characteristics of glucoamylase from aspergillus terreus

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Journal of Applied Bacteriology 1991, 71, 144-146 ADONIS 002188479100103N Characteristics of glucoamylase from Aspergillus terreus S. Ali and 2. Hossain Department of Biochemistry, University of Dhaka, Bang/adesh 3399/05/90: accepted 26 September 1990 s. ALI AND z. HOSSAIN. 1991. Glucose was the only product of starch hydrolysis liberated by glucoamylase. The enzyme was a glycoprotein with an isoelectric point at pH 3.4 and was optimally active at pH 4.0 and 60°C. It was remarkably stable over a wide range of pH and at elevated temperatures. Divalent Mg2 and Ca2 slightly stimulated glucoamylase activity. The enzyme exhibited specificity for substrates containing a( 1 -+ 4) glucosidic linkages and the K m for starch hydrolysis was 4-0 g/l. INTRODUCTION lsoelectric focussing Glucoamylase (1,'f-rZ-D-glUCan glucohydrolase, E.C. 3.2.1.3) is an exo-acting enzyme that catalyses the production of b-glucose from the non-reducing ends of glucose contain- ing di-, oligo- and polysaccharides. The enzyme occurs almost exclusively in filamentous fungi and far less in bac- teria and yeasts (Fogarty & Kelly 1980). The purification of glucoamylase from Aspergillus terreus has recently been reported (Ali et al. 1990a). Similarities and differences were noted in various aspects of its proper- ties and characteristics compared with glucoamylase from different Aspergillus species (Charuchinda et al. 1984; Punpeng et al. 1984). T h e present investigation was under- taken to study the physico-chemical properties of gluco- amylase from A. terreus. MATERIALS AND METHODS Organism and growth Aspergillus terreus G T C 826 was grown in rice-bran liquid medium under static condition as described by Ali et al. (1989). Enzyme assay Purification of glucoamylase was carried out by DEAE- cellulose and Concanavallin A-Sepharose column chroma- tography (Ali el al. 1990a). Enzyme activity was determined by the method of Ali et al. (1990b). One unit of glucoamy- lase activity was defined as that producing 1 pmol of glucose cquivalent/min. Specific activity was defined as the unit of enzyme/mg of protein. Correspondence to : Dr S. Ah, Department of Biochemistry, University of Dhaka, Dhaka-1000, Bangladesh. Isoelectric focussing of the enzyme was performed in a polyacrylamide gel (5% w/v) rod with Pharmalyte (pH 3-10) on a Pharmacia FBE 3000 apparatus. The gels were fixed in a 10% TCA-5% sulphosalicylic acid mixture for 1 h and then stained with Commassie Brilliant Blue R. Glucoamylase activity was also detected in parallel gels after cutting the gels into 0-5 cm segments followed by elu- tion with 400 p150 mmol/l citrate-phosphate buffer (pH 4.0). Periodate Schlff staining Periodate Schiff staining was carried out on 7.5%" (w/v) polyacrylamide slab gel using Schiff s reagent (Sigma). Protein measurement Proteins were estimated as described by Lowry et al. (195 1) with bovine serum albumin as standard. RESULTS AND DISCUSSION Glucose was found to be the sole product at all stages of starch hydrolysis and no oligosaccharide was detected in the course of the reaction (Fig. 1). The isoelectric point of the enzyme was pH 3.4 which revealed that enzyme was an acidic protein. Glucoamylase from A. terreus contained 7.5% carbohydrate (Ali et al. 1990a) in the present investi- gation. Periodate Schiff staining further confirmed that the enzyme was a glycoprotein. Glucoamylase was maximally active at pH 4.0. The enzyme was fairly stable over the pH range 3G7.0 but not at pH 2.0 and 9.0. Optimum temperature for the action of the enzyme was 60°C. The enzyme was stable at tem- peratures between 40 and 60°C with essentially no loss of

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Page 1: Characteristics of glucoamylase from Aspergillus terreus

Journal of Applied Bacteriology 1991, 71, 144-146 ADONIS 002188479100103N

Characteristics of glucoamylase from Aspergillus terreus

S. Ali and 2. Hossain Department of Biochemistry, University of Dhaka, Bang/adesh

3399/05/90: accepted 26 September 1990

s. A L I AND z. HOSSAIN. 1991. Glucose was the only product of starch hydrolysis liberated by glucoamylase. The enzyme was a glycoprotein with an isoelectric point at pH 3.4 and was optimally active a t pH 4.0 and 60°C. It was remarkably stable over a wide range of p H and at elevated temperatures. Divalent Mg2 + and Ca2 + slightly stimulated glucoamylase activity. T h e enzyme exhibited specificity for substrates containing a( 1 -+ 4) glucosidic linkages and the K m for starch hydrolysis was 4-0 g/l.

