Studies on the Mechanism of Enzymatic Hydrolysis of Cellulosic Substances T. K. GHOSE and V . S. BISARIA, Biochemical Engineering Research Centre, Indian Institute of Technology, Delhi, Hauz Khas, N e w Delhi-110029, Zndia

Summary Most cellulosic substances contain appreciable amounts of cellulose and hemicellulose, which on enzymatic hydrolysis mainly yield a mixture of glucose, cellobiose, and xylose. In this paper, studies on the mechanisms of hydrolysis of bagasse (a complex native cellulosic waste left after extraction of juice from cane sugar) by the cellulase enzyme components are described in light of their adsorption characteristics. Simultaneous adsorption of exo- and endoglucanases on hydrolyzable cellulosics is the causative factor of the hydrolysis that follows immediately after. It supports the postulate of synergistic enzyme action proposed by Eriksson. Xylanase pretreatment enhanced the hydrolysis of bagasse owing to the creation of more accessible cellulosic regions that are readily acted upon by exo- and endoglucanases. The synergistic action of the purified exoglucanase, endoglucanase, and xylanase has been found to be most effective for hydrolysis of bagasse but not for pure cellulose. Significant quantities of glucose are produced in p-glucosidase-free cellulase action on bagasse. Individual and combined action of the purified cellulase components on hydrolysis of native and delignified bagasse are discussed in respect to the release of sugars in the hydrolysate.

INTRODUCTION Enzymatic hydrolysis of native mixed cellulosic materials yields a mixture of glucose, cellobiose, and xylose plus minute amounts of cellodextrins. Bagasse has been chosen as a model substrate because of its abundance. The cellulose of bagasse is not effectively hydrolyzed by Trichoderma reesei (formerly called T . viride) cellulase owing to restricted availability of the cellulosic surface, which is crosslinked with hemicellulose to the enzymes. Enrichment of the Trichoderma cellulase with the xylanase component of Aspergillus wentii has been shown to increase the hydrolysis rate of bagasse. Since adsorption of enzymes on cellulosic surfaces is a prerequisite step for their hydrolysis, detailed studies of the process may lead to a better understanding of the mechanism of enzymatic *st

Biotechnology and Bioengineering, Vol. XXI, Pp. 131-146 (1979) @ 1979 John Wiley & Sons, Inc. 0006-3592/79/0021-0131$01.OO

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GHOSE A N D BISARIA

degradation processes. Here, the adsorption characteristics of purified cellulase components on bagasse are presented and discussed in the light of their role on the hydrolysis process. The action of enzyme components, when present alone and in combination, on the hydrolysis of bagasse is studied with special reference to the role of xylanase action on bagasse breakdown. The nature of adsorption of cellulose and its degradation by T. reesei have been adequately explained in a recent report from this Centre.3 MATERIALS AND METHODS

Cellulose used in the studies was the microcrystalline cellulose powder obtained from Cellulose Products of India Ltd., Ahmedabad. The bagasse used was obtained from a local sugar mill as a waste residue left after extraction of the juice from sugar cane (45% a-cellulose, 25% hemicellulose, and 20.5% lignin). One lot of this material was dried and stored in a cool dry place for use throughout the studies.

Enzyme Production Trichoderma reesei QM 9414 (obtained from the U.S. Army Natick Laboratories, Mass.) was grown on Mandels-Weber4 medium. Aspergillus wentii Pt 2804 (obtained from Institute of Microbiology, Swiss Federal Institute of Technology, Zurich, Switzerland) was grown on the following medium that contained in (glliter): fine straw, 20; malt sprouts, 10; K,HPO,, 1.4; MgS04.7H20, 0.76; NH,NO,, 1.0; and in (mg/liter): CaCO,, 5 ; NaCI, 5 ; ZnS04-7H20, 17.6; FeS0,-7H20, 18.2, and MnS0,.5H20, 15.8. The pH was adjusted to 5.4 and the culture was grown a t 24°C for five days. The cultures of T. koningii IMI 73022 and Fusarium solani IMI 95994 were obtained from Commonwealth Mycological Institute, Kew, Surrey, England. The medium used for the production of cellulases from the organisms was that described by Wood.5 Preparation of Mixed Enzyme The enzyme solutions were prepared by separately growing the cultures of T. reesei QM 9414, which produces significant amounts of endo- and exoglucanases, and A. wentii Pt 2804, which produces a high amount of xylanase enzyme, on their respective production media. The culture filtrates were separately precipitated by acetone (1 : 3 v/v) at S°C for 30 min and the precipitate was resuspended in

