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International Journal of Environment Science and Technology
Center for Environment and Energy Research and Studies (CEERS)
ISSN: 1735-1472
EISSN: 1735-2630
Vol. 11, No. 1, 2014, pp. 119-126
Bioline Code: st14013
Full paper language: English
Document type: Research Article
Document available free of charge

International Journal of Environment Science and Technology, Vol. 11, No. 1, 2014, pp. 119-126

 en Heterologous expression of thermostable esterase gene from Geobacillus thermoleovorans check for this species in other resources YN under different expression promoters
Soliman, N. A.; Abdel-Fattah, Y. R.; Mostafa, H. E. & Gaballa, A.

Abstract

The gene encoding extracellular thermostable carboxylesterase (estA) from Geobacillus thermoleovorans check for this species in other resources YN was fused to six histidines and expressed under different promoters. Three different clones carrying this gene were constructed in host cell Escherichia coli check for this species in other resources BL21 (DE2) using three expression systems pET-17b, pBluescript II KS(+) and pCYTEX-P1 (pET-estA T7 promoter; pBlueestA 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 lmol/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 lmol/ 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.

Keywords
BIOFLO III bioreactor; Encoding gene; Escherichia coli; Induction; Six histidines

 
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