INTRODUCTION lsoelectric focussing

Glucoamylase (1,'f-rZ-D-glUCan glucohydrolase, E.C. 3.2.1.3) is an exo-acting enzyme that catalyses the production of b-glucose from the non-reducing ends of glucose contain- ing di-, oligo- and polysaccharides. The enzyme occurs almost exclusively in filamentous fungi and far less in bac- teria and yeasts (Fogarty & Kelly 1980).

The purification of glucoamylase from Aspergillus terreus has recently been reported (Ali et al. 1990a). Similarities and differences were noted in various aspects of its proper- ties and characteristics compared with glucoamylase from different Aspergillus species (Charuchinda et al. 1984; Punpeng et al. 1984). The present investigation was under- taken to study the physico-chemical properties of gluco- amylase from A . terreus.

MATERIALS AND METHODS

Organism and growth

Aspergillus terreus G T C 826 was grown in rice-bran liquid medium under static condition as described by Ali et al. (1989).

Enzyme assay

Purification of glucoamylase was carried out by DEAE- cellulose and Concanavallin A-Sepharose column chroma- tography (Ali el al. 1990a). Enzyme activity was determined by the method of Ali et al. (1990b). One unit of glucoamy- lase activity was defined as that producing 1 pmol of glucose cquivalent/min. Specific activity was defined as the unit of enzyme/mg of protein.

Correspondence to : Dr S. Ah, Department of Biochemistry, University of Dhaka, Dhaka-1000, Bangladesh.

Isoelectric focussing of the enzyme was performed in a polyacrylamide gel (5% w/v) rod with Pharmalyte (pH 3-10) on a Pharmacia FBE 3000 apparatus. The gels were fixed in a 10% TCA-5% sulphosalicylic acid mixture for 1 h and then stained with Commassie Brilliant Blue R. Glucoamylase activity was also detected in parallel gels after cutting the gels into 0-5 cm segments followed by elu- tion with 400 p150 mmol/l citrate-phosphate buffer (pH 4.0).

Periodate Schlff staining

Periodate Schiff staining was carried out on 7.5%" (w/v) polyacrylamide slab gel using Schiff s reagent (Sigma).

Protein measurement

Proteins were estimated as described by Lowry et al. (195 1) with bovine serum albumin as standard.

RESULTS AND DISCUSSION

Glucose was found to be the sole product at all stages of starch hydrolysis and no oligosaccharide was detected in the course of the reaction (Fig. 1). The isoelectric point of the enzyme was pH 3.4 which revealed that enzyme was an acidic protein. Glucoamylase from A. terreus contained 7.5% carbohydrate (Ali et al. 1990a) in the present investi- gation. Periodate Schiff staining further confirmed that the enzyme was a glycoprotein.

Glucoamylase was maximally active at pH 4.0. The enzyme was fairly stable over the pH range 3G7.0 but not at pH 2.0 and 9.0. Optimum temperature for the action of the enzyme was 60°C. The enzyme was stable at tem- peratures between 40 and 60°C with essentially no loss of

Page 2: Characteristics of glucoamylase from Aspergillus terreus

C H A R A C T E R I Z A T I O N OF GLUCOAMYLASE 145

Fig. 1 Thin layer chromatogram of hydrolysis product of soluble starch by glucoamylase from Aspergillus terreus at different reaction times. Glucose (G) and maltose (M) were used as standards. The corresponding time of hydrolysis (h) is indicated at the bottom of each lane. Samples were run on silica gel plates developed with n-propanol : ethyl acetate : water (4 : 5 : 1 by vol.) and detected as described by Trevelyn et at. (1950)

activity in 30 min. The effects of various monovalent, diva- lent and trivalent ions were studied. Among the metal ions tested, Ag' and Hg2+ had a pronounced inhibitory effect on the enzyme. Mg2 + and Ca2 + ions stimulated glucoamy- lase activity 15%) and 7% respectively. Substrate specificity of the glucoamylase was tested with a-methyl glucoside, sucrose, starch, maltose and maltotriose (Table 1). The enzyme could hydrolyse different substrates in the order : starch, maltotriose and maltose. The Lineweaver-Burk plot allowed the calculation of Km equal to 4.0 g/l (Fig. 2).