ENZYMATIC HYDROLYSIS OF CELLULOSICS

I33

O.05M citrate buffer, pH 4.8,so as to obtain enzyme solutions with the same protein levels. These solutions were mixed in 1 : 1 proportion (subsequently referred to as mixed enzyme) and used to determine the adsorption behavior of its components on fresh and spent bagasse. Protein Analysis

Protein was measured by the method of Folin6 using bovine serum albumin as a standard. Sugar Analysis

Reducing sugars were estimated by either the dinitrosalicylic acid' or by the Nelson-SomogyiR methods. Glucose and xylose were measured by the glucose oxidase9 and by the modified orcinol" methods, respectively. Total carbohydrate was determined by the phenol sulfuric acid method of Dubois et al." The amount of cellobiose and cellodextrins, reported as "cellobiose," was calculated by subtracting the amount of glucose plus xylose from total carbohydrate. Enzyme Activities

Enzyme activities were defined in terms of international units (IU) ( I pmol sugar formed/min). Exoglucanase (1-4,P-glucan-4cellobiohydrolase) activity was measured using dewaxed cotton (DP-3000) as a ~ u b s t r a t eThe . ~ measurement of exoglucanase activity depends on the synergistic action of both exo- and endoglucanases for solubilization of cotton. Hence, a sufficient quantity of purified endoglucanase (1-4, /?-glucan-4-glucanohydrolase)was added to the purified exoglucanase protein for its estimation. Endoglucanase and xylanase activities were assayed using carboxymethylcellulose (degree of substitution = 0.5) and xylan (from larch saw dust), respectively, as ~ u b s t r a t e s . P-Glucosidase *~~~ activity was measured with o-nitrophenyl-P-D-glucopyranoside as a substrate. l3 Preparation of Delignified Bagasse

Bagasse was delignified by mixing it with 1% NaOH in a ratio of I :7 (w/w) and heating the suspension at 80°C for 3 hr. l 2 The excess alkali was removed by repeated washings with distilled water until the pH was neutral. The delignified bagasse thus obtained was air dried at 40°C.

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GHOSE A N D BISARIA

Purification of Cellulase Components

Exo- and endoglucanases were purified by DEAE-Sephadex chromatography from a 20- 80% (NH,),SO, precipitated, desalted, and freeze-dried culture filtrate of T . koningii IMI 73022. The endoglucanase fraction, free from exoglucanase or P-glucosidase activities, was eluted in the starting buffer (0.1M formate-NaOH buffer, pH 4.8). Exoglucanase was desorbed from the Sephadex by shifting the pH from 4.8 to 3.5 using a linear gradient and was then found to be free from P-glucosidase activity but contained traces of endoglucanase activity; the specific activity of this purified exoglucanase against CMC being only 4.1 x lop3 IU/mg protein. The endoglucanase fraction contained a high-molecular-weight (47,000) and a low-molecular-weight (13,000) component as seen by Sephadex G- 100 chromatography. The low-molecular-weight component, the removal of which does not affect the kinetics of ~olubilization,'~ was separated by chromatography of the fraction on Biogel P-100 column and the pure high-molecular-weight endoglucanase component was used for subsequent studies. Xylanase was isolated from the culture filtrate of T. reesei by double chromatography on a Biogel P-100 column (4 1.5 x 41 cm).? The component had no action on the hydrolysis of cotton cellulose, CMC, or cellobiose as shown by the analysis of reducing sugars by the Nelson-Somogyi method. P-Glucosidase was purified by precipitating the culture filtrate of F . solani IMI 95994 by (NH,),SO, between the limits of 2 0 4 0 % saturation and carrying out gel filtration of this enzyme solution on a Sephadex G-100 column (4 2.5 x 80 cm) with 0.1M acetate-NaOH buffer, pH 5.0. Owing to the high molecular weight (400,000 daltons) of P-glucosidase compared to exo- and endoglucanases (45,000 and 37,000 daltons, respectively), it was obtained in pure form in the volume corresponding to the void volume of the bed. Adsorption of Cellulase Components

