Biochemical characterization and mutational improvement of a thermophilic esterase from Sulfolobus solfataricus P2.
A thermophilic esterase, SsoPEst, from Sulfolobus solfataricus P2 was cloned and expressed in E. coli AD494 (DE3). Gene sequencing indicated the encoded 353 amino acids had less than 32% identity with reported esterases. The recombinant enzyme hydrolyzed p-nitrophenyl esters but not tributyrin or tricaprylin, exhibiting the highest specific activity (1.1 U/mg) with p-nitrophenyl caprylate. The enzyme was optimally active at pH 5.5 and 80 degrees C. It retained 50% activity after 1 h incubation at 80 degrees C. Activity was significantly inhibited by PMSF. Five SsoPEst mutants were generated by site-directed mutagenesis. One mutant had a higher specific activity of 2.8 U/mg at 37 degrees C and 14 U/mg at 80 degrees C than the wild-type enzyme which exhibited 0.7 U/mg at 37 degrees C and 3.8 U/mg at 80 degrees C against p-nitrophenyl butyrate.
Shang
Yu-Shuan
YS
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhang
Xian-En
XE
Wang
Xu-De
XD
Guo
Yong-Chao
YC
Zhang
Zhi-Ping
ZP
Zhou
Ya-Feng
YF
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D004950:Esterases