Isolation and overexpression of a gene encoding an extracellular β- (1,3-l,4)-glucanase from Streptococcus bovis JB1

Abstract

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against β-(1,3-1,4)glucans. A gene was isolated encoding a specific β-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant β- glucanase activity was obtained when the cloned β-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The β-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JBL. The β-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a V(max) of 338 μmol of glucose equivalents per rain per mg of protein with barley β-glucan as the substrate. The S. bovis β-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.