Studies on Mold Dextranases. Part II. Dextranase Production by a Strain of Aspergillus carneus. and Juichiro FUKUMOTO

Transcription

1 [Agr. Biol. Chem., Vol. 35, No. 11, p `1732, 1971] Studies on Mold Dextranases Part II. Dextranase Production by a Strain of Aspergillus carneus By Daisuke TSURU,* Nobutsugu HIRAOKA and Juichiro FUKUMOTO, Tsuneyo HIROSE Faculty of Science, Osaka City University, Sumiyoshi-Ku, Osaka Received April 16, 1971 Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28 Ž for 5 to 6 days in medium containing 1% dextran, 1% NaNO3 or polypeptone, 0.2% yeast extracts, 0.4% K2HPO4 and small amounts of inorganic salts, ph 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus. In a previous paper of this series," we reported Penicillium luteum ATCC 9644 to produce dextranase in amounts equivalent to or greater than those found in Pen. funiculosum2,3) and Pen. lilacium.2,3) In order to obtain the microorganisms which are much more active in producing dextranase than molds so far reported, about 400 strains isolated from soil were subjected to screening. Nine of them secreted dextranase, but only one strain was found to exceed Pen. luteum ATCC 9644 in enzyme-forming ability. The strain was identified as Asp. carneus. In the present paper, culture conditions for the production of dextranase by the mold and some general properties of the mold are described. MATERIALS AND METHODS Cultures. About 400 strains were isolated from soil and subjected to the examination of the dextranase- TABLE I. CHEMICAL COMPOSITION OF MEDIA USED FOR THE SCREENING OF DEXTRANASE-FORMING MICROORGANISMS Microorganisms were aerobically grown in 20 ml of medium per 100 ml Erlenmyer flask at 28 C. 1) J. Fukumoto, H. Tsuji and D. Tsuru, J. Biochem., 69, 1113 (1971). 2) T. Kobayashi, Nippon Nogeikagaku Kaishi, 352 (1954) ) H. M. Tsuchiya, A. Jeanes, H. M. Bricker and C. A. Wilham, J. Bacteriol., 64, 513 (1952). * To whom requests for reprints should be addressed. The present address is: Faculty of Pharmacological Sciences, Nagasaki University, Bunkyo-Machi 1-14, Nagasaki City 852. a) Commercial preparation for industrial use.b ) Defatted cotton seeds debris c) Corn steep liquor. * Concentration, 0%.

2 a 1728 D. TSURU, N. HIRAOKA, T. HIROSE and J. FUKUMOTO forming ability in media containing dextran as the carbon source. Three kinds of media were chosen, the composition of which was presented in Table I. The medium, 20 ml, in a 100 ml Erlenmyer flask was inoculated with spores or vegetative cells of respective microorganisms and aerobically incubated at 28 C for five days on a reciprocating shaker. Leuconostoc mesenteroides IAM 1046 was kindly supplied from Institute of Applied Microbiology, Tokyo University. Three strains of dextran-forming bacteria, which seemed to belong to Leuconostoc, were isolated from commercial malts by the method recommended by Misaki and his co-workers.4) The agar medium for isolating dextran-forming bacteria was composed of 2.0% sucrose, 0.5% polypeptone, yeast extracts, 0.1A% K2HPO4, 0.1% MgSO4 E7H2O, 0.002% NaCl and agar, ph 7.0. After cultivation for 24 hr at 25 C on the plate agar medium, the colonies which significantly swelled out were isolated. For the production of dextran, the bacteria isolated were grown at 25 C for 15 hr in a liquid medium containing 1000 sucrose, polypeptone, yeast extracts, 0.1% K2HPO4, 0.1% MgSO4 E7H2O, 0.003% NaCl and % FeSO4 E7H2O, ph 7.0, as reported by Misaki et al.,41 and the dextran accumulated was purified by repeated fractionations by ethanol as described by Kobayashi.5) Analytical methods. Reducing sugars were measured according to the method of Schaffer-Somogyi6) and calibrated against glucose. Total sugars were determined by Phenol-H2SO4 method of Dubois et al.7) Assay of dextranase activity was carried out as follows: Chemicals. Dextran T-2000 (molecular weight: 2,000,000) and commercial dextran preparations for industrial use were purchased from Pharmacia Fine Chemicals, Uppsala, through Seikagaku Kogyo Co. Ltd., Tokyo. All the chemicals and salts were of reagent grade and used without further purificatioṇ RESULTS AND DISCUSSION Comparison of dextranase forming ability among various strains isolated from soil Of 400 strains isolated, only 9 strains were found to secrete dextranase, as shown in Table II. Two of them belonged to mold, one of them to yeast and the remainder were bacteria, though identification of these strains remained to be done. For comparison, enzyme produc tion by Pen. luteum ATCC 9644 was also ex amined under the same condition. A strain belonging to Aspergillus was most active in producing dextranase, the ability being much higher than that of Pen. luteum ATCC TABLE II. SCREENING OF DEXTRANASE-FORMING MICROORGANISM Microorganisms isolated from soil or stocked at our laboratory were aerobically grown at 28 C for 4 to 5 days in three kinds of media shown in Table I. Dextran T-2000, g, was dissolved in 100 ml of 50 mm acetate buffer solution, ph 5.5. To 4 ml of the dextran solution at 40 C was added one ml of enzyme, and the mixture was incubated for 10 min.at 40`C. The reducing sugars liberated were determined by the method of Schaffer and Somogyi.6/ One unit of dextranase activity was defined as the enzyme quantity which liberated reducing sugar equivalent to one p mole of glucose per minute under the condition. 4) A. Misaki, S. Yukawa, T. Asono and M. Isono, Ann. Refit. Takeda Res. Lab., 25, 42 (1966). 5) T. Kobayashi, Nippon Nogeikagaku Kaishi, 25, 421 (1951). 6) P. A. Schaffer and M. Somogyi, J. Biol. Chem., 100, 695 (1933). 7) M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Reberts and F. Smith, Anal. Chem., 28, 350 (1956). ) Stocked cultures in our laboratory

3 Studies on Mold Dextranases. Part II 1729 Effect of carbon source on the enzyme production by the Aspergillus isolated Microbial dextranases have been known to be formed induciblly in general. A strain of Aspergillus chosen here also produced dextranase only when it was grown in media containing dextran as the carbon source. When other carbohydrates were added to the medium to gether with dextran, they were rather inhibitory or retarded the period of the enzyme produc tion. The results are shown in Fig. 1. In this FIG. 2. Effect of Dextran Concentration on Dextra nase Production. The medium used was the basal one shown in Fig. 1, except that dextran concentration was varied as indicated. Cultivation temperature was 28 Ž. Dextran concentration: medium containing 1.5% dextran. However, longer period of cultivation was necessary for the enzyme production, and viscous culture broth was obtained under the condition. To FIG. 1. Effect of Some Carbohydrates on Dextranase Production Induced by Dextran. The medium used was composed of 1% dextran (T-2000), 0.5% NaNO3, 0.40% K2HPO4, 0.2% yeast extracts, 0.02% each of MgSO4 E7H2O and KCl, 0.001% FeSO4 E7H2O, ph 8.0. To this medium, 0.50 carbohydrates indicated were added. Cul tivation temperature was 28"C A: Control (dextran) B: A+Lactose C: A+Starch D: A+Fructose experiment, the medium containing 1 0 dext ran, 0.5% NaNO3, 0.4% KSHPO4, 0.2% yeast extracts, 0.02% each of MgSO4 E7H2O and KCl, 0.001% FeSO4 E7H2O, ph 8.0, was used as the basal one. Effect of dextran concentration on the enzyme production was also examined. The results are illustrated in Fig. 2, indicating the maximal production of the enzyme to occur in the avoid these defects, and from economical point of view, the medium containing 1% dextran was usually used for later experiments and for large scale production of dextranase. The dextrans of Leuconostoc mesenteroides IAM 1046 and of an unidentified strain No. 2 isolated from a commercial malt were also used as the carbon source and their inducibilities were compared with those of the commercial pre parations for the enzyme production. The result is shown in Fig. 3. Dextran IAM was found to be about three times much more effective than dextran T-2000 for the enzyme production. The former dextran has been assumed to be composed of 66% of ƒ -1,6-, 19% of ƒ -1,4- and 15% of ƒ -1,3-glucosidic linkages" 8) A. Hattori and K. Ishibashi, Presented at Ann. Meet. Agr. Chem. Soc. Japan, April 2 (1970).