Bagasse (500 mg) was contacted with 10 ml enzyme solution in 0.05M citrate buffer (pH 4.8) for 15 min, and the amount of original activity remaining in the free solution was measured after removal of solids by vacuum filtration. RESULTS AND DISCUSSION

It has been dernon~trated'~ that the cellulase components isolated from many organisms act synergistically. This is characteristic of

ENZYMATIC HYDROLYSIS OF CELLULOSICS

135

cellulase components that are released freely into the culture broth. The purified cellulase components isolated from culture filtrates of three different organisms for the present studies are, therefore, also expected to behave in a similar fashion. It has been reported in a previous paperP that adsorption of the mixed enzyme components decreased with an increase in temperature from 5 to 50°C and with an increase or decrease of pH from 4.8. The specific adsorption of enzyme components (activity adsorbed/mg protein), on the other hand, was found to be maximum at 50°C and pH 4.8 (the conditions optimum for cellulose hydrolysis), suggesting that the active enzyme components were held more strongly onto the cellulosic substrate compared to nonactive ones. The interactions of the components present in the mixed enzyme, however, have not been identified in these studies. The four purified components, exoglucanase, endoglucanase, xylanase, and /3-glucosidase, were studied to determine the extent of their adsorption when present alone and in combination (Figs. 1 and 2 ) . Purified xylanase was adsorbed more rapidly than endo- and exoglucanases.

Fig. 1 . Adsorption of individual cellulase components o n bagasse at 5°C when present alone. Purified cellulase components (10 ml) ((0)exoglucanase, 3.8 x IUiml; (0) endoglucanase, 18.3 IUlml; (0) xylanase, 0.4 IUlml, and P-glucosidase, 0.26 IUlml) in 0.05M citrate buffer, pH 4.8, were individually contacted with 500 mg bagasse at 5°C and the activities were determined at various time intervals.

CHOSE A N D BISARIA

136

0

5

-

15 20 Time, rnin

I0

25

30

Fig. 2. Adsorption of cellulase components of bagasse at 5°C when present together. Experimental details same as in Fig. I , except that the enzyme solution contained all the four purified components in the mixture.

p-Glucosidase was not adsorbed at all up to 30 min. The adsorption of the components increased when present alone, suggesting that the cellulosic surface contains some common sites at which all of these components can be attached. Because of the appearance of sugars in solution owing to cellulose hydrolysis at higher temperatures, it has not been possible to obtain an equilibrium activity distribution between the solid cellulosic and liquid phases. However, because of the high initial adsorption rate, a contact time of 15 min was found to be sufficient by which maximum adsorption could take place. The enzyme started returning into the solution as a result of hydrolysis.15 Hence, for adsorption studies, lower temperatures were generally employed and the amount of adsorption after a 15 min contact period was taken as the maximum adsorbed value. The adsorption behavior can be represented by a first-order kinetics: dC,/dt

=

k(C,

-

C,)

where C , is the concentration of adsorbed enzyme at any time, t (IUig substrate); C , is the saturation concentration of adsorbed enzyme, taken as the amount of enzyme adsorbed after 15 min contact period (IU/g substrate), and k is the adsorption rate constant.

ENZYMATIC HYDROLYSIS OF CELLULOSICS

I37

Integrating

k = ( l / t >InI[Cs/(C, -

c, )I

From the plot of In k vs. 1/T (Fig. 3), values of the activation energy for different components have been computed as: exoglucanase, 1142 cal/g mol; endoglucanase, 807 cal/g mol, and xylanase, 365 caVg mol. The activation-energy values signify the physical nature of adsorption and the difference in the characteristic affinity of these components with bagasse. Adsorption Behavior on Fresh a n d Spent Bagasse

Following hydrolysis the spent bagasse had a different noncellulosics-to-hydrolyzable cellulosics ratio compared to the freshly ground (150-170 mesh) bagasse. A 5% bagasse suspension in contact with the mixed enzyme (6.4 X I U h l exoglucanase; 26.0 IU/ml endoglucanase, and 12 I U h l xylanase) for 48 hr produced 54% conversion based on initial hydrolyzable cellulosics (cellulose plus hemicellulose). The solids were separated by centrifugation and washed five times with equal volumes of citrate buffer to remove all traces of adsorbed enzyme. After drying overnight in a 40°C oven under vacuum, various amounts of these spent cellulosics

Fig. 3. Arrhenius plot for adsorption of cellulasr components. (c)Xylanase. 365 calig mol; ( 0 ) endoglucanase, 1142 calig mol; (0) endoglucanase, 807 calig rnol.

