Gene cloning and characterization of a novel thermophilic esterase from Fervidobacterium nodosum Rt17-B1.
A bioinformatic screening of the genome of the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 for esterhydrolyzing 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 (pNPC(2)) as substrate. The catalytic efficiency (k(cat)/K(m)) toward p-nitrophenyl acetate (C(2)) was approximately 120-fold higher than toward p-nitrophenyl butyrate (C(4)). No significant esterase activity was observed for the substrates with a chain length > or =C(8). The monomeric enzyme has a molecular mass of 27.5 kDa and exhibits optimal activity around 75 degrees C, at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70 degrees 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 alpha/beta-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.
Yu
Shanshan
S
Jilin University, Changchun, China.
Zheng
Baisong
B
Zhao
Xinyu
X
Feng
Yan
Y
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology; D002384:Catalysis; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000235:genetics*; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004950:Esterases