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KMID : 0545119950050010014
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Journal of Microbiology and Biotechnology
1995 Volume.5 No. 1 p.14 ~ p.17
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High-Level Expression of Pseudomonas sp. LBC505 Endoglucanase Gene in Escherichia coli
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CHUN, YANG WOO
KIM, YANG WOO/CHUNG, YOUNG CHUL/KIM, KYEONG SOOK/SUNG, NACK KIE
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Abstract
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Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLC1. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Escherichia coli expression vector, pAS1, containing the leftward promoter P_L of bacteriophage lambda. The level of genec expression was controlled by the thermal inactivation of the heat-sensitive lambda cl857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to 37℃ after induction at 42℃ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. E.coli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLC1. To enhance the expression level of endoglucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pAS1 resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.
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