ISOLATION AND OVEREXPRESSION OF A GENE ENCODING AN EXTRACELLULAR BETA-(1,3-1,4)-GLUCANASE FROM STREPTOCOCCUS-BOVIS JB1

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular en zyme active against beta-(1,3-1,4)-glucans. A gene was isolated encodi ng a specific beta-(1,3-1,4)-glucanase that corresponds to this size a nd belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned be ta-glucanase gene was reintroduced into S, bovis JB1 by use of constru cts based on the plasmid vector pTRW10 or pIL253, The beta-(1,3-1,4)-g lucanase gene was also expressed upon introduction of the pTRW10 const ruct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SSI although extracellular activity was 8- to 50-fold lower than t hat in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the cu lture supernatant of S. bovis JB1 carrying pTRWL1R showed a K-m of 2.8 mg per mi and a V-max of 338 mu mol of glucose equivalents per min pe r mg of protein with barley beta-glucan as the substrate. The S, bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacter ium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.