World Journal of Microbiology & Biotechnology 17: 747750, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

747

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, Tehran 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 produced 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 mutational and lethal effects of these mutagens on 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 nitrous acid, which produced 6.73 times more dextrinizing and 5.13 times more saccharogenic activity than the parent strain. In general, the mutants obtained by nitrous acid and u.v. were more potent than those obtained by MNNG.

Introduction Aspergillus oryzae is a well-known fungus, which has been widely used to obtain many kinds of hydrolytic enzymes, like a-amylase, protease and nuclease. To prepare these extracellular enzymes on 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 linkages randomly 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 presence of 2deoxy-D -glucose is a way to obtain more efcient enzyme-producing cells (Annos & Blaschek 1991; Cheng & Yang 1995). In this study, u.v. light (Calam 1970; Fantini 1975; Crueger & Crueger 1984), nitrous acid (Sinha & Chakrabarty 1977; Carlton 1981) and N-methyl-Nnitro-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 -glucose was used for selection of mutants, most of the isolates showed either increased or at least unaltered levels of a-amylase production as compared to the parent strain.

Materials and methods Microorganism As parent strain, the a-amylase-producing strain of A. oryzae PTCC 5164, was used. Freeze-dried conidiospores of the fungus were used to inoculate potato dextrose agar (Oxoid) plates containing per l:200 g potato, 20 g glucose, 20 g agar. After maturation of conidiospores, indicated by formation of a homogeneous green mat, covering the surface of the plates, the conidia were washed with phosphate buer (0.2 M, pH 6.5) containing 0.1% (v/v) Tween 80, and used for mutagenesis. Random mutagenesis U.V. treatment Washed conidial suspension of about 106 conidia/ml were plated on a solid dened medium, which contained per l: 5.0 g soluble starch, 3.0 g NaNO3, 0.5 g KCl, 1.0 g KH2PO4, 0.5 g MgSO4 7H2O, 0.01 g FeSO4 7H2O, 1.0 ml Tween 80, 17 g agar, pH 5.5 (Sinha & Chakrabarty 1978). All the plates were 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

748 2-deoxy-D -glucose (0.2% w/v), was shown to be inhibitory for growth of the parent strain. To the above medium, 0.08% (w/v) sodium deoxycholate (Merck, Germany) was added in order to reduce the size of colonies, and hence their more precise isolation (Hopwood 1970). The plates were exposed to short wavelength u.v. light, (280 nm) from a distance of 20 cm by using a PhilipsTM 30 W germicidal u.v. lamp, till a survival of about 0.1% was obtained (Sinha & Chakrabarty 1977; Baltz 1986). MNNG treatment Washed suspension of A. oryzae conidia was centrifuged for 5 min at 3000 g. To the pellet was added 10 ml citrate buer (0.1 M, pH 5.0) containing 4 mg MNNG per millilitre (Alikhanian 1962; Adelberg 1965), where a concentration of 106 conidia/ml, was obtained. After 1050 min., the reaction was stopped by adding cysteine at a concentration of 100 mg/ml and pelleting the conidia by centrifugation for 5 min at 3000 g. After appropriate dilution of conidia in citrate buer, they were plated on agar medium supplemented with 0.2% (w/v) 2-deoxy-D -glucose and 0.8% sodium deoxycholate (w/v). Nitrous acid treatment A 0.07 M solution of NaNO2 in acetate buer (0.2 M, pH 4.5) was added to the washed and centrifuged conidia of A. oryzae (Sinha & Chakrabarty 1977; Carlton & Brown 1981). The solution was thoroughly shaken for 210 min. At given times between 2 and 10 min 1 ml of solution was withdrawn and diluted 5fold in phosphate buer (0.2 M, pH 7.1) to stop the reaction. The conidia were cultured on agar medium containing 0.2% (w/v) 2-deoxy-D -glucose and 0.8% sodium deoxycholate (w/v). Mutagen-free plates were treated similarly as control during above experiments. Selection of mutants After 72 h of incubation in 26 C, those colonies showing a halo zone diameter/colony diameter (H/C ratio) larger than that of the parent strain, on starchcontaining medium, were selected for the second step of screening (Hopwood 1970; Fantini 1975; Jacobson 1984; Queener & Lively 1986). Determination of enzymatic activity Both the saccharogenic and dextrinizing activities of the mutants in liquid culture were determined in the second step of screening. Hundred millilitre of liquid culture media, as described above, but without agar, were inoculated by 1 ml of 106 conidia/ml of mutant and parent strains and incubated on shaker-incubator (IRCI-U, Clim-O-Shake, Adolf Kuhner, Switzerland), at 26 C, 150 rev/min for 72 h. Clear supernatant was obtained by centrifugation of cultures and used as crude enzyme source.

