Biotechnology Letters Vol 4 No 10 633-638

L©CALIZATIO[~ AND RELEASE MECHANISM OF CELLULASES IN Trichoderma reesei QM 9414

t~eera Nanda, V.S.Bisaria and T.K.Ghose* Biochemical Engineering Res. Centre

Indian Institute of Technology, Delhi I{auz Khas, New Delhi-llO O16

(India)

SUMMARY:

A significant increase in the extracellular yield of 8-glu- cosidase was observed when Trichoderma reesei QH 9414 was cultivated on a cellulose medium containing chitin. tte asurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while 8-glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degra- ding enzymes (produced from Trichoderma) led to a release of B-glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chiti- nase and other cell wall lytic enzymes which promote re- lease of the intramural 8-glucosidase into the medium.

INTRODUCTION:

T.reesei, though the best-known producer of cellulase, is

deficient in ~-glucosidase. T.reesei cellulase prepara-

tions have 0.2 - 0.3 13-glucosidase units per FP unit (Ryu

and Mandels, 1980) which falls short of the optimum ratio

of 1.5 B-glucosidase units per unit of FPA required for

maximal cellulose hydrolysis (Han and Smith, 1978). Whe-

ther this observed deficiency is due to inadequate syn-

thesis or a limited secretion of B-glucosidase is inves-

tigated. The cell walls of bacteria and fungi pose a

physical barrier against the release of some enzymes which

after crossing the plasma membrane get trapped in the space

between the cell wall and the membrane (Bull, 1972). For

instance, in N.crassa the cell walls allow only a selec-

tive secretion of lower molecular weight isozymes of in-

vertase (Gratzner and Shenan, 1969) and in Aspergillus

oryzae, the cell wall has specific binding sites for amy-

lase (Yabuki and Fukui, 1970).

Chitin, a polymer of N-acetylglucosamine(B,l+4 linkages)

is present in the cell walls of all fungi; T.reesei

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contains 22% (w/w) of the total dry cell wall material as

chitin (Benitez et el., 1975) and is a known producer of

chitinase enzyme (de Vries, 1972).

We report here some observations on co-induction of cellu-

lase and chitinase by culturing T.reesei on a medium con-

taining both cellulose and chitin as substrates. Also,

some observations on cellular localization of cellulase

components and the release of ~-glucosidase from the

mycelium by the action of cell wall degrading enzymes are

presented.

MATERIALS AND METHODS:

Substrates: Microcrystalline cellulose powder (MCCP) was procured from Cellulose Products of India Ltd., Ahmedabad; chitin from Sigma Chemical Co., USA; carboxymethyl cellu- lose (CMC) from Dr. Bender and Hobeins AG, Switzerland and laminarin (B,I÷3 glucan) from Koch and Light, U.K.

Culture Techniques: The organism Trichoderma reesei QM 9414 was maintained on PDA slants at 4 ° . A spore suspen- sion in distilled water was used to inoculate iO0 ml of Mandels medium (Andreotti et el., 1977) containing 0.2% glucose. The 36 h old glucose culture was used as a seed culture for further experiments. The basal medium used was Mandels medium containing either MCCP (1%) or MCCP supplemented with varying concentrations of chitin. For enzyme localization the organism was cultivated on 1% CMC.

Subcellular localizatio n of cgllulase com~0nents: At appro- priate time intervals, CMC grown mycelium was harvested and fractionated to obtain:

(a) The cell wall and (b) intracellular component by ultra- sonic treatment (6~ peak, I0 minutes in 0.05 M citrate buffer, pH 4.8). The residue on centrifugation consisted of pure cell wall fractions and the supernatant of intra- cellular fluid.

(c) Periplasmic fluid: obtained by the osmotic shock treatment as described by Wiley (1970).

(d) Extracellular component: the culture filtrate.

Cell Wall Lytic Enzymes: Enzyme preparations containing B,(I+3) glucanase and chitinase activities were obtained by cultivating T.reesei on a medium containing chitin only and chitin plus autoclaved mycelium of T.reesei as the sole carbon sources.

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Enzyme release from the mycelium: CMC grown mycelium was washed repeatedly in citrate buffer and 30 mg (wet wt.) was suspended in 15 ml of lytic enzyme preparation. The suspension was incubated for 8 hour. The samples were centrifuged and supernatant analysed for B-glucosidase activity.

Enzyme assays: Filter paper activity, endoglucanase, B-glu- cosidase and ~,(i÷3) glucanase were assayed following the procedure of Mandels (Andreotti, et al., 1977). Chitinase assay was according to the method given by Jeuniax (1966). All activities are reported as international units/ml of the enzyme.

RESULTS:

(a) Effect of addition of chitin to cellulose medium: As

shown in Fig.l(a & b) addition of chitin to the cellulose

medium led to earlier and increased production of FPA and

B-glucosidase. The effect of chitin was more pronounced

on ~-glucosidase: the final activity of 8-glucosidase in

the mixed substrate containing 0.25% chitin was twice as

much as in the medium containing cellulose only, while the

final FPA of the culture broth of chitin supplemented

medium was only a little higher than the cellulose culture.

When Trichoderma was grown on media containing only chitin

or its monomer,N-acetylglucosamine, no cellulase or 8-glu-

cosidase activity appeared. Clearly, these components had

no inducing effect on cellulase synthesis.