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138

were contacted with 10 ml fresh mixed enzyme and the initial rates of hydrolysis at 50°C were measured (Fig. 4). The initial rates with fresh substrate under similar conditions are also shown on the same figure for comparison. The ratio of noncellulosics (30%) to hydrolyzable cellulosics (70%) in the fresh bagasse was 0.43.The fraction of hydrolyzable cellulosics in the spent bagasse (54% hydrolyzed) was 0.7,( 1 0.54)= 0.332.The noncellulosics-to-hydrolyzable-cellulosicsratio in the spent bagasse was, therefore, 0.98, about 2.2 times more than that in fresh bagasse. The decrease in hydrolytic breakdown of spent bagasse could be attributed to the increase in noncellulosicsto-hydrolyzable-cellulosics ratio. Lignin physically hinders the enzyme molecules to come in contact with the cellulose or hemicellulose chain. An increase in lignin with respect to hydrolyzable cellulosics would obviously make it more difficult for the enzyme molecules to penetrate the solid matrix and physically come in contact with the p- 1 ,4-glycosidic linkages. In order to find the mode of participation of the cellulase components in the hydrolysis of spent bagasse, various quantities of bagasse were contacted with the fresh enzyme to measure the ad-

“1

t 36-

-E

23.0-

.c L:

;2 4 3

l.2p 81

fj 1.8-

h

0.6

I

I

I

-re

Conc.,mqlml,

Fig. 4. Initial rate of hydrolysis as a function of spent cellulosics (bagasse) conFresh cellulosics; (-&) spent cellulosics. centration. (--o--)

ENZYMATIC HYDROLYSIS OF CELLULOSICS

139

sorption of the enzyme components. All three components were found to be adsorbed at concentrations up to 7% bagasse. Results of such adsorption studies at 5% spent cellulosics concentration are shown in Table I. The adsorbed values of enzyme components on fresh bagasse are also shown in the same table for comparison. It is to be noted that the percent adsorption of initial activity was significantly less on spent bagasse compared to fresh bagasse, whereas the values remained more or less the same for per gram hydrolyzable cellulosics. Simultaneous adsorption of exo- and endoglucanases on hydrolyzable cellulosics corroborates the mechanism of synergistic enzyme action postulated by Eriksson, Pett e r ~ s o n , and ' ~ Wood and McCrae. l 8 Furthermore, the cellulosics in spent bagasse have a higher crystalline-to-amorphous ratio, and the preferential adsorption of endoglucanase (13.7%) compared to exoglucanase (5.4%) suggests that this is the enzyme that enters first and causes disorganization in the cellulosic chain. The results are in sharp contrast to the ones mentioned by Mitra and Wilke,Ig where they observed validity of the mechanism of cellulose degradation proposed by Reese et aI.?OThe spent bagasse was milled in a Wiley mill, and the extent of adsorption of the mixed enzyme components on them was determined. Higher adsorption of exoglucanase on milled spent bagasse (Table I) compared to that on spent bagasse was probably due to the increased contents of cellulosic chain ends created as a result of milling. This is in line with the hydrolytic nature of exoglucanase, which cleaves cellobiose units successively from the cellulosic chain ends.

