Molecular Colning and Ewpression of the $\alpha$-L-Arabinofuranosidase Gene of Bacillus stearothermophilus in Escherichia coli

Bacillus stearothermophilus로부터 $\alpha$-L-Arabinofuranosidase 유전자의 클로닝 및 Escherichia coli에서의 발현

  • Eom, Soo-Jung ;
  • Kim, Hee-Sun ;
  • Cho, Ssang-Goo ;
  • Choi, Yong-Jin
  • 엄수정 (고려대학교 자연과학대학 유전공학과) ;
  • 김희선 (고려대학교 자연과학대학 유전공학과) ;
  • 조쌍구 (고려대학교 자연과학대학 유전공학과) ;
  • 최용진 (고려대학교 자연과학대학 유전공학과)
  • Published : 1994.12.01

Abstract

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.

Keywords

$\alpha$-Arabinofuranosidase;Bacillus stearothermophilus;molecular cloning

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