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KMID : 0545119970070010008
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Journal of Microbiology and Biotechnology
1997 Volume.7 No. 1 p.8 ~ p.12
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Overproduction of Escherichia coli D-Xylose Isomerase Using λPL Promoter
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PARK, HEUL DONG
JOO, GIL JAEJoo, Gil Jae/RHEE, IN KII
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Abstract
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In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketolisomerase, EC 5.3.1.5) gene (xylA) was fused to λP_L, promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the λP_L, promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of λN and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive λcI857 gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.
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