glucan cellobiohydrolases ( E C 3.2.1.91) ( C B H ) , which together comprise a system capable of rapidly and effectively converting cellulose to glucose. Each of these enzymes is a glycoprotein and catalyzes both hydrolytic and transglycosylation reactions. Although there have been several reported isolations of multiple forms of these enzymes, the structural, functional, and biosynthetic relationships between them remain unclear (3,15,16, 17). A central goal of cellulase research has been to determine the role of each enzyme (or enzyme form) and to establish the number of com ponents necessary and sufficient for the effective conversion of cellulose to glucose. This report reviews molecular aspects of these enzymes and presents information regarding the catalytic properties and induction of some enzymes elaborated by the strain Trichoderma reesei Q M 9414. The sequential and cooperative roles of the enzymes are shown in Figure 1. Endoglucanases, often called cellulases or carboxymethylcellulases, provide nonreducing chain ends from which the exoCBH's cleave cellobiosyl residues. The cellobiose, which is inhibitory to the depolymerizing enzymes, is cleaved to glucose by the β-glucosidase. Since the product of one enzyme may become the substrate for another, enzyme purification is an essential prerequisite to any clear description of the system. ^ CELLULOSE

ENDOGLUCANASES • ΕΧΟ-CELLOBIOHYDROLASES

OLIGOSACCHARIDES

Figure 1.

^GLUCQSIDASE

OLIGOSACCHARIDES + GLUCOSE

^

Cooperative action of enzymes of the cellulase system in hydro lytically converting cellulose to glucose

Our overall strategy for purification is shown in Figure 2. When applied to a commercial cellulase preparation, an initial affinity column of microcrystalline cellulose results in separation of the enzymes into two classes. Elution with buffer removes low-affinity components collectively termed the "buffer fraction"; subsequent elution with distilled water removes tightly bound components comprising the "water fraction." The buffer fraction contained a β-glucosidase and two endoglucanases, which were further purified by ion exchange chromatography. Those enzymic components eluted with water included the cellobiohydrolase and two tightly bound endoglucanases, which were purified by ion exchange chromatography and preparative gel electrophoresis.


12.

239

Trichoderma Cellulase System

G R i T Z A L i AND BROWN

CRUDE

ENZYME

1

MICROCRYSTALLINE CELLULOSE

1 LOW AFFINITY ' B U F F E R FRACTION' ENZYMES


HIGH AFFINITY 'WATER FRACTION' ENZYMES

BATCH

1

BATCH

SEPARATION

Τ

1

SEPARATION

Ε Χ Ο - CELLOBIOHYDROLASE

1 CHROMATOGRAPHY!

CHROMATOGRAPHY

ιf /3-GLUCOSIDASE

ENDOGLUCANASES Π Ι

Figure 2.

ENDOGLUCANASES I

AND EZ

AND E

Schematic diagram of the general procedures used in purify ing cellulase system components (1, 2, 4)

The categories of substrates which are used for assays of cellulase enzymes are shown in Table I. The use of crystalline, insoluble forms of cellulose as substrates makes assays difficult and has led to such trivial names as "Avicelase" activity. These assays are useful as indications of the capacity of an enzyme system to degrade native cellulose and indicate the presence of C B H enzyme which cannot be assayed in the presence of endoglucanases or β-glucosidase. The susceptibility to enzymatic attack generally increases with the hydration of the polymer chains that accom-

Table I.

Cellulase Substrates

Insoluble Polymers hydrocellulose Avicel α-cellulose filter paper swollen cellulose Walseth cellulose Soluble Polymers derivatized cellulose carboxymethylcellulose Oligomers cellooligosaccharides

cellopentaose



240


panies loss of crystallinity. Seldom are the specific molecular changes caused by enzymic action measured; instead, reducing-sugar production or changes of viscosity or turbidity are reported, leading to nonlinear kinetics. Among the cellulase substrates, only the cellooligosaccharides have specific (i.e., not statistically described) ιηοΐβομ^ properties. It has been useful to compare the activities on oligomers of enzymes that are involved in depolymerization and saccharification of polymeric sub strates. For such studies, oligosaccharides of >99% purity are required to permit evaluation of initial reaction products. A typical separation of soluble cellooligosaccharides is shown in Figure 3. To identify the linkage cleaved in cellooligosaccharides, we employed reduction by sodium borohydride to provide the corresponding alditols. Sugar alcohols subse quently formed by enzymic action will be indicative of origination from the position usually occupied by the reducing end. The entire series of normal and reduced oligosaccharides was separated isocratically by high performance liquid chromatography ( H P L C ) (IS).

I

Ο

I

4

I

I

8

ι

I

I

12

I

16

I

I

20

I

I

24

TIME (min) Figure 3. Separation of cellooligosaccharides using the Waters Associates column method (18). The solvent system employed was acetonitrile-water (72:28) at aflowrate of 4.0 mL/min.


