反应详情

温度

27 °C

A liquid culture medium, consisting of 4.0% (w/v) of “PINE-DEX #4™”, a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST™”, a yeast extract commercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in an amount of 100 ml each, sterilized by autoclaving at 121° C. for 20 min, cooled, and then seeded with a stock culture of Bacillus globisporus C9 strain (FERM BP-7143), followed by culturing under rotary-shaking conditions at 27° C. and 230 rpm for 48 hours for a seed culture. About 20 L of a fresh preparation of the same liquid culture medium as used in the above seed culture were placed in a 30-L fermentor, sterilized by heating, and then cooled to 27° C. and inoculated with 1% (v/v) of the seed culture, followed by culturing at 27° C. and pH 6.0 to 8.0 for 48 hours under aeration-agitation conditions. After completion of the culture, the resulting culture, which had about 0.45 unit/ml of α-isomaltosylglucosaccharide-forming enzyme, about 1.5 units/ml of α-isomaltosyl-transferring enzyme, and about 0.95 unit/ml of a cyclotetrasaccharide-forming activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant. When measured for enzymatic activity, the supernatant contained about 0.45 unit/ml of α-isomaltosylglucosaccharide-forming enzyme, i.e., a total enzymatic activity of about 8,110 units; about 1.5 units/ml of α-isomaltosyl-transferring enzyme, i.e., a total enzymatic activity of about 26,900 units; and about 0.95 unit/ml of cyclotetrasaccharide-forming enzyme, i.e., a total enzymatic activity of about 17,100 units. These activities were assayed as follows: The activity of α-isomaltosylglucosaccharide-forming enzyme was assayed by dissolving maltotriose in 100 mM acetate buffer (pH 6.0) to give a concentration of 2% (w/v) for a substrate solution, adding a 0.5 ml of an enzyme solution to a 0.5 ml of the substrate solution, enzymatically reacting the mixture solution at 35° C. for 60 min, suspending the enzymatic reaction by boiling the solution for 10 min, and quantifying maltose, among the isomaltosyl maltose and maltose formed mainly in the reaction mixture, on HPLC disclosed in Experiment 1. One unit activity of α-isomaltosylglucosaccharide-forming enzyme is defined as the enzyme amount that forms one micromole of maltose per minute under the above enzymatic reaction conditions. Throughout the specification, the enzymatic activity of α-isomaltosylglucosaccharide-forming enzyme means the unit(s) assayed as above.

1. extraction, Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST™”, a yeast extract
2. temperaturecooled
3. customby culturing under rotary-shaking conditions at 27° C.
4. wait230 rpm for 48 hours for a seed culture
5. customAbout 20 L of a fresh preparation of the same liquid culture medium
6. temperatureby heating
7. customby culturing at 27° C.
8. waitpH 6.0 to 8.0 for 48 hours under aeration-agitation conditions
9. customof α-isomaltosylglucosaccharide-forming enzyme, about 1.5 units/ml of α-isomaltosyl-transferring enzyme
10. waitabout 0.95 unit/ml of a cyclotetrasaccharide-forming activity, was centrifuged at 10,000 rpm for 30 min
11. customto obtain about 18 L of a supernatant
12. customof α-isomaltosylglucosaccharide-forming enzyme, i.e.
13. customand about 0.95 unit/ml of cyclotetrasaccharide-forming enzyme, i.e.
14. customThe activity of α-isomaltosylglucosaccharide-forming enzyme
15. customto give a concentration of 2% (w/v) for a substrate solution
16. customat 35° C.
17. customfor 60 min
18. customthe enzymatic reaction
19. customfor 10 min