TABLE I Adsorption of Cellulase Components on Fresh and Spent Bagassea Activity adsorbedb/g hydrolyzable cellulosics Sample No. 1

2 3

exoglucanase Substrate fresh bagasse' spent bagasse milled, spent bagasse

endoglucanase (IU/g)

xylanase

62.9 (34.3%)d 245.7 (33.1%) 134.3 (39.2%) 221.0 (13.7%) 114.0 (15.3%) 21.46 (5.4%) 36.6 (9.2%) 169.6 (10.5%) 120.7 (16.2%)

a The mixed enzyme derived from T . reesei and A . wenrii containing 6.4 x IUiml exoglucanase, 26.0 IU/ml endoglucanase, and 12.0 IU/ml xylanase was used. Adsorbed activities determined after 15 min contact at 5°C. Bagasse (150- 170 mesh) 5% suspension. Figures in parentheses represent the percent of initial enzyme activity adsorbed.

GHOSE AND BISARIA

I40

Mechanism of Xylanase Action in the Hydrolytic Breakdown of Bagasse The nature of xylanase action was investigated by hydrolyzing bagasse by endo- and exoglucanases in the presence of xylanase and by hydrolyzing xylanase-pretreated bagasse with endo- and exoglucanases. After treating bagasse with the xylanase component for different periods of time at 5OoC, the adsorbed xylanase was removed by repeated washings with O.05M citrate buffer, pH 4.8. The washed bagasse was hydrolyzed by the purified exo- and endoglucanases at 50°C, and the initial rates of hydrolysis were determined by measuring the reducing sugars produced in the first hour (Table 11). The results with xylanase-pretreated cellulose are also shown in Table I1 for comparison. When only exo- and endoglucanases were used for bagasse hydrolysis, no xylose was found in the reaction products. However, when xylanase was used to hydrolyze bagasse along with exo- and endoglucanases, much higher quantities of reducing sugars were produced (Table 111). Comparison of the data reveals that the production of reducing sugars (as glucose plus “cellobiose”) was about 20% higher when all the three components were present together compared to the 60

TABLE I1 Effect of Xylanase Pretreatment on the Hydrolysis of Bagasse by Exo- and Endoglucanasesa

Treatment

Substrate

1 ) none 2) with xylanase at 50°C for: i) 30 min ii) 60 min iii) 90 min

Bagasse

1) none 2) with xylanase at 50°C for 60 min

Cellulose

RSb produced in first hour (mg glucose/ml) 1.33

1.48 1.64 1.73 1.44 1.44

-

Activities of the purified components were: exoglucanase, 3.8 X 1U/ml; endoglucanase, 18.3 IU/ml, and xylanase, 0.4 IUlml. Exo- and endoglucanases were used to hydrolyze a 5% suspension of bagasse or cellulose (80-100 mesh) at 50°C in O.05M citrate buffer, pH 4.8. ‘I Reducing sugars. a

141

ENZYMATIC HYDROLYSIS OF CELLULOSICS TABLE 111 Synergistic Effect of Xylanase Action with Exo- and Endoglucanases for Bagasse Hydrolysis" RSb produced in first hour

xylose ExoExo-

+ endoglucanase

+ endoglucanase + xylanase

glucose

"cellobiose"

(mdm1)

Enzyme

0.32

0.97 1.47

0.4 0.58

~~

Activities of the purified enzyme components were: exoglucanase, 3.8 IU/ml, endoglucanase, 18.3 lU/ml, and xylanase, 0.4 1Uiml. Enzyme was used to hydrolyze a 5% suspension of bagasse (80-100 mesh) at 50°C in 0.05M citrate buffer, pH 4.8. Reducing sugars. a

x

min xylanase-pretreated bagasse by exo- and endoglucanases (cf. Table 11). It is quite clear from these studies that xylanase pretreatment had a definite increased effect on hydrolysis of bagasse. This suggests that xylanase helps in creating more accessible cellulosic regions, which could be acted upon readily by exo- and endoglucanases by hydrolyzing the hemicellulose component of the bagasse thereby resulting in higher sugar production. Hydrolysis of pure cellulose, however, was not affected by the xylanase pretreatment because of the absence of hemicellulose. Furthermore, xylanase action was most effective when acting synergistically with other hydrolytic components, i.e., exo- and endoglucanase for enzymatic degradation of bagasse. Rolc of PClucosidase o n the Hydrolysis of Cellulose A 5% suspension of cellulose (150-170 mesh) was hydrolyzed with exo- and endoglucanases in the presence and absence of pglucosidase to determine its role on the formation of hydrolysis products. In the absence of p-glucosidase, most cellobiose appeared in solution in about 20 min with a slow and steady increase in its concentration for a subsequent period of time. The glucose concentration was also found to increase at a slower rate (Fig. 5). In the presence of p-glucosidase, however, cellobiose accumulated and then gradually decreased after 40 min. Glucose concentration was increased by /3-glucosidase. Increased production of reducing sugars in the presence of P-glucosidase was due to the cellobiose removal, which is a potent inhibitor of e x o g I u c a n a ~ e . ~When ~ - ~ ~ differing concentrations of cellulose were hydrolyzed with exo- and endo-

