[Directed evolution of Thermophilic esterase from the archaeon Aeropyrum pemix K1].
Thermophilic esterase (APE1547) from Aeropyrum pernix K1 was subjected to error-prone PCR (epPCR) to enhance activity. For the screening of mutants, an efficient and reliable assay suitable for high throughput screening was developed based on the enzyme thermostability. Two successive rounds of random mutagenesis by epPCR resulted in a four amino acid substitution variant M020 with significantly increased activity (six-fold under the screening condition. Further assay for the purified enzymes showed that the mutant possess 1.5-fold higher specific activity and nearly 4-fold higher expressed level than the wild-type. The mutant has an optimal activity at pH 8.5, corresponding to an alkaline shift of 0.5 pH unit compared to the wild type. The structure analysis suggests that R526S may contribute to the enhanced activity and the shift of pKi.
Wang
Qiu-Yan
QY
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China. wangqiuyan@mail.jlu.edu.cn
Yang
Guang-Yu
GY
Liu
Yan-Li
YL
Wang
Yan-Ping
YP
Feng
Yan
Y
D041522:Aeropyrum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics
D019843:Archaeal Proteins; D004950:Esterases