title: Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6.
abstract: In this paper, a molecularly directed evolution-based approach was applied to modify the nitrilase from Rhodococcus rhodochrous tg1-A6 for improving properties in catalyzing nitriles. In the process of error-prone polymerase chain reaction (PCR) with the wild-type nitrilase gene acting as the template, a library of the randomly mutated nitrilase gene was constructed. Since the pH value of catalyzing solution decreased when glycolonitrile was used as the substrate of nitrilase, a high-throughput strategy based on the color change of a pH-sensitive indicator was established for rapid screening of the mutated nitrilase. After three rounds of random mutation and screening about 5000 clones, a variant (Mut3) with 5.3-fold activity of the wild-type counterpart was obtained. Five amino acid substitutions (D27E, N97K, L246F, D108E, and S111R) were found in the variant Mut3. The properties of three mutated enzymes obtained in the three-round mutation were investigated. In the conversion of glycolonitrile, the variant (Mut2) accumulated the highest concentration of glycolic acid at 10.6 g l(-1), a much higher value than the wild type (3.2 g l(-1)).
authors.affiliation: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, People's Republic of China. luohui@ustb.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Luo
authors.affiliation: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, People's Republic of China.|Department of Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, People's Republic of China.
authors.forename: Jinwei
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, People's Republic of China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China.
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D019020:Directed Molecular Evolution; D007700:Kinetics; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000097:Acetonitriles; D000619:Aminohydrolases; C021867:nitrilase; C035740:glycolonitrile
keywords: Directed evolution; Glycolic acid; Glycolonitrile; High-throughput screening; Nitrilase