GHOSE AND BISARIA

142 2801

1

$”’

260

0 Tim, mm-

Fig. 5. Glucose and “cellobiose” formation in cellulose hydrolysis. Purified exIUlml) and endoglucanase (5.94 IUlml) in 0.05M citrate oglucanase (1.56 x buffer, pH 4.8, were used to hydrolyze a 5% suspension of cellulose at 50°C, in the absence of P-glucosidase. In another suspension, p-glucosidase (0.26 IUlml) was mixed with the exo- and endoglucanases. (m) Cellobiose without P-glucosidase; ( 0 ) glucose with P-glucosidase; (m) cellobiose with P-glucosidase; (0) glucose without Pglucosidase.

glucanases free of p-glucosidase, the formation of glucose and “cellobiose” increased with an increase in cellulose concentration (Table IV). Since p-glucosidase is primarily responsible for hydrolysis of cellobiose into glucose, no glucose should have appeared in the hydrolysate in its absence. However, glucose production was more than 13% of the total sugars produced, which suggests that the appearance of glucose in the absence of p-glucosidase was either due to the combined action of exo- and endoglucanases or due to the presence of some other route.23 Individual and Cooperative Action of Purified Cellulase Components on Hydrolysis of Fresh and DeligniJied Bagasse

Hydrolysis of a 1% suspension of bagasse was carried out with different combinations of purified cellulase components in citrate buffer (O.O5M, pH 4.8) at 50°C. The effect of each component on the hydrolysis was determined with respect to the release of sugars in the hydrolysis products in the first 4 hr. The same experiment

ENZYMATIC HYDROLYSIS OF CELLULOSICS

143

TABLE IV Effect of Cellulose Concentration on the Rate of Hydrolysis using Cellulase Free of p-Glucosidase”

Sample No. 1 2 3 4 5 6 7

Hydrolysis in 4 hr Cellulose conc. total sugars glucose “cellobiose” (I*.g/ml)

2 4 6 8 10 12 14

288 3 00 342 414 504 540 648

40 48 53 60 64 72 78

248 252 289 354 440 468 570

IU/ml a Purified enzyme solution containing 1.56 X exoglucanase and 5.94 IU/ml endoglucanase activity were used to hydrolyze cellulose at 50°C in 0.05M citrate buffer, pH 4.8.

was repeated for the hydrolysis of delignified bagasse in order to find the effect of lignin on hydrolysis (Table V). The increased production of reducing sugars in the case of delignified bagasse suggests that lignin was associated with cellulose and hemicellulose in the native bagasse in such a way that the enzyme action is physically hindered resulting in decreased hydrolysis rates. The presence of P-glucosidase did not affect the hydrolysis in the absence of exo- and endoglucanases. It, however, resulted in a higher appearance of glucose as a result of cellobiose cleavage into its monomer in presence of exo- and endoglucanases. CONCLUSION Adsorption of cellulolytic components on cellulosic materials is a prerequisite phenomenon for their hydrolysis. According to Eriksson’s postulate,16the degradation process is based on the association of more than one enzyme. This has been confirmed in the present investigation based on the mechanism of action rendered by the adsorption process. Simultaneous adsorption of all three enzyme components on fresh as well as spent bagasse (Table I) provides evidence for this confirmation. Preferential adsorption of endoglucanase compared to exoglucanase suggests that it is the enzyme that initiates the attack on native cellulose and creates more reactive cellulose to be subsequently acted upon by exoglucan-

Sample

xyl + exo xyl + endo xyl + P-glu xyl + exo + endo xyl + exo + p-glu xyl + endo + p-glu xyl + exo + endo + p-glu exo + endo + p-glu

