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

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• 作者：Hui Luo ; Jinwei Ma ; Yanhong Chang ; Huimin Yu…
• 刊名：Applied Biochemistry and Biotechnology
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：178
• 期：8
• 页码：1510-1521
• 全文大小：625 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Biochemistry
  
• 出版者：Humana Press Inc.
• ISSN：1559-0291
• 卷排序：178

文摘

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).KeywordsNitrilaseDirected evolutionGlycolonitrileHigh-throughput screeningGlycolic acid