World Journal of Microbiology and Biotechnology 6, 19-22

Induction of glucoamylase production by non-starchy carbohydrates in Aspergillus terreus

Showkat Ali, Zahurul Hossain, Saleh Mahmood and Rashidul Alam

Glucoamylase production in Aspergi l lus terreus was induced, in order, by glucose, cellobiose, sorbitol, sucrose, a-methyl mannoside and a-methyl glucoside. Opti- mal induction was at 38~ pH 4.0 and with 8 mg glucose/ml. Cycloheximide at 10 i~g/ml completely inhibited induction indicating de novo protein synthesis was involved in induction of glucoamylase.

La production de gluco-amylase est induite chez Asperg i l lus terreu=, on ordre d6croi- ssant, par le glucose, le celloboise, le sorbitol, le sucrose, I'a-methylmannoside et I'e-methylglucoside. L'induction est optimum & 38~ pH 4.0 eten pr6sence de 8 mg de glucose par ml. La cycloheximide

10 lag par ml inhibe compl6tement I'induction, ce qui implique une synth&se de prot6ine de novo dans I'induction de la gluco-amylase.

The authors are at the Department of Biochemistry, University of Dhaka, Dhaka- 1000, Bangladesh. S. Ali is the Corre- sponding Author.

�9 1990 Rapid Communications of Oxford LtcL

A number of bacteria and fungi during their growth on starchy substrates, synthesize and excrete amylase into the medium (Saito et al. 1975, Pestana & Castillo 1985, Kanlayakrit et al. 1987, Ali et al. 1989). These enzymes are important in view of their potential biotechnological application in starch- processing industries.

Many authors have reported that a-amylase and glucoamylase are produced constitutively at relatively low activities and can be induced by different sugars such as maltrose, maltooligosaccharides, glucose n-methyl glucoside, dextrin and specially starch in bacteria, yeast and fungi (Windish & Mhatre 1965, Yabuki et al. 1977, Manjunath et aL 1983, Junichi et aL 1985, De Mot & Verachtert 1986). However, the mechanism and condition of glucoamylase induction using Aspergillus terreus not yet been established. In the present paper induction of glucoamylase production by non-starchy carbohydrates in Aspergillus terreus is described.

Materials and Methods

Organism ./tspergillus terreus GTC826, obtained from West German Type Culture Collection (DSM), was used in this study. Long term stock cultures were preserved at 4~ on agar slants and subcultured at two month intervals.

Culture Media Unless otherwise stated, the organism was grown in stationary culture flasks containing different carbohydrates and basal medium having the following composition (g/l): (NH4)2SO4, 3; peptone, 2; KH2PO4, 1; MgSO4.7H20, 0.3% and CaC12.2H20, 0.3.

Inoculum To prepare non-induced mycelia, 0.5 ml of conidial suspension was inoculated into 50 ml of the above described basal medium containing 10 g arabinose/1 as carbon source at 37~ for 72 h. During cultivation, glucoamylase was not

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S. All, et al.

produced. The mycelial preparation was washed with sterile distilled water aseptically and inoculated into 250 ml conical flask containing induction medium (basal medium containing different carbohydrates).

Preparation of Substrates Solutions of monosaccharides and disaccharides were sterilized by membrane filtration (pore size 0.45 lim) whereas polysaccharides were autoclaved. All sugars were added aseptically to the culture medium to give the desired concentration.

Preparation of Crude Enzyme and Assay Samples were taken at intervals and the supernatant fluid used for assaying glucoamylase activity. Activity was determined by measuring the reducing sugar produced from soluble starch after 10 min at 60~ The reaction mixture contained 1% (w/v) starch, 50mM acetate buffer (pH 3.0), 25 til enzyme solution in one ml. The quantity of sugar liberated was estimated as described by Nelson (1944) and Somogyi (1952). One unit of enzymic activity is defined as producing one t~mol glucose/min.

Results and Discussion

Condition for induction of Glucoamylase by Glucose The following cultural conditions gave maximum induction of glucoamylase in Aspergillus terreus using D-glucose as carbon source: pH4, at 35 to 40~ with 8.0 mg glucose/ml (Fig. 1a-c). There was a lag period of 4 h before induction of glucoamylase began (Fig. ld). After 4 h, a large increase in enzyme activity was observed and reached a maximum after about 48 h. A similar pH, temperature, substrate concentration and time dependency on glucoamylase induction was observed using sorbitol, sucrose and cellobiose. We have therefore used these conditions in our subsequent investigation.