The enzyme properties showed marked similarities with various Aspergillus species reported by many other investi-

-4 0 4 8 12 16

(O/O) [*I Fig. 2 Lineweaver-Burk plot for the determination of Km value of glucoamylase. The reaction was carried out at 60"C, using 50 mmol/l citrate phosphate buffer (pH 4.0), 4 U glucoamylase and different concentrations of starch

Table 1 Substrate specificity of glucoamylase from Aspergilhs terreus*

Substrate (0.5%) - ~-

Specific activity

Starch Cellobiose Sucrose a-methyl glucoside Maltose Maltotriose

188-0 0.0 0.0 0.0

63.3 98.5

* The amount of reducing sugar was determined by the glucose oxidase-peroxidase method (Killburne & Taylor 1969). Results are the average of three determinations.

gators in respect of metal ion requirement (Manjunath et al. 1983), optimum temperature and pH (Saha & Ueda 1981; Punpeng et al. 1984), pH and heat stabiiity (Klesov & Gerasimas 1979; Tani et al. 1986) and substrate specificity (Attia & Ali 1977; Bartoszweick 1986).

REFERENCES

A L I , S . , MAHMOOD, S . , A L A M , R . & H O S S A I N , Z . (1989) Culture condition for production of glucoamylase from Asper- gillus terreus from rice bran by Aspergillus terreus. MIRCEN Journal of Applied Microbiology und Biotechnology 5, 525-532.

A L I , S . , H O S S A I N , Z . , M A H M O O D , S . & A L A M , K . (1990a) Purification of glucomylase from Aspergillus terreus. World Journal of Microbiology and Bintechnology 6, 16- 18.

A L I , S . , H O S S A I N , Z . , M A H M O O D , S. & A L A M , R . (1990b) Induction of glucoamylase production by non-starchy carbohydrates in Aspergillus terreus. World Journal of Mtcro- biology and Biotechnology 6, 19-22.

A T T I A , R . M . & A L I , S . A . (1977) Utilization of agricultural wastes by Aspergillus awamori for the production of glucoamylase. Zentrablatt f ; r Bakteriologie, Parusitenkrankheiten Infektionskrankheiten und Hygiene 132, 322-325.

Page 3: Characteristics of glucoamylase from Aspergillus terreus

146 S . ALI AND Z . H O S S A I N

B A R T O S Z W E I C K , K . (1986) Glucoamylase of Aspergillus niger. Acta Biochim Poland 33, 17-29.

C I I A w u c I I I N 1) A , A ., L A o P A I B o O N , R . , C H A I SR I SOOK , C . , LOTONG, N. & YAMAMOTO, K . (1984) Glucoamylase of citric acid producing mutant of Aspergillus niger. In Microbial Utilization uf Renewable Resources ed. Taguchi, H. Vol. 4, pp. 101-106. Osaka, Japan: Osaka University.

FOGARTY, W.M. & KELLY, C . T . (1980) Amylases and amy- loglucosidases and related glucanases. In Microbial Enzymes and Bioconversions (Economic Microbiology) ed. Rose, A.H. Vol. 5, pp. 115-170. New York: Academic Press.

K I I . I . R U R N E , D . M . & TAYLOR, R.M. (1969) Effect of sulfhydryl reagents on glucose determination by the glucose oxidase method. Analytical Biochemistry 27, 555-558.

K L E S O V , A . A . & G E R A S I M A S , V . B . (1979) Substrate ther- mostabilization of soluble and immobilized glucoamylase. Bio- khimiu 44, 1084-1092.

I . O W R Y , O . , R O S E B R O U G H , N . , F A R R , A . L . & R A N D A L L , P. (1951) Protein measurement with folin phenol reagent.

Journal of Biological Chemistry 193, 265-275. MANJUNATH, P. , S H E N O Y , B.C. & R A G H U V E N D R A RAO,

M . R. (1983) Fungal glucoamylases. Journal of Applied Bio- chemistry 5, 235-260.

PUNPENG, P . , H A R A , T. & HAYASHIDA, S . (1984) Glu- coamylase from an acid tolerant strain of black Aspergillus. In Annual Reports of ZCME ed. Taguchi, 11. Vol. 7, pp. 291-302. Osaka, Japan : Osaka University.

S A H A , B.C. & U E D A , S . (1981) Inhibition of raw starch digestion by one glucoamylase preparation of black Aspergillus at high enzyme concentration. StarchlStrake 33,313-316.

T A N I , Y., VONGSUVANLERT, V . & KUMUNUANTA, J . (1986) Raw cassava starch digestive glucoamylase of Aspergillus spp. N-2 isolated from cassava starch. Journal of Fermentation Technulogy 64, 405-413.

T R E V E L Y N , W . E . , PROCTER, D . P . & H A R R I S O N , J . S . (1950) Detection of sugar on paper chromatogram. Nuture (London) 166,444445.