NDLI: Heterologous expression of thermostable esterase gene from Geobacillus thermoleovorans YN under different expression promoters

Content Provider	Springer Nature Link
Author	Soliman, N. A. Mostafa, H. E. Abdel Fattah, Y. R. Gaballa, A.
Copyright Year	2013
Abstract	The gene encoding extracellular thermostable carboxylesterase (estA) from Geobacillus thermoleovorans YN was fused to six histidines and expressed under different promoters. Three different clones carrying this gene were constructed in host cell Escherichia coli BL21 (DE2) using three expression systems pET-17b, pBluescript$^{®}$ II KS(+) and pCYTEX-P1 (pET-estA T7 promoter; pBlue-estA T7 promoter and pBlue-estA T7 promoter and Lambda pL promoter). The efficiency of expression of the three constructs was evaluated, where the highest esterase expression (589 μmol/ml) using p-nitrophenyl laurate as substrate (pNP-laurate: C18H27NO4) was measured under the control of T7 promoter in expression vector pET-17b after 4 h of the induction. A 1.5-fold increase in enzyme activity was measured with specific activity 1,043 μmol/mg protein on growing the clone expressed the target gene under T7 promoter (pET-estAT7) in 2-L working volume BIOFLO III bioreactor under optimal conditions. Recombinant expression of the tested thermostable esterase was monitored by sodium dodecyl sulfate polyacrylamid gel electrophoresis, zymogram and activity measurements with α-naphthyl acetate (C12H10O2) and Fast Red. The SDS-PAGE analysis of E. coli BL21(DE3)/pET-estA lysates indicated the presence of a protein band (29 kDa) related to the targeted expressed gene.
Starting Page	119
Ending Page	126
Page Count	8
File Format	PDF
ISSN	17351472
Journal	International Journal of Environmental Science & Technology
Volume Number	11
Issue Number	1
e-ISSN	17352630
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-10-08
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Ecotoxicology Induction Escherichia coli Encoding gene BIOFLO III bioreactor Six histidines Water Management Waste Water Technology Aquatic Pollution Environment Water Pollution Control Environmental Chemistry Environmental Science and Engineering Soil Science & Conservation
Content Type	Text
Resource Type	Article
Subject	Environmental Chemistry Environmental Engineering Agricultural and Biological Sciences

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