Enzyme preparation from extract of wheat bran koji by alcohol precipitation

A method for the preparation method of enzymes for shoyu making was studied. When enzyme proteins were extracted with water from a column of wheat bran koji culture of Aspergillus oryzae 460, the tailing phenomenon resulted in low recovery. However, a better yield was obtained by the use of the solution passed at the latter stage of extraction as the extraction liquid for the succeeding extraction. Thus, in case of leucine aminopeptidase (Leu-Gly-Gly as substrate) the extraction yield reached 89%. Considering the extraction yield, and the amount of alcohol required to precipitate enzyme proteins, an appropriate volume of extract was found to be 1.5 to 2 times the amount of wheat bran koji. Moreover, less sporulation of koji culture due to a shortening of the culture time to 48 h, or 38 h by the use of germinated spores as seed culture, resulted in less water repellence, and a higher yield of extraction of enzymes than in the old culture (58.5 h). The temperature of the mixture of extract and alcohol should be kept at 5°C to increase the yield of leucine aminopeptidase and acid carboxypeptidase. By the concentration of enzyme proteins through ultrafiltration, the use of alcohol could be reduced, and an enzyme preparation with high specific activity and recovery could be obtained.