GRiTZALi

AND BROWN

Trichoderma Cellulose System

241


12.

Figure 4. Polyacrylamide-disc gel electrophoretic patterns obtained for forms of CBH I from Trichoderma (2). The protein samples applied to these gels were, from left to right: 20 pg CBH I (A), 15 \x.g CBH I (B), 33 μg CBH I (C), and 40 μg CBH I (D). Forms A, B, and C were puri fied from a commercial T. viride cellulase preparation and Form D was purified from a culture of T. reesei CM 9123 grown on purified cellulose.


242



1,4-fi-T>-Glucan Cellobiohydrolases CBH's are the most abundant components of the Trichoderma cellulase system. They are glycoprotein exoglucanases that produce cellobiose. Four forms of C B H previously have been purified (2) and characterized. Forms A, B, and C were derived from a commercial T. viride cellulase preparation, whereas Form D was produced by T. reesei Q M 9123 grown in submerged culture. Polyacrylamide disc gel electrophoretic patterns obtained from the four CBH's are shown in Figure 4. These acidic proteins have similar amino acid compositions, molecular weights, ultraviolet absorption spectra, and all cysteine residues linked by disulfide bonds. Some of the properties of these enzymes are summarized in Table II. It was of interest to determine if the small differences in amino acid composition between C B H D from T. reesei and the CBH's from T. viride were significant. The principal structural differences between the four C B H forms are the respective neutral carbohydrate content and composition (2). In each case mannose was found to be the principal neutral sugar component. Analysis of the oligomeric carbohydrate side chains of C B H C, after their removal by ^-elimination, revealed that the mannosyl units are linked by 1 - » 6 glucosidic bonds, at least some of which have anomeric carbon atoms with a-configuration (19). For each C B H C molecule, there are about 17 side chains of one to five monosaccharide units in length, which are linked to the protein by O—mannosyl bonds to serine and threonine residues. Immunological studies revealed cross reactivity between all four of these C B H forms and small spurs of "nonidentity" for forms A, B, and D when tested Table II.

Properties of the Forms of Cellobiohydrolase I Forms A

Molecular weight (by thin-layer gel-filtration chromatography )

53,000 ± 5,000

Neutral carbohydrate content (wt %)

1.4

Glucosamine (wt % as iV-acetylglucosamine)

0.8

Thermal stability (activity remaining after 30 min at 60°C) Cellobiose produced from swollen cellulose (wt % of soluble products after 30 min incubation at 40°C) Specific activity (/miol reducing sugar released from swollen cellulose/min/mg protein)

64 > 90

1.01



12.

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AND BROWN

243

against antiserum to form C. The β-glucosidase and C B H II described in this report do not react with antiserum to C B H C whereas at least one endoglucanase (IV) does. Thus CBH's A, B, C, and D are structurally related forms of C B H I. The activity of the CBH's increases with chain length of oligomeric substrates up to cellotetraose, but remains essentially constant for cellotetraose, cellopentaose and cellohexaose, indicating that this enzyme may have a limited number of subsites. Examination of the frequency of bond cleavage of normal and reduced oligosaccharides by C B H reveals that: (1) there is a lack of absolute specificity for terminal cellobiosyl groups, and (2) there is little tendency to cleave bonds linking the sorbityl residue at the former reducing end of the substrate (20). Endo-1 >4-fi-T>-glucanases Of the four glycoprotein endoglucanases purified from T. viride, three were obtained in quantities large enough to provide some structural information (3,4). Endoglucanases I and II have a low affinity for cellulose, whereas Endoglucanases III and IV bind tightly to a column of microcrystalline cellulose. These enzymes are distinguished from the other types of enzymes in the cellulase system by their ability to reduce the viscosity of CM-cellulose solutions. A summary of some of the properties of the endoglucanases is presented in Table III. Endoglucanase II has the highest specific activity (expressed in terms of change in specific fluidity/min/mg protein). Disc gel electrophoresis of the endo glucanases yields a broad but distinctly different band for each enzyme. Each has a similar p H optimum and is labile above p H 8.0. EndoglucaCellobiohydrolase I of Trichoderma

(2)

Cellobiohydrolase I Forms B

C

D

53,000 ± 5,000

53,000 ± 5,000

53,000 db 5,000

5.8

10.4

6.7

0.8

0.5

0.5

68

65

65

> 90

> 90

> 90

0.67

0.56

0.53


244


Table III.