XYI

Enzyme

\ 0.1 0.05 0.164 0.04 0.03 0.078 0.07

0.14 0.17 0.08 0.22 0.19

(mdml) -

“cellobiose”

0.04

glucose

0.14 0.18

0. I65 0.14

0.1

0.1 0.136 0.14

xylose

-

0.15 0.195 0.09 0.246 0.225

0.046

-

glucose

-

0.19 0.053 0.04 0.095 0.082

0.12 0.06

(mdml) -

“cellobiose”

Delignified bagasse

0.18 0. I95 0.21 0.1 0.23 0.205 0.21 0.25 -

xylose

IUiml exoglucanase, 2.33 IUiml endoglucanase, 0.4 IUiml xylanase and 0.26 IU/ml pa Purified enzyme components had 1.56 x glucosidase activity. Enzyme(s) was used to hydrolyze a 1% suspension of bagasse (150-170 mesh) in 0.05M citrate buffer, pH 4.8 at 50°C for 4 hr. xyl = xylanase, exo = exoglucanase; endo = endoglucanase: p-glu = P-glucosidase.

3 4 5 6 7 8 9

2

1

No.

Substrate

Bagasse

TABLE V Effect of Purified Cellulase Components on Hydrolysis of Bagasse with Respect to the Release of Sugarsa

*e

CA

W

0

->

Rm *z

n z

ENZYMATIC HYDROLYSIS OF CELLULOSICS

I45

ases. 1 4 ~ 1 7 ~ 1These 8 results appear to invalidate the C , concept originally propounded by Reese.20 The increase in the adsorption of purified enzyme components individually as against the adsorption exhibited by their mixture indicates that the cellulosic surface contains some common sites at which at least a part of the cellulase components can be adsorbed. P-Glucosidase is not required to initiate the degradation process as it is not adsorbed. The activation-energy values indicate the difference in the characteristic affinity of these components in respect to the substrate; the greater adsorption on bagasse corresponds to a lower activation-energy value of a specific component. Pretreatment by pure xylanase has been shown to create more available cellulosic surfaces, which can be readily acted upon by exo- and endoglucanases thereby resulting in enhanced rates of production of reducing sugars. The xylanase action is most effective when present together with exo- and endoglucanases. Thus, all three components act synergistically and are therefore, required in combination for manifestation of a maximum rate of hydrolysis. Appearance of glucose in the hydrolysis products of cellulose breakdown by exo- and endoglucanases in the absence of p-glucosidase indicates the presence of some route other than the hydrolysis of cellobiose. One possibility is through the action of p-1,.l-glucan glucohydrolase acting on the nonreducing ends of the free chains created by endoglucanase. This h y p ~ t h e s i showever, ,~~ needs to be confirmed. The increased production of reducing sugars by the hydrolysis of delignified bagasse compared to the fresh native bagasse via single and multiple actions of cellulase components suggests that lignin reacts with cellulose and hemicellulose in a manner that is unfavorable for enzyme action. Little is known to clarify this situation adequately. The authors are grateful to the Swiss Federal Institute of Technology, Zurich for the grant received in connection with the studies.

References I . T. K . Ghose, V. S. Bisaria, and C. P. Dwivedi, in Abstracis of the V Iniernutionul Fermeniuiion Symposium, Berlin (Springer-Verlag, Berlin, 1976), p . 439. 2. V. S. Bisaria and T. K . Ghose, in Proceedings o f t h e Symposium on Bioconversion of C~llitlo.sicSubstances into Energy, Cheniicals and Microbiul Proiein, T. K . Ghose. Ed. (IIT, Delhi. 1977). p. 155. 3. A. Binder and T. K. Ghose. Biotechnol. Bioeng., (20, 1187 (1978). 4. T. K. Ghose. A. N . Pathak, and V. S. Bisaria, in Proceedings of ihe Sym-

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Accepted for Publication April 17, 1978

Studies on the mechanism of enzymatic hydrolysis of cellulosic substances.

Studies on the Mechanism of Enzymatic Hydrolysis of Cellulosic Substances T. K. GHOSE and V . S. BISARIA, Biochemical Engineering Research Centre, Ind...
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