Effect of Various Carbohydrates on Glucoamylase Production Various non-starchy carbohydrates were tested for their influence on

m

E

> .

E

8 L9

15 30 45 60 Temperature (~

m

I I I I 2 4 6 8

pH

C

2.5 5,0 7.5 10.0 o-glucose

d

16 32 48 64 Time (hour)

Figure 1. Effect of (a) temperature, (b) pH, (c) D-glucose concentration and (d) time course of induction of glucoamylase by o-glucose in Aspergillus terreus.

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Induction of glucoarnylase

Table 1. Induction of glucoamylase production by various carbohydrates in Aspergillus terreu$.

Carbohydrates Glucoamylase activity (U/ml)

Monosaccharides D-glucose 6.2 o-mannose 0.0

a-Methyl glucoside 1.0 a-Methyl mannoside 1.4

D-galactose 0,0 D-arabinose 0.0 D-xylose 0.0 D-fructose 0.0 D-sorbitol 4.4 L-sorbose 0.0

Disaccharides Ceilobiose 6.4 Lactose 0.0 Sucrose 3.3

Trisaccharide Raffinose 0.0

Polysacchar ide Cellulose 0.0

The glucoamylase activity was measured after 48 h incubation at 38~ containing 8 mg of various inducers/ml.

O _ O

o

E

~ o e~

e -

c O i i ~ / _ o o r

I 2.5 5.0 7.5 10 100

Cycloheximide (tag/ml)

Figure 2. Effect of cycloheximide on glucoamylase induction by D-glucose in Aspergillus terreus. Cycloheximide was added at zero time of induction.

glucoamylase production in Aspergillus terreus (Table 1). Synthesis of glucoamylase was strongly suppressed by monosaccharides such as mannose, galatose, arabinose, xylose and sorbose and by the disaccharide, lactose; the trisaccharide, raffinose and the polysaccharide, cellulose. Fructose, a-methyl rnannoside and el-methyl glucoside had little effect on glucoamylase production.

Inhibition of Glucoamylase Induction by Cyclohexirnide Cycloheximide at 10/ag/ml completely inhibited the induction of glucoamylase by glucose (Fig. 2) and also by the other inducing sugars in a similar manner. It is therefore evident that glucoamylase induction involves de novo synthesis.

R e f e r e n c e s

ALl, S., MAHMOOD, S., ALAM, R. & HOSSAIN, Z. 1989 Culture condition for production of glucoamylase from rice bran by Aspergillus terreus. MIRCENJournal of Applied Microbiology and Biotechnology. 5, 525-532.

DE MOT, R. & VERACHTERT, H. 1986 Enhanced production of amylase and glucoamylase by amylolytic yeasts using cyclodextrin as carbon source. Applied Microbiology and Biotechnology, 24, 459-462.

Ju-NICHI, A., FREDRICO, B.W., KAZUKAI, O. & SUSUMU, H. 1985 Raw starch digesting enzyme of Aspergillus spp. K-27. Denpu Kagaku 32, 128-135.

KANLAYAKRIT, W., ISHIMATSU, K., NAKAO, M. & HAYASHIDA, S. 1987 Characteristics of raw starch-digesting glycoamylase from thermophilic Rhizomucorpusillus. Journal of Fermentation Technology 65, 379-385.

MANJUNATH, P., SHENOY, B.C. & RAGHUVENDRA RAO, M.R. 1983 Fungal glucoamlyases. Journal of Applied Biochemistry 5, 235-260.

NELSON, N. 1944 A photometric adaption of the Somogyi for the determination of glucose. Journal of Biological Chemistry 153,375-380.

PESTANA, F. & CASTELLO, J. 1985 Glucoamytase production by Aspergillus awamori on rice flour medium and partial characterisation of the enzyme. MIRCENJournal of Applied Microbiology and Biotechnology 1,225-237.

SAITO, N. & YAMAMOTO, K. 1975 Regulating factors affecting a-amylase production in Bacillus licheniformis. Journal of Bacteriology 121,848-856.

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SOMOGYI, M. 1952 Notes on sugar determination.Journal of BiologicalChemistry 195, 19-23.

WINDISH, M.W. & MHATRE, N.S. 1965 Microbial amylases. Advances in Applied Microbiology 7,273-298.

YABUKI, M., ONO, N., HOSHINO, K. & FUKUI, S. 1977 Rapid induction of a-amylase by nongrowing mycelia of Aspergillus oryzae. Applied and Environmental Microbiology 34, 1-6.

(Received lO July 1989; accepted 4 August 1989)

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