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

The gene encoding extracellular thermostable carboxylesterase (estA) from Geobacillus thermoleovorans

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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; 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