Directed evolution and characterization of a novel D-pantonohydrolase from Fusarium moniliforme.
D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of D-pantoic acid. The Fusarium moniliforme D-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type D-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved D-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved D-pantonohydrolase were also studied and compared.
Liu
Zhiqiang
Z
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi, People's Republic of China.
Sun
Zhihao
Z
Leng
Yong
Y
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005670:Fusarium / Q000201:enzymology*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013237:Stereoisomerism; D013379:Substrate Specificity; D014162:Transfection
D006885:Hydroxybutyrates; C016031:pantoic acid; C005412:pantolactone; D006867:Hydrolases; D015107:4-Butyrolactone