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Improving the Thermostability and Catalytic Efficiency of the Subunit‐Fused Nitrile Hydratase by Semi‐Rational Engineering
Author(s) -
Xia Yuanyuan,
Cui Wenjing,
Cheng Zhongyi,
Peplowski Lukasz,
Liu Zhongmei,
Kobayashi Michihiko,
Zhou Zhemin
Publication year - 2018
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201701374
Subject(s) - thermostability , nitrile hydratase , chemistry , nitrile , rational design , protein engineering , catalysis , combinatorial chemistry , biocatalysis , enzyme , organic chemistry , stereochemistry , materials science , reaction mechanism , nanotechnology
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 k cat / K m 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.