title: [Directed evolution of D-lactonohydrolase by error prone PCR and DNA shuffling].
abstract: D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.
authors.affiliation: Laboratory of Biocatalysis, School of Biotechnology, Southern Yangtze University, Wuxi 214036, China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.forename: Zhi-Hao
authors.initials: ZH
authors.lastname: Sun
authors.forename: Pu
authors.initials: P
authors.lastname: Zheng
authors.forename: Yong
authors.initials: Y
authors.lastname: Leng
authors.forename: Jia-Nan
authors.initials: JN
authors.lastname: Qian
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D002265:Carboxylic Ester Hydrolases; C501958:D-lactonohydrolase, Fusarium moniliforme