Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6

• Published in: Applied biochemistry and biotechnology 2016-04, Vol.178 (8), p.1510-1521
• Main Authors: Luo, Hui, Ma, Jinwei, Chang, Yanhong, Yu, Huimin, Shen, Zhongyao
• Format: Article
• Language: English
• Subjects: Acetonitriles - chemistry; Amino Acid Substitution - genetics; Amino acids; Aminohydrolases - chemistry; Aminohydrolases - genetics; Biochemistry; Biotechnology; Catalysis; Chemistry; Chemistry and Materials Science; Directed Molecular Evolution; Kinetics; Molecular biology; Mutation; Rhodococcus - enzymology; Rhodococcus - genetics; Rhodococcus rhodochrous; Substrate Specificity
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-015-1964-9

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 ).