M. Azin and E. Noroozi Saccharogenic activity a-Amylase activity which resulted in production of reducing sugars (as maltose) in the product molecules, was measured by the dinitrosalicylic acid (DNS) method, according to Bernfeld (1955), after 6 min, where 1% (w/v) soluble starch in phosphate buer (0.2 M, pH 7.0) at 37 C was used as substrate. Each unit of saccharogenic activity was dened as the amount of enzyme that produced 1 lmol maltose per minute. Dextrinizing activity Dextrinizing activity of the enzyme, which resulted in the production of short dextrin molecules, was measured at 37 C, using 1% (w/v) potato starch in acetate buer (0.2 M, pH 5.0). Since the activity of the enzyme was to be evaluated against raw starch, potato starch was used instead of a soluble starch. Reaction was stopped by placing the reaction tubes in a boiling water bath, after which, by using the iodine method, the extent of dextrinizing activity was calculated. Each unit of dextrinizing activity was dened as the amount of enzyme activity that in one minute causes 10% reduction in colour density (660 nm), which was generated by iodine solution (Yamamoto 1988).

Results and discussion During three stages of mutagenesis, lethal doses and frequency of selected mutants were calculated as presented in Figure 1. As shown, the best mutation frequencies, which have been calculated as ratio of surviving cells, in the presence of 2-deoxy-D -glucose after mutagenesis, to the total number of cells at the beginning of treatment, has been observed after 2, 40 and 4 min of exposure of the conidia to u.v. light, MNNG and nitrous acid, respectively. Although H/C ratio measurement is not a precise means for nal judgment about the a-amylase production ability of the isolates, it proved useful in the rst screening step, where large number of colonies should have been screened for their potential ability to digest starch. Since the rst screening step was performed in the presence of 2-deoxy-D -glucose, the isolated colonies produced mostly bigger H/C ratios than the parent strain. As a result, after treatment of parent conidiospores by nitrous acid, u.v. light and MNNG, respectively, 65.3, 52.8 and 39.4% of isolates, showed bigger H/C ratios than the parent. 5.5 and 1.4% of the isolated mutants resulted from nitrous acid treatment showed respectively, 30 and 40% bigger H/C ratios, than that of the parent. The best mutants obtained at each step, were determined based on their enzyme activity in liquid culture, in the secondary screening step. Table 1 shows the results thus obtained. Both the saccharogenic and dextrinizing activities of the mutants were measured, and as is

Random mutagenesis of A. oryzae

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Figure 1. Comparison of eect of dierent mutagens on the surviving fractions' percent (S, d) of A. oryzae conidia and mutant frequencies' percent (M, h) obtained.

Table 1. Saccharogenic and dextrinizing activities of the parent strain and mutants, obtained by dierent methods. Mutagen agent Mutant PTCC 5164 (parent) N11 N13 U.v. light P22 P24 A2 P122 P124 Nitrous acid H2 H4 H8 MNNG Saccharogenic Dextrinizing activity (U/ml) activity (U/ml) 39 90 130 87 100 150 126 115 70 110 200 (2.31) (3.33) (2.23) (2.56) (3.84) (3.23) (2.94) (1.8) (2.82) (5.13)
a

References
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1.41 5.11 6.45 5.18 6.55 7.18 4.75 4.57 4.02 6.77 9.49 (3.62) (4.57) (3.67) (4.65) (5.10) (3.37) (3.24) (2.85) (4.80) (6.73)

a The numbers in parentheses, show the rate of increase in amylolytic activity, relative to the parent strain.

shown, mutant H8, with respectively 5.13 and 6.73 times more saccharogenic and dextrinizing activity, than the parent strain, was the most potent strain isolated. 2-Deoxy-D -glucose is a suitable antimetabolite for isolation of a-amylase derepressed mutants as is shown here and by other authors (Annos & Blaschek 1991; Cheng & Yang 1995). The highest percentage occurrence of mutagenesis was observed after treatment of conidia by nitrous acid for 4 min. In these circumstances, 5.5% of survivors produced the enzyme at an elevated level. Acknowledgements This work was nanced by Iranian Research Organization for Science and Technology. The technical assistance of Mr Davood Zareh is gratefully appreciated.

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Queener, S.W. & Lively, D.H. 1986 Screening and selection for strain improvement. In: Manual of Industrial Microbiology and Biotechnology, eds. Demain, A.L. & Solomon, N.A. pp. 155169. American Society for Microbiology. ISBN 0-914826-73-5. Sinha, S. & Chakrabarty, S.L. 1977 Relative eectiveness of mutagens on amylase production in Aspergillus wentii. Indian Journal of Experimental Biology 15, 10681070. Sinha, S. & Chakrabarty, S.L. 1978 Production of amylase in a submerged culture of Aspergillus wentii. Folia Microbiologica 23, 611.

M. Azin and E. Noroozi

Uhlig, H. 1998 Amylases. In: Industrial Enzymes and their Applications, pp. 4060. John Wiley and Sons, Inc. ISBN 0-47119660-6. Webb, J.L. 1966 Enzyme and Metabolic Inhibitors vol. II, pp. 386403. Academic Press. Inc. Yamamoto, T. 1988 Assay method of amyloclastic activity. In: Handbook of Amylases and Related Enzymes. pp. 247248. The Amylase Research Society of Japan. Japan. Pergamon Press. ISBN 0-08-036141-2.