(b) Subcellular localization: The activity of 8-glucosidase

and endo-glucanase was measured in the extracellular fluid

and the intracellular compartments (cell wall, cytoplasmic

and periplasmic fractions). The relative distribution of

the enzymes in these :fractions was followed with respect to

time (Fig.2). Enzyme activities were computed in terms of

IU/mg mycelium dry weight. The points represent average of

triplicate runs. As ;is clear from the figure, B-glucosidase

was primarily cell wall bound. At all stages of growth, con-

siderably more enzyme was associated with the cell wall than

in any other fraction. In the initial phase of growth, ratio

635

of B-glucosidase activity retained on the cell wall to the

activity appearing extracellularly was approximately 6

(Fig.3). After the end of the growth phase, the ratio of

the cell wall bound to free enzyme decreased to 2 (Fig.3).

Unlike Kubicek (1981), we did not find any CMCase activity

in the mycelium at any stage of growth.

(c) Effect of cell wgl! degradin ~ enzymes on the release of cell wall bound B-@lucosidage: Results are tabu- lated in Table-i

Table-l: Effect of autol[tic enzyme preparation on ~L~lucos~dase release from the mycelium

Substrate used for enzyme induction

8,(l+3) gluca- nase

(IU/ml)

Chitinase 8-glucosidase

( i U / m l ) x l O 2 r e l e a s e d f r o m the mycelium

(IU/ml) x 102

1% (w/w) chitin

i% (wet weight) autoclaved T.reeseimycelium + 0.5% chitin

0.25 1.O 2.7

0.352 1.3 9.2

The lytic enzyme prepared by cultivating Trichoderma on

its own autoclaved mycelium was more effective for the

release of 8-glucosidase than the enzyme produced on

chitin alone. The former enzyme preparation was also

found to have a higher 8,1+3 glucanase and chitinase act-

ivities. More work with purified chitinase and laminari-

nase is in progress to elucidate the exact nature of bind-

ing of 8-glucosidase with the cell wall.

It is clear that the cell wall bound 8-glucosidase is dis-

located due to action of chitinase and/or 8,(1+3) glucanase

present in the lytic enzyme preparations.

Discussion: The relative lack of ~-glucosidase in the cul-

ture filtrate of T.reesei grown on cellulose is due to its

being retained by the mycelium cell wall.

636

,% 1.2 E

~, 0.8 m 8

(~1 (b)

1.2

~ 0.8

"0 .4

4. 5 8 10 02 /+ 5 5 10 time, d~ys t ime, days

FIG. 1. EFFECT OF ADDITION OF CHITIN TO 1% CELLULOSE MEDIUH ON (o) ~8-GLUCOSIDASE & ( b ) FPA SYNTHESIS.

no chit in, e--e 1% chit in, ~ 0.5 % chitin, ~ 0.25% c hifin, 0.1% chitin

=-6

2

-01

b E

T - - i

O ~J

0 0

FIG. 2

ell ~tdl b~/ -cJlucosi d / ~ / /

/ 8

//// / 6 /

extmeellutor / = _ / e 6, ,~-glucosidase / " / /

/ / ~ / " periptosmic / / ,B- glucosidose _

bound " /i[._____.,m.- ~ ~,enc:loglucan&se

" ~ ? - - ~ - ? - - - , T o 6, 6 8 10 2

l ime, days

01STRIBUTION OF hR-GLUCOSIOASE AND ENDOGLUCANASE IN SUBCELLULAR FRACTIONS OFT.REESEI

i , i

Ecw- cell well bound #-glucosidase IU mycellial dry wf{mg)

Ecf- extmcellular #-g[ucosidase IU/n~/celial dry ~.(mg )

1'0 time, days

FIG. 3. RELATIVE /3-GLUCOSIDASE ACTIVITY BETWEEN CELL WALL BOUND & EXTRACELLUL AR FRACTIONS-

637

Our observations on the increase in ~-glucQsidase !yields

on growing the fungus on a mixture of cellulose plus

chitin can be explained in the light of the observed

release of intramural 8-glucosidase by cell wall lytic

enzymes. Presence of chitin in the medium induced the

synthesis of chitinase and other cell wall lytic enzymes

which in turn affected the cell wall bound B-glucosidase.

Preliminary results of these studies were presented at 34th ~mnual Meeting of Indian Institute of Chemical Engineers in December 1981 at Madras.

Acknowled~emegt: The authors are thankful to Dr. E.T. Reese of U.S. Army Natick Development Centre & Prof. K.S. Gopalkrishnan of this Centre for their useful suggestions.

BIBLIOGRAPHY:

Andreotti, R.E., Mandels, M. and Roche, C. (1977), Proc. Bioconversion Symp. (Ed. T.K.Ghose, IIT Delhi), p.249

Benitez, T., Villa, T.G. and Garacia Acha (1975) Arch. Microbiol., 105, 277

Bull, A.T. (1972), J. Appl. Chem. Biotech., 2~, 261

de Vries (1972), J.Gen. Microb£ol., 73, 13

Gratzner, H.Go and Sheenan, A. (1969), J.Bact., 97, 544

Han, Y.W. and Smith, S.K. (1978), Utilization of Agricul- tural Crop Residues, US Dept. Agr. Res. Service, ARS,W-53

Jeuniax, C. (1966) "Chitinases" Methods in Enzymology, Vol. VIII, (Ed. Neufield E.F. and Ginsberg, V), p.644

Kubicek, C.P. (1981), Eur.J.Appl.Microbiol.Biotech.,13, 226

Ryu, D.D.Y. and Mandels, M. (1980), Enz. Microb. Tech.,~, 81

Wiley, W.R. (1970), J. Bact., iO3, 655

Yabuki, M. and Fukui, S. (1970), J.Bact., 104, 138

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