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Improving the Thermostability and Catalytic Efficiency of the Subunit‐Fused Nitrile Hydratase by Semi‐Rational Engineering
ChemCatChem ( IF 3.8 ) Pub Date : 2018-02-14 , DOI: 10.1002/cctc.201701374 Yuanyuan Xia 1 , Wenjing Cui 1 , Zhongyi Cheng 1 , Lukasz Peplowski 2 , Zhongmei Liu 1 , Michihiko Kobayashi 3 , Zhemin Zhou 1
ChemCatChem ( IF 3.8 ) Pub Date : 2018-02-14 , DOI: 10.1002/cctc.201701374 Yuanyuan Xia 1 , Wenjing Cui 1 , Zhongyi Cheng 1 , Lukasz Peplowski 2 , Zhongmei Liu 1 , Michihiko Kobayashi 3 , Zhemin Zhou 1
Affiliation
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Nitrile hydratase (NHase, EC 4.2.1.84) is a key enzyme in the hydration of nitriles to their corresponding amides and is widely used in the industrial production of highly purified acrylamide and nicotinamide. However, the poor thermostability of NHase is the major factor preventing its extensive industrial application. Here, a semi‐rational design approach based on the pmut scan application of Rosetta 3.4 and molecular dynamics (MD) simulations was used to improve the thermostability of a subunit‐fused nitrile hydratase from Pseudomonas putida NRRL‐18668 (Fus‐NHase). A small mutant library was constructed, and three mutants, B‐M150C, B‐T173Y, and B‐S189E, with half‐life increases at 50 °C of 32 %, 7 %, and 107 %, respectively, were obtained. Additionally, the kcat/Km values of B‐M150C, B‐T173Y, and B‐S189E were 1.1‐, 1.5‐, and 2.2‐fold higher, respectively, than that of Fus‐NHase. This study provides an effective strategy for improving protein thermostability and catalytic efficiency.
中文翻译：
通过半定量工程改进亚基融合腈水合酶的热稳定性和催化效率
腈水合酶（NHase，EC 4.2.1.84）是腈水合成相应酰胺的关键酶，广泛用于高纯度丙烯酰胺和烟酰胺的工业生产中。但是，NHase的热稳定性差是阻止其广泛工业应用的主要因素。在这里，基于Rosetta 3.4的pmut扫描应用和分子动力学（MD）模拟的半定量设计方法被用来提高恶臭假单胞菌NRRL-18668（Fus -NHase）的亚单位融合腈水合酶的热稳定性。构建了一个小的突变体文库，获得了三个突变体B-M150C，B-T173Y和B-S189E，它们在50°C下的半衰期分别增加了32％，7％和107％。另外，kB-M150C，B-T173Y和B-S189E的cat / K m值分别比Fus -NHase高1.1倍，1.5倍和2.2倍。这项研究为提高蛋白质的热稳定性和催化效率提供了有效的策略。
更新日期：2018-02-14
中文翻译：
通过半定量工程改进亚基融合腈水合酶的热稳定性和催化效率
腈水合酶（NHase，EC 4.2.1.84）是腈水合成相应酰胺的关键酶，广泛用于高纯度丙烯酰胺和烟酰胺的工业生产中。但是，NHase的热稳定性差是阻止其广泛工业应用的主要因素。在这里，基于Rosetta 3.4的pmut扫描应用和分子动力学（MD）模拟的半定量设计方法被用来提高恶臭假单胞菌NRRL-18668（Fus -NHase）的亚单位融合腈水合酶的热稳定性。构建了一个小的突变体文库，获得了三个突变体B-M150C，B-T173Y和B-S189E，它们在50°C下的半衰期分别增加了32％，7％和107％。另外，kB-M150C，B-T173Y和B-S189E的cat / K m值分别比Fus -NHase高1.1倍，1.5倍和2.2倍。这项研究为提高蛋白质的热稳定性和催化效率提供了有效的策略。
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