title: Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.
abstract: Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(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 beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(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 JB1. The beta-(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 Vmax of 338 mumol of glucose equivalents per min per 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 bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.
authors.affiliation: Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom.
authors.forename: M S
authors.initials: MS
authors.lastname: Ekinci
authors.forename: S I
authors.initials: SI
authors.lastname: McCrae
authors.forename: H J
authors.initials: HJ
authors.lastname: Flint
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D044644:Endo-1,3(4)-beta-Glucanase; D013293:Enterococcus faecalis / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016985:Streptococcus bovis / Q000201:enzymology* / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D044644:Endo-1,3(4)-beta-Glucanase