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

M S Ekinci, S I McCrae, H J Flint

Research output: Contribution to journalArticle

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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-SSI 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 mi and a V-max of 338 mu mol 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.

Original languageEnglish
Pages (from-to)3752-3756
Number of pages5
JournalApplied and Environmental Microbiology
Volume63
Issue number10
Publication statusPublished - Oct 1997

Keywords

  • ACID-SEQUENCE SIMILARITIES
  • MOLECULAR-CLONING
  • ESCHERICHIA-COLI
  • RUMINOCOCCUS-FLAVEFACIENS
  • ALPHA-AMYLASE
  • EXPRESSION
  • 4-GLUCANOHYDROLASE
  • IDENTIFICATION
  • CLASSIFICATION
  • CONSTRUCTION

Cite this

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1. / Ekinci, M S ; McCrae, S I ; Flint, H J .

In: Applied and Environmental Microbiology, Vol. 63, No. 10, 10.1997, p. 3752-3756.

Research output: Contribution to journalArticle

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AU - Flint, H J

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N2 - 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-SSI 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 mi and a V-max of 338 mu mol 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.

AB - 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-SSI 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 mi and a V-max of 338 mu mol 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.

KW - ACID-SEQUENCE SIMILARITIES

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KW - ESCHERICHIA-COLI

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KW - EXPRESSION

KW - 4-GLUCANOHYDROLASE

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KW - CONSTRUCTION

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