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

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 Ò 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 a-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.

The Genus Geobacillus and Their Biotechnological Potential

“…Carboxylesterases generally require no cofactor and exhibit high regiospecificity and stereospecificity due to the presence of a unique small acyl binding pocket, which optimally fits to the acyl moiety of their substrates (Panda & Gowrishankar, 2005). To date, thermostable carboxylesterases have been isolated and characterized from G. stearothermophilus ATCC12980 and ATCC7954 (Ewis, Abdelal, & Lu, 2004), G. thermoleovorans YN (Soliman, Abdel-Fattah, Mostafa, & Gaballa, 2014), G. thermodenitrificans T2 (Yang et al, 2013), G. thermodenitrificans CMB-A2 (Charbonneau, Meddeb-Mouelhi, & Beauregard, 2010), G. kaustophilus HTA426 (Montoro-García et al, 2009), G. caldoxylosilyticus TK4 (Yildirim, Colak, Col, & Canakci, 2009), G. thermoglucosidasius EAEC, and many other thermophilic strains attributed to the Geobacillus genus (Ayna, 2013;€ Ozbek, Kolcuo glu, Konak, Colak, & € Oz, 2014;Tekedar & Şanlı-Mohamed, 2011;Zhu et al, 2012). p0345…”