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Acta Biochimica et Biophysica Sinica
Year: 2010  |  Volume: 42  |  Issue: 4  |  Page No.: 288 - 295

Gene cloning and characterization of a novel thermophilic esterase from Fervidobacterium nodosum Rt17-B1

S Yu, B Zheng, X Zhao and Y. Feng    

Abstract:

A bioinformatic screening of the genome of the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 for ester-hydrolyzing enzymes revealed a putative bacterial esterase (FNE) encoded by Fond_1301 with typical GDSL family motifs. To confirm its putative esterase function, the FNE gene was cloned, functionally expressed in Escherichia coli, and purified to homogeneity. Recombinant FNE exhibited the highest esterase activity of 14,000 U/mg with p-nitrophenyl acetate (pNPC2) as substrate. The catalytic efficiency (kcat/Km) toward p-nitrophenyl acetate (C2) was approximately 120-fold higher than toward p-nitrophenyl butyrate (C4). No significant esterase activity was observed for the substrates with a chain length ≥C8. The monomeric enzyme has a molecular mass of 27.5 kDa and exhibits optimal activity around 75°C, at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70°C, but less stable compared with some other hyperthermophilic esterases. A structural model was constructed using acetylesterase from Aspergillus aculeatus as a template. The structure showed an /β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine, which was verified by site-directed mutagenesis. Sequence analysis showed that FNE was only distantly related to other esterases. A comparison of the conserved motifs shared with GDSL proteins revealed that FNE could be grouped into GDSL family and was further classified as SGNH hydrolase.

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