Endoglucanases of Trichoderma viride

(3,4)

Endoglucanase II Molecular weight

52,000

49,500

1010

60

250


Specific activity (/*mol reducing sugar/min/mg protein) substrate: CM-cellulose phosphoric-acid swollen cellulose Avicel Stability Neutral carbohydrate content (wt %)

IV

37,000

Specific activity (Δ specific fluidity/ min/mg protein) substrate: CM-cellulose

p H optimum

III

28 9.6 0.1

28 9.9 0.1

9 7.4 0.1

4.5

4.5

4.5

labile at p H > 8.0

labile at p H > 8.0

labile at p H > 8.0

4.5

15.0

15.2

nase II has a distinctly lower molecular weight as determined by sedimentation equilibrium ultracentrifugation. The amino acid composi tions are similar, particularly those of Endoglucanases II and IV. Since the former exceeds the latter only with respect to apparent leucine and and methionine content there is a real possibility of a precursor/product (IV/II) relationship. The high affinity forms, Endoglucanases III and IV, have the highest neutral-carbohydrate content, about 14^15% by weight as determined by the phenol-sulfuric acid method of Dubois et al. (21) or by gas chromatography of derivatives of the neutral sugars (22). A useful comparison of the modes of action among endoglucanases is the plot of decrease in specific fluidity vs. concomitant production of reducing sugar (3). Endoglucanases II and IV, like liquefying amylases, effect a large change in specific fluidity relative to Endoglucanase III, which is analogous to "saccharifying" amylases. C B H C, by comparison, has essentially no endoglucanase activity and must be assayed at a concentration ten-fold higher than that of the endoglucanases in order to cause a measurable increase in fluidity. The endoglucanases exhibit higher maximum velocities on longer cellooligosaccharides, as expected for "endo" action. These enzymes have little or no xylanase or amylase activity but display the highest specific activity (pmol reducing sugar released/min/mg protein) when acting on CM-cellulose. Activity on swollen cellulose is much greater than that on Avicel. The transglycosylation activity of Endoglucanase IV is demon-



12.

GRiTZALi

AND BROWN


245

strated by the formation from cellopentaose of hexa-, hepta-, and octasaccharides in addition to the hydrolytic products cellobiose and cellotriose. Since both the shorter and longer oligosaccharide products are substrates for the endoglucanases, the pattern of products from the cleavage of longer oligomers (e.g. cellohexaose) changes with time as the sugars produced initially are hydrolyzed further (3). Thus the array of products observed depends on the extent of the reaction. The initial reaction rate may be measured as the decrease in substrate concentration per unit time. To elucidate the specificity of bond cleavage, one must note the array of products formed after only a few percent of substrate has reacted. Endoglucanase III, unlike Endoglucanases II and IV, cleaves or transfers cellobiosyl units from the nonreducing ends of substrates. The use of reduced oligosaccharides to permit identification of the bond cleaved was illustrated by the action of Endoglucanase IV on cellotetraitol and cellopentaitol (3). In order to demonstrate directly the transfer of cellobiosyl units by Endoglucanase III and to confirm the origin of these units as the nonreducing ends of oligomers, cellotetraitol and cellopentaitol were again employed as substrates (3). The latter yielded cellotriitol whereas from the former was produced cellobiitol together with cellotriose and cello triitol without corresponding quantities of cellobiose, glucose, or glucitol. Rechromatography of the cellotetraitol mixture revealed a hexaitol peak which could serve as an intermediate (formed by cellobiosyl transfer) by cleavage to equal amounts of G and G H . This would account for the production of these trisaccharides without concomitant production of glucose or glucitol. To determine the roles of the endoglucanases in the degradation of insoluble cellulose, we incubated each of them with filter paper discs for two days (3). As seen in Figure 5, only Endoglucanases III and IV reduced the discs to short fibers. This demonstrates the importance of the high-affinity enzymes that, although less active in degrading C M cellulose, are effective in degrading pure cellulose. 3

3

β-Glucosidase The β-glucosidase is a glycoprotein which cleaves ^-linked glucose oligomers as well as aryl-/?-D-glucosides (1,20) and transfers glucosyl units. Since the majority of the glucosidase and cellobiase activities from a commercial T. viride cellulase preparation copurified, it was con cluded that one enzyme is responsible for these two types of activity. Two minor components having aryl glucosidase activity are removed during the purification process. The amino acid composition of the β-glucosidase reveals that it is less acidic and more hydrophobic than



246


Figure 5. Reaction mixtures demonstrating short-fiber-forming activities of endo-l,4^-O-glucanases. The reaction mixtures are shown after two weeks incubation at 40 °C with 20 mgfilterpaper (3). Represented from left to right are a control without enzyme, Endoglucanase IV (235 μg/ mL), Endoglucanase III (87 μg/mL), and Endoglucanase II (75 μg/mL).

Figure 6. Transglycosylation activity of β-glucosidase. The reaction mixture initially contained 3% cellobiose, 0.0009% β-glucosidase, and 0.05M sodium acetate buffer, pH 5:0. Incubation was car ried out at 45°C in a recipro cating shaker. Each sample was boiled forfiveminutes and analyzed by HPLC (G = glucose, G = cellobiose, G = trisaccharides.) t

2

3

TIME (HRS.)


12.

GRiTZALi


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1 σ» 8 Ε < oc α >ι ο m oc

The Cellulase System of - American Chemical Society

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