Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength.
The enzymatic approach to the treatment of cellulosic wastes depends on the availability of cost-effective means for the production of cellulases. We have engineered an excretion construct, tacIQpar8cex, to investigate the extracellular production of a Cellulomonas fimi exoglucanase (Exg) in Escherichia coli. The overall yield of Exg expressed by the culture of JM101 (tacIQpar8cex) was 2-11 times higher than that obtained using other systems. Over 20% of the activity was detected in the medium. When the culture was induced with IPTG, the overall production of Exg dropped dramatically. The lower yield was found to be caused by both rapid cell death and plasmid curing. A derivative of tacIQpar8cex containing the weaker lacUV5 promoter, designated lacUV5par8cex, was constructed to enhance excretion of Exg from strain JM101. Even with IPTG induction, the JM101 (lacUV5par8cex) culture was found to show a high level of cell viability and plasmid stability as well as the ability to provide efficient expression and excretion of Exg. Upon IPTG induction for 12 h, the activity and specific activity of the excreted Exg obtained from the lacUV5par8cex construct were 143 U ml-1 and 793 U mg-1 protein, respectively, which are 2-5 times higher than that detected from the tacIQpar8cex construct and from the best construct expressing the same gene reported previously.
Lam
T L
TL
Department of Biochemistry, Hong Kong University of Science & Technology, Kowloon, Hong Kong.
Wong
R S
RS
Wong
W K
WK
D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase