Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution
The short-chain dehydrogenase/reductase (SDR) from <i>Empedobacter brevis</i> ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (<i>R</i>)-1-phenylethanol, but its industrial application is restricted by its insufficient t...
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MDPI AG
2022-09-01
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| author | Hui Zhang Bei Wang Shengli Yang Hongwei Yu Lidan Ye |
| author_facet | Hui Zhang Bei Wang Shengli Yang Hongwei Yu Lidan Ye |
| author_sort | Hui Zhang |
| collection | DOAJ |
| description | The short-chain dehydrogenase/reductase (SDR) from <i>Empedobacter brevis</i> ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (<i>R</i>)-1-phenylethanol, but its industrial application is restricted by its insufficient tolerance to acetophenone. In this paper, we developed a chromogenic reaction-based high-throughput screening method and employed directed evolution to enhance the acetophenone tolerance of EbSDR8. The resulting variant, M190V, showed 74.8% improvement over the wild-type in specific activity when catalyzing the reduction of 200 mM acetophenone. Kinetic analysis revealed a 70% enhancement in its catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>). Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance, and the result implied that the M190V mutation is conducive to the binding and release of coenzyme. Aside from the improved catalytic performance when dealing with a high concentration of acetophenone, other features of M190V, such as a broad pH range (6.0 to 10.5), low optimal cosubstrate concentration (1% isopropanol), and a temperature optimum close to that of <i>E. coli</i> cells (35 °C), also contribute to its practical application as a whole-cell catalyst. In this study, we first designed a directed evolution means to engineer the enzyme and obtained the positive variant which has a high activity under high concentrations of acetophenone. After that, we optimized the catalytic performance of the variant to adapt to industrial applications. |
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| spelling | doaj.art-2dfd9e51c2cb424aac4473d4010aa1662023-11-23T15:31:54ZengMDPI AGCatalysts2073-43442022-09-01129107110.3390/catal12091071Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed EvolutionHui Zhang0Bei Wang1Shengli Yang2Hongwei Yu3Lidan Ye4School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, ChinaCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, ChinaCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, ChinaInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, ChinaInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, ChinaThe short-chain dehydrogenase/reductase (SDR) from <i>Empedobacter brevis</i> ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (<i>R</i>)-1-phenylethanol, but its industrial application is restricted by its insufficient tolerance to acetophenone. In this paper, we developed a chromogenic reaction-based high-throughput screening method and employed directed evolution to enhance the acetophenone tolerance of EbSDR8. The resulting variant, M190V, showed 74.8% improvement over the wild-type in specific activity when catalyzing the reduction of 200 mM acetophenone. Kinetic analysis revealed a 70% enhancement in its catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>). Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance, and the result implied that the M190V mutation is conducive to the binding and release of coenzyme. Aside from the improved catalytic performance when dealing with a high concentration of acetophenone, other features of M190V, such as a broad pH range (6.0 to 10.5), low optimal cosubstrate concentration (1% isopropanol), and a temperature optimum close to that of <i>E. coli</i> cells (35 °C), also contribute to its practical application as a whole-cell catalyst. In this study, we first designed a directed evolution means to engineer the enzyme and obtained the positive variant which has a high activity under high concentrations of acetophenone. After that, we optimized the catalytic performance of the variant to adapt to industrial applications.https://www.mdpi.com/2073-4344/12/9/1071acetophenone tolerancewhole-cell catalystdirected evolution(<i>R</i>)-1-phenylethanolshort-chain dehydrogenase/reductase |
| spellingShingle | Hui Zhang Bei Wang Shengli Yang Hongwei Yu Lidan Ye Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution Catalysts acetophenone tolerance whole-cell catalyst directed evolution (<i>R</i>)-1-phenylethanol short-chain dehydrogenase/reductase |
| title | Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution |
| title_full | Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution |
| title_fullStr | Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution |
| title_full_unstemmed | Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution |
| title_short | Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution |
| title_sort | enhancing acetophenone tolerance of anti prelog short chain dehydrogenase reductase ebsdr8 using a whole cell catalyst by directed evolution |
| topic | acetophenone tolerance whole-cell catalyst directed evolution (<i>R</i>)-1-phenylethanol short-chain dehydrogenase/reductase |
| url | https://www.mdpi.com/2073-4344/12/9/1071 |
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