Subsite mapping of purified glucoamylase I, II, III produced by Arthrobotrys amerospora ATCC 34468 D. Norouzian 1; *, K. Rostami 2 , D. Nouri Inanlou 1 and M. Saleh 1 1 Pilot Biotechnology Dept., Pasteur Institute of Iran, Tehran, Iran 2 Biotechnology Center, IROST, Iran *Author for correspondence: Fax: 6465132, E-mail: dnsa@Institute.Pasteur.ac.ir Received 29 July 1999; accepted 6 January 2000 Keywords: Arthrobotrys amerospora, glucoamylases, subsite mapping Summary Arthrobotrys amerospora ATCC 34468 produced glucoamylase in a medium containing maize starch as carbon source. On native PAGE, crude glucoamylase showed three isoenzymes which were designated as Glu I, Glu II, Glu III according to their electrophoretic mobility. These were purified by column chromatography techniques. The energy of binding for each glucoamylase was calculated using Hiromi’s kinetic based calculation. At subsite 1, the binding energies for Glu I, II and III were found to be negative. Introduction Arthrobotrys amerospora is a nematode-trapping fungus. The genus Arthrobotrys is widely distributed in nature (Mankau 1980). One strain, Arthrobotrys amerospora produced glucoamylase with no detectable transglucos- idase activity in a semi-synthetic medium containing maize starch as carbon source (Jaar et al. 1993). Glucoamylase (1,4-a-D-glucan glucohydrolase, EC 3.2.1.3) is an important biocatalyst used in hydrolysis of liquefied starch to D-glucose. The hydrolysis involves successive cleavage of glycosidic bonds starting from the non-reducing end by multichain attack. According to Hiromi et al. (1973), the cleavage of various a-glucosidic bonds occurs at the same active site in the enzyme molecule. A major problem in the starch-processing industry where glucoamylase is employed is the poor D-glucose yields, caused by the formation of dierent iso-oligosaccharides (di-tri & tetrasaccharide) during the process of starch hydrolysis. Based on kinetic studies of glucoamylases of Aspergillus saitoi (Takashi et al. 1984), Aspergillus oryzae (Kazuhisa et al. 1988), Aspergillus niger (Meagher et al. 1989); (Christensen et al. 1997), the following mechanism has been proposed: (1) glu- coamylase has 7 subsites; (2) glycosidic linkages of maltooligomers are cleaved between subsite 1 and 2; (3) subsite anities are greatest at subsite 2; (4) the anity of an individual site decreases from subsite 3 to 7; (5) the subsite anity at site 1 for glucoamylase is very low while for a-glucosidase it is high. In this paper we report purification of glucoamylase produced by Arthrobotrys amerospora using column chromatography, employing electrophoresis techniques for establishing purity of each iso-enzyme and measure- ment of V max ; K m with respect to maltooligosaccharides (maltose to maltoheptaose) containing a-1-4 linkages. The free energy binding of each D-glucosyl residue in each subsite in the glucoamylase active site is calculated by the Hiromi et al. (1973) method. As there is no report on glucoamylase of Arthrobotrys amerospora therefore we decided to study the kinetic behaviour of this enzyme and compare the results with the available ones. Materials and Methods Chemicals Starch, maltooligosaccharide (G 2 to G 7 ), glucose oxi- dase, horse-radish peroxidase, polyethylene glycol 20000, molecular weight marker, Coomassie brilliant blue R250, biotin, Tris were from Sigma Chemical Company USA. o-Dianisidine and TEMED were from Kochlight, England. Riboflavin and acrylamide were from Pfizer, India. 3,5-Dinitrosalicylic acid was from Centron, India. Bis-acrylamide and bromophenol blue were from BDH, England. Glucose was from Merck, Germany. Sephadex G-25, G100 and DEAE-Sephadex A-50 were from Pharmacia, Sweden. Other reagents used were of analytical grade. Organism Arthrobotrys amerospora ATCC 34468 was obtained from American Type Culture Collection, Maryland, USA. It was sub-cultured every 4–6 weeks on corn meal agar. World Journal of Microbiology & Biotechnology 16: 155–161, 2000. 155 Ó 2000 Kluwer Academic Publishers. Printed in the Netherlands.