4 1730 D. TSURU, N. HIRAOKA, T. HIROSE and J. FUKUMOTO TABLE III. EFFECT OF NITROGEN SOURCE ON DEXTRANASE PRODUCTION The Aspergillus was grown at 28 C on a recipro cating shaker in the medium shown in Fig. 1, except that nitrogen sources indicated were used. FIG. 3. Comparison of Dextranase Production In duced by Three Kinds of Dextrans. The basal medium shown in Fig. 1 was used. and was slowly hydrolyzed by the Aspergillus dextranase presented here." Dextran T-2000, on the other hand, contains more than 95% of ƒ -1,6-glucosidic linkage"' and was hydrolyzed very rapidly by the enzyme." The effectiveness of dextran No. 2 lay between that of two dextrans mentioned above. The culture broth from medium containing dextran IAM was, however, quite viscous and there were some troubles during the purification of dextranase.9) Thus, the commercial dextran preparation for industrial use* was used for large scale produc tion of the enzyme. Effect of nitrogen source on enzyme production Dextranase production in media containing various nitrogen sources was summarized in 9) J. Fukumoto, N. Hiraoka and D. Tsuru, Pre sented at Ann. Meet. Agr. Chem. Soc. Japan, April 4 (1971). 10) M. Nomoto, Personal communication. * Showed almost the same effect as that of dextran T-2000 on the enzyme production. Table III. The chemical composition of media used here was the same as that of basal one adopted in Fig. 1, except that NaNO3 was replaced by various organic and inorganic nitrogen sources. The results indicate that 100 polypeptone is most effective and 1% NaNO3 follows it but ammonium nitrate is completely inert for the enzyme production. Effects of ph and temperature on enzyme production The enzyme production in media of various initial ph values is illustrated in Fig. 4. The mold produced greater amounts of dextranase in media of higher initial ph values. As shown in Fig. 5, the enzyme production was observed at earlier period of cultivation at 28 C, but the final amounts of enzyme accumulated were greater at 25 C cultivation than at 28 C. These results suggest that cul tivation of the mold at 28 C for the first 2 days and at 25 C for the additional 3 to 4 days is most suitable for the enzyme production. In

5 Studies on Mold Dextranases. Part II 1731 TABLE IV. CHEMICAL COMPOSITION OF MEDIA AND CULTURE CONDITION USED FOR THE DEXTRANASE PRODUCTION BY Aspergillus ISOLATED FIG. 4. Dextranase Production in Media of Various Initial ph Values. IAM: Dextran IAM C.P.. Dextian for industrial use. * NaNO3 FIG. 5. Effect of Temperature on Dextranase Pro duction. fact, we could obtain the culture broth showing 140 units dextranase per ml or more by cul tivating the mold under the condition of me dium B shown in Table IV The time course of the growth of mold and the enzyme pro duction is summarized in Fig. 6. Identification of the Aspergillus isolated In Fig. 7, the microphotograph of a strain of Aspergillus used here is shown. The mold forms pale grayish pink spores when grown on FIG. 6. Time Course of Cell Growth and Dextra nase Production. Culture condition is shown in Table V-B.

6 1732 D. TSURU, N. HIRAOKA, T. HIROSE and J. FUKUMOTO TABLE V. COLONY CHARACTERISTICS OF THE Aspergillus TABLE VI. MICROSCOPIC CHARACTERISTIC OF THE Aspergillus FIG. 7. Microphotograph of the Strain of Aspergillus which Showed High Dextranase Production. a Koji agar medium. It also accumulates grayish green pigment in the culture broth when grown in media containing dextran at ph values higher than 8. From the compara tive taxonomic experiments, the mold was tentatively identified as a strain of Aspergillus candidus. More detailed taxonomic reinvestiga tions were done by the staffs of Central Research Laboratory of Kyowa Hakko Kogyo Co., Ltd., and the mold was finally identified by them to be a strain belonging to Aspergillus carneus. The morphological characteristics of the mold are summarized in Tables V and VI. Acknowledgements. The authors wish to express their gratitude to Dr. S. Kinoshita and other staffs of Central Research Laboratory of Kyowa Hakko Kogyo Co., Ltd. for performing the identification of the mold used. Thanks are also due to Dr. A. Misaki for his valuable advices for isolating dextran-forming bacteria.