Production of ${\beta}-Galactosidase$ with High Transgalactosylation Activity by Bacillus sp. A4442 Mutant

변이주 Bacillus sp. A4442에 의한 갈락토스 전이활성이 높은 ${\beta}-Galactosidase$의 생산

In an attempt to improve the productivity of ${\beta}-galactosidase$ from Bacillus sp. A1, which was isolated from soil and has remarkably higher transgalactosylation activity than lactose hydrolysis activity, a chemical mutation procedure using N-methyl-N'-nitro-N-nitrosoguanidine followed by selection was conducted. The final selection, designated as Bacillus sp. A4442, turned out to show a substantially increased enzyme productivity. Catabolite repression by glucose and lactose requirement as an inducer for the enzyme biosynthesis, which were shown in the parent strain, was markedly diminished; instead it was found out that galactose acts as another inducer. Because pH of medium, one of the most important factors for cell growth as well as enzyme production, is closely related with the sugar concentration during culture, it was kept in the optimum range of $6.5{\sim}7.5$; for this the initial glucose concentration was adjusted to be 0.5% which was thereafter maintained by the controlled pumping-in of lactose using the pH-stat technique. By doing so, we were able to increase the productivity of ${\beta}-galactosidase$ with high transgalactosylation activity up to $44\;unit/m{\ell}-broth$.

토양으로부터 분리된 Bacillus sp.A1 균주는 가수분해활성보다는 전이활성이 훨씬 높은 ${\beta}-galactosidase$를 생산하기 때문에 산업적 응용 가능성이 있으나, glucose에 의한 catabolite repression을 보일 뿐만 아니라 lactose를 inducer로 요구한다는 결정적인 단점이 있어 갈락토올리고당의 제조에 직접 사용하기에는 적합하지 않았다. 따라서 galactose 전이효소의 생산성 제고를 꾀하기 위하여 N-methyl-N'-nitro-N-nitrosoguanidine를 이용한 3단계 변이를 시도하여 A4442변이주를 선발하였다. 이 변이주의 효소생산 능력은 괄목할 만큼 향상하였으며(약 20배 내외) catabolite repression과 lactose 요구성이 상당히 해제되었음을 확인하였다. 아울러 갈락토스가 새로운 inducer로 작용하는 것도 관찰하였다. 변이주를 이용한 발효시에 당의 농도와 배양액의 pH는 상호연관되어 효소의 생산에 영향을 끼쳤다. pH stat 기법을 이용하여 배양중 당의 농도를 0.5% 이하로 조절할 때 pH는 $6.5{\sim}7.5$ 범위내로 유지되었으며 효소활성은 $44\;unit/m{\ell}-broth$로 높게 나타났다.