Cloning of the Bacillus pumilus beta-xylosidase gene (xynB) and its expression in Saccharomyces cerevisiae.
A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the beta-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2P-MF alpha 1S-xynB-ADH2T was designated XLO1. Both plasmids pDLG11 and PDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional beta-xylosidase. The total beta-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45-50 degrees C and pH 6.6 respectively. The enzyme was thermostable at 45 degrees C; however, at 60 degrees C most of the Xlo1 was inactive after 5 min.
La Grange
D C
DC
Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
I S
IS
van Zyl
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WH
D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase