title: Improvement of stability of nitrile hydratase via protein fragment swapping.
abstract: Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase fragment from Comamonas testosteroni 5-MGAM-4D and two fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of PpNHase. Seven chimeric NHases were designed using STAR (site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. All constructed chimeric NHases showed 1.4- to 3.5-fold enhancement in thermostability and six of them become more tolerant to high-concentration product. Notably, one of these NHases, 3AB, exhibited a 1.4±0.05-fold increase in activity compared to the wild-type PpNHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type PpNHase. The 3AB constructed in this study is useful for further industrial application, and the method for designing the chimeric protein using homologous protein fragment swapping without a decrease in activity may be a strategy to improve the stability of other enzymes.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Youtian
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Institute of Applied Biochemistry and the Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Electronic address: kobay@agbi.tsukuba.ac.jp.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China. Electronic address: zhmzhou@jiangnan.edu.cn.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D006836:Hydro-Lyases; C048831:nitrile hydratase
keywords: Enzyme stability; Fragment swapping; Homologous recombination; Molecular dynamics; Nitrile hydratase