Overproduction of Escherichia coli D-Xylose Isomerase Using ${\lambda}P_L$ Promoter

  • Park, Heui-Dong (Department of Food Science and Technology, Kyungpook National University) ;
  • Joo, Gil-Jae (Department of Agricultural Chemistry Kyungpook National University) ;
  • Rhee, In-Koo (Department of Agricultural Chemistry Kyungpook National University)
  • 발행 : 1997.02.01

초록

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ 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 ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ 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 ${\lambda}cI857$ 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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