Effect of cultural conditions on production of cellulases in trichoderma longibrachiatum

Soc.84 2, 251-258 1985 Printed in Great BritainEFFECT OF CUL TURAL CONDITIONS ON PRODUCTION OF CELLULASES IN TRICHODERMA LONGIBRACHIATUM Department of Biology, Guru Nanak Dev University,

Trans Br mycol Soc.84 (2), 251-258 (1985) Printed in Great Britain

EFFECT OF CUL TURAL CONDITIONS ON PRODUCTION OF

CELLULASES IN TRICHODERMA LONGIBRACHIATUM

Department of Biology, Guru Nanak Dev University, Amritsar-is joo e, India

The production of cellulase components which include FP activity, CM-ase and p-glucosidase

on carboxymethyl cellulose (CMC) was investigated The relative distribution of cell free and cell associated enzymes varied with the age of the culture The optimal pH of the medium for synthesis of enzymes in extracellular, cytosol and cell debris associated states was between 4'5 and 5"0 with optimal temperature being 27°C Shake cultures gave comparatively low yields of enzymes as compared to stationary cultures When the medium was supplemented with 1%lactose, maximum production of cellulolytic activities in the culture fiitrate was achieved, that is, 8'1, 0·6 and 0'13 units per ml of CM-ase, FP activity and ,a-glucosidase respectively There was a varied response in the induction of cell associated enzymes by different substrates An increase in substrate concentration (CMC and lactose) had no significant effect on production of extracellular enzymes

In recent years, attention has been focused on

decreasing the cost of the enzymatic hydrolysis of

cellulosic plant material, either by screening

microbial mutants (Montenecourt & Eveleigh,

1977; Farkas et al., 1981) or by manipulation of

cultural conditions to improve cellulase enzyme

levels Improvement in cellulase productivity has

also been obtained by increasing cellulose

concen-tration, addition of glucose to cellulose medium in

order to increase cell mass and continuous culture

methods (Wilke, Yang & Von Stockar, 1976;

Peitersen, 1977) Temperature profiling and pH

cycling have also led to increased production of

cellulase in Trichoderma species (Mukhopadhyay&

Malik, 1980)

Trichoderma longibrachiatumRifai has proved to

be a good cellulase producer and a potential source

of single cell protein (Sidhu & Sandhu, 1980;

Sandhu &Kalra, 1982) The aim of the present

study was to characterize the effect of culture

conditions on the complete cellulolytic enzyme

complex in this species

MATERIALS AND METHODS

Organism Trichoderma longibrachiatumRifai (ATCC 44788)

used in the present study was isolated from

degrading Mangijera wood The stock culture was

maintained in soil at 4 °C and subcultured in

Vogel's glucose agar whenever required

Influence of shake or stationary cultures

Vogel's medium (25 ml) (Sandhu & Kalra, 1982) supplemented with 1% carboxymethyl cellulose (CMC) (Sigma) was dispensed in 100 ml Erlen-meyer flasks The flasks were autoclaved at 10 p.s.i for 20 min Each flask was inoculated with a spore suspension to give a final concentration of

5 x 106ml? of the medium and incubated at 27° as stationary or shake cultures Six flasks per treatment were analysed daily for 10 days

Incubation temperature and initial pH of medium

The medium was inoculated with test organisms and incubated at 15°,22°,27°,32°,37° and 42° The initial pH was adjusted with 0'1 N-NaOH or 0'1 N-HCl to different values ranging from 2 to 8 After inoculation cultures were incubated at 27° Flasks were analysed after 5 days of incubation as stationary cultures

Effect of various soluble substrates

Carboxymethyl cellulose in Vogel's medium was replaced by 1% lactose, maltose, sucrose or cellobiose as a sole source of carbon Sugars were sterilized by seitz filtration before adding to the autoclaved medium Complex compounds like yeast and malt extract at a concentration of 1% were used separately and in combination for cellulase production The effect of substrate concentrations on cellulase production was studied

RES ULTS

Influence of carbon source

Among the carbon sources tested the highest extracellular enzyme activities were recorded with lactose followed by CMC and malt extract (Fig 4)

FP activity and CM-ase in the cytosol fraction were found to be maximum on malt extract whereas p-glucosidase was maximum on maltose and CMC Cellobiose gave maximum mycelial dry weight and was found to be the best source for cell debris enzymes except for p-glucosidase which had a higher activity in yeast extract No CM-ase activity was detected on sucrose (Fig.4b). Of the different concentrations of CMC, t :5% gave the highest

Influence of pH and temperature

Maximum activity in the culture filtrate was recorded at pH 5"0 The pH values lower than 4 '0 and higher than 5'5 had an adverse effect on cellulase production (F ig.2a-c) The cytosol and

cell debris enzymes were maximum between pH 4-5 and negligible at pH 3 '0 and 7'0 except for p-glucosidase (F ig.zc-c) The enzymes in all three fractions were h igher at 27° but best growth was supported at 32° with reasonable amounts of enzymes also (F ig.3 a-c ) At minimal and maximal

temperatures tested, i.e 15° and 42°, the enzyme components in the extracellular and cytosol fractions were present but were absent in cell debris excepting p-glucosidase

Effe ct of shake and stationary culture on enzyme

production

The production of the three components of cellulase in the culture filtrate was higher in stationary culture being 0'275, 1'64 and 0'10 units ml-1 and 0'17, 1'0 and 0'08 units rnl'? in shake culture for FP activity, CM-ase and p -glucosidase respectively (F ig.1a,d,g) The enzyme activities in

the cytosol and cell debris fractions appeared earlier and were comparable under the two culture conditions (F ig 1b, c, e,f,h,1) Enzyme activities

have also been expressed for different fractions as activity per flask (T able1).The relative proportions

of the enzyme in the above two fractions were found

to be highest for p-glucosidase followed by CM-ase and FP activity The activities in the cytosol disappeared after the eighth day but persisted in the cell debris beyond the tenth day The maximum mycelial mass was obtained on 4 and 6 days of incubation in shake and stationary culture respect-ively The pH of the medium showed a steady increase towards neutrality after an initial decline

Fractionation of samples

Each sample was filtered through a sintered glass

funnel at 4° The resulting culture filtrate was

centrifuged at 8500g for 15 min at 4° and stored

for analysis of enzyme activity and unused

substrate The cell mass was washed with 0'1 M

acetate buffer at pH 5"0 and dried between folds of

filter paper The above mycelium was frozen,

crushed with chilled acid washed sand to prepare

cytosol extracts and for cell debris fraction, and

macerated without sand Both fractions were

suspended in buffer and centrifuged at 22000 g for

30 min at 4° The supernatant was taken as the

cytosol fraction while the pellet of cell debris

without sand was resuspended in5ml of buffer and

stored at - 15° if not immediately analysed

flasks wer e incubated at 27° for 5 days as stationary

cultures

Dry weight determinations

Samples were transferred to dried and preweighed

sintered glass funnels and washed thoroughly with

cold deionized water These were dried at 75° to a

constant weight

Enzyme unit

One unit of enzyme activity is equivalent to 1pM

of product released per min Cytosol and cell debris

enzymes have been expressed in terms of specific

activity as units mg? protein for cytosol and units

mg? dry weight for cell debris The sol-able protein

content was estimated by the method of Lowry

et al (1951).

Estimation of enzymes

Filter paper activity (FP activity) and

carboxy-methyl cellulase (CM -ase) were estimated as

described earlier (Sand hu & Kalra, 1982) using

filter paper strips and CMC as substrates For

p-glucosidase, to 0 '5 ml of the diluted enzyme

sample was added 0'5 ml of 5

msr-p-nitrophenyl-p-D-glucopyranoside (PN P G) (Sigma) The

mix-ture was incubated at 37° for 30 min The reaction

was terminated by adding 4 ml of 0'2 M-NaOH/

glycine buffer at pH 10·6 and the absorbance read

at 420nm

Carboxymethyl cellulose in the culture filtrate

was determined with anthrone reagent (U pd egraff,

1969)

D K Sandhu and M. K Kalra 253

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Fig 1 Production ofFPactivity (a-c), CM-ase (d-f),p-glucosidase (g-l) in shake ( -) and stationary ( )

cultures on ';;0CMcellulose.

Table1 Distribution of enzymes in three fractions of eellulases

Enzyme distribution (un its per flask)

Enzyme fraction

Extracellular

Cytosol

Cell-debris

FP activity

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Fig 3 Effect of temperature of incubation on growth and

cell-debris are plotted as activity x

D K Sandhu and M K Kalra 255

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Fig 4 Effect of various carbon sources on growth and cellulase production in extracellular (~), cytosol (.), cell-debris (0) fractions; FP activity and growth(a),CM-ase(b),p-glucosidase(c).CM-ase and FP activity

in cell-debris are plotted as activity x 10-'.

yield of all the extracellular enzymes (Fig 5a-c).A

proportional increase in cytosol and cell debris

enzymes was recorded with increase in the

substrate concentration All the cellulase

compon-ents in the three fractions reached their plateaux at

1-1'5%of lactose concentration after which the

activities were almost constant (Fig.6a-c).Growth expressed as dry weight increased with increase in substrate concentration both in the case of CMC and lactose, the increase being relatively small above one per cent (Figssa, 6a).

