Random mutagenesis and use of 2-deoxy- D -glucose as an antimetabolite for selection of a-amylase-overproducing mutants of Aspergillus oryzae Mehrdad Azin* and Elham Noroozi Biotechnology Center, Iranian Research Organization for Science and Technology, P.O. Box 15815-3538, Teh 15819, Iran *Author for correspondence: Tel./Fax: 98-21-8838350, E-mail: azin@irost.com Received 27 March 2001; accepted 24 July 2001 Keywords:a-Amylase, Aspergillus oryzae, 2-deoxy- D -glucose, N-methyl-N¢-nitro-N-nitrosoguanidine, nitrous acid, random mutagenesis, u.v. light Summary By using 2-deoxy- D -glucose, selection of different mutants of Aspergillus oryzae PTCC 5164, which were produc by random mutagenesis by u.v. radiation,nitrous acid and N-methyl-N¢-nitro-N-nitrosoguanidine (MNNG), was studied. 2-Deoxy- D -glucose, a well-known antimetabolite, was used to isolate derepressed mutants. The mutati and lethaleffectsof these mutagenson conidia of A. oryzae were compared and the frequency distribution of isolated mutants, in the presence of 2-deoxy- D -glucose, was determined. Potent mutants, which produced higher dextrinizing and saccharogenic activities, were isolated. The best strain was a result of mutagenesis by nitro which produced 6.73 times more dextrinizing and 5.13 times more saccharogenic activity than the parent str general, the mutantsobtained by nitrousacid and u.v. were more potent than those obtained by MNNG. Introduction Aspergillus oryzae isa well-known fungus, which has been widely used to obtain many kinds of hydrolytic enzymes, like a-amylase, proteaseand nuclease.To prepare these extracellular enzymeson a commercial scale, many attempts have been made to select superior strains of the fungus. a-Amylase from A. oryzae hydrolyses a-1,4-glycosidic linkagesrandomly throughout the starch molecule, and is a widely used industrial enzyme (Fogarty & Kelly 1990;Uhlig 1998). Selection of derepressed mutants of amylase-overproducing strains in the presenceof 2- deoxy- D -glucoseis a way to obtain more ef®cient enzyme-producing cells(Annos& Blaschek 1991; Cheng & Yang 1995). In thisstudy, u.v. light (Calam 1970; Fantini 1975; Crueger & Crueger 1984), nitrousacid (Sinha & Chakrabarty1977;Carlton 1981)and N-methyl-N¢- nitro-N-nitrosoguanidine (MNNG) (Adelberg 1965; Carlton & Brown 1981; Baltz 1986),were used to mutagenize A. oryzae conidiospores, which were then cultured in the presence of 2-deoxy- D -glucose to select derepressed a-amylase mutants (Webb 1966; Queener & Lively 1986).Since 2-deoxy- D -glucosewas used for selection of mutants, most of the isolates showed either increased orat least unaltered levelsof a-amylase production ascompared to the parent strain. Materials and methods Microorganism Asparent strain,the a-amylase-producing strain of A. oryzaePTCC 5164, wasused. Freeze-dried conidio- sporesof the funguswereused to inoculatepotato dextroseagar (Oxoid) platescontainingper l:200 g potato,20 g glucose, 20 g agar.After maturation of conidiospores, indicated by formation ofa homoge- neous green mat, covering the surface of the plates, the conidia were washed with phosphate buer (0.2 M, pH 6.5)containing 0.1% (v/v)Tween Ò 80, and used for mutagenesis. Random mutagenesis U.V. treatment Washed conidialsuspension ofabout10 6 conidia/ml were plated on a solid de®ned medium, which contained per l: 5.0 g soluble starch, 3.0 g NaNO 3 , 0.5 g KCl, 1.0 g KH 2 PO 4 , 0.5 g MgSO 4 á 7H 2 O, 0.01 gFeSO 4 á 7H 2 O, 1.0 ml Tween Ò 80, 17 g agar, pH 5.5 (Sinha & Chakra- barty 1978). All the plateswere supplemented with 0.2% (w/v) 2-deoxy- D -glucose(Sigma,France),which was sterilized by passing through 0.2 lm pore sized cellulose acetate membrane ®lters(Sartorius, Germany), to allow isolation of derepressed mutants. This concentration of World Journal of Microbiology & Biotechnology 17: 747±750, 2001. 747 Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.