84

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Fig 6 Production of cellulase and growth at different

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Fig 5 Production of cellulase and growth at different

cytosol (.), cell-debris (0) fractions; FP activity and

DISCUSSION

Extracellular enzyme production studies in other

fungi (Boretti et ai., 1973; D'Souza & Furtado,

1977) has been shown as linear with growth The

enzymes in the cytosol and cell debris fraction contributed minor proportions of FP activity and eM-ase but reasonable amounts of p-glucosidase However, relative proportions of the three fractions varied during growth as the cell associated (cytosol and cell debris) reaction was higher in the early phase compared to the late growth phase, thus indicating synthesis and release of these enzymes into the medium which added to the concentration

D K Sandhu and M K Kalra 257

of extracellular enzymes (Berg&Pettersson, 1977;

Kubicek, 1981, 1983) Regarding distribution of

the enzymes in the three fractions during the late

exponential growth phase, most of the FP activity

and CM-ase (97'8, 99'1%, respectively) were

located extracellularly Measurement of

fJ-gluco-sidase showed up to 33%of the total activity to be

associated with the mycelium throughout the

growth period Halliwell & Lovelady (1981) have

reported that 99'0% of CM-ase in T koningii

Oudem is extracellular whereas 90% of

fJ-glucosidase is associated with cells The active

release of CM-ase and FP activity into the medium

in early stages of cultivation ofT reesei Simmons

was reported by Vaheri, Vaheri&Kaupinen (1979)

with fJ-glucosidase being detected mainly in the cell

debris which was not released until the cells had

autolysed In different Trichoderma species, which

are the most promising fungi for industrial

production of cellulases, most of the ,a-glucosidase

activity has been shown as localized within the cells

(Berg&Pettersson, 1977; Kubicek, 1981) except in

the cases ofT harzianum Rifai and T pseudokoningii

Rifai where high levels of extracellular enzymes

have been reported (Sidhu, 1983) Studies on fungi

other thanTrichoderma, which include Penicillium

janthinellum Biourge and Sporotrichum

pulverulen-tum Burds., have also shown the cell free

occurrence offJ-glucanases and fJ-glucosidases with

only small amounts associated with the mycelium

(Eriksson& Hamp, 1978; Rapp, Grote& Wagner,

1981)

The cultural conditions have a marked effect on

the production of enzymes in all three fractions In

shake flasks the enzymes reached their maximum

values earlier but the activity was low compared

with stationary cultures This low activity may be

due to inactivation of enzymes by shaking, as

reported earlier (Reese& Mandels, 1980)

Maxi-mum production of cell free and cytosol enzymes

was obtained at pH 5'0 except for CM-ase in

cytosol fraction which along with all cell debris

activities showed a pH optimum at4'5 Growth and

enzyme production was markedly inhibited when

the initial pH was below4'0and above6'0.This pH

range has also been reported as the most favourable

hydrogen ion concentration for cellulolytic enzyme

synthesis in several other fungi namely,Sclerotium

rolfsii Sacco (Shewale & Sadana, 1978), T reesei

(Andreottiet al., 1980; Mukhopadhyay& Malik,

1980), Pellicularia filamentosa (Pat.) Rogers

(Tani-guchietal., 1980), Eupenicilliumjavanicum (Beyma)

Stolk&Scott (Tanakaet al., 1980) and Aspergillus

terreus Thom (D'Souza & Volfova, 1982) As

reported in studies on T viride Pers., production

of maximum cell mass may not produce maximum

cellulase yield (Andreottiet al., 1980;

Mukhopad-hyay, 1982) The observations made in the present study also show similar characteristics Here the growth was maximum at 32° while optimal production of all cellulase components in the three fractions was maximum at 27°

Of the different carbon sources lactose gave the highest yield ofcellulase enzymes in the extracellular medium although best growth was supported by cellobiose In earlier studies onTrichoderma species

lactose has been shown to be a good inducer (Andreotti et al., 1980; Kubicek, 1983) In

Penicillium species too, it produced higher amounts

of fJ-glucosidase but gave low yields of other cellulase components (Lakshmikanthan & jagan-nathan, 1980) Carboxymethyl cellulose proved to

be less effective in inducing the enzymes compared with lactose and was inhibitory at higher concen-trations This may be due to release of high levels

of glucose in the culture broth which is a known repressor of cellulase Increasing the concentration

of lactose above 1% did not bring about a significant increase in enzyme production in the culture filtrate The three cellulolytic enzymes in the cytosol and cell debris showed differential induction by various substrates This location of enzymes in association with mycelium or cell free state has previously been shown to depend upon the carbon source (Berg, 1975; Berg&Pettersson, 1977; Chaudhury& Tauro, 1982) In the present study the induction pattern of extracellular and cell-associated enzymes by various carbon sources suggests that different control mechanisms may be operative in the synthesis of each component enzyme of cellulase

The authors acknowledge the receipt of a fellowship from Council of Scientific and Industrial Research, New Delhi to one of us (M K K.) during the course of this study

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(Receive d for publication31May 1984 )