|本期目录/Table of Contents|

 SHEN Ruihua,GUO Junling,ZHOU Zhemin.Recent advances in stability reconstruction of nitrile hydratase[J].Chinese Journal of Bioprocess Engineering,2020,18(03):269-276.[doi:10.3969/j.issn.1672-3678.2020.03.001]





Recent advances in stability reconstruction of nitrile hydratase
1.江苏昌九农科化工有限公司,江苏 南通 226413; 2.江南大学 生物工程学院,江苏 无锡 214122
SHEN Ruihua1GUO Junling2ZHOU Zhemin2
1. Jiangsu Changjiu Agrochemical Co.,Ltd.,Nantong 226413,China; 2.School of Biotechnology,Jiangnan University,Wuxi 214122,China
腈水合酶 生物催化 稳定性改造 蛋白质工程 工业应用
腈水合酶(nitrile hydratase,NHase,EC是一种金属酶,能催化腈类物质转化为相应的酰胺类物质,广泛应用于高附加值酰胺类化合物的生产。但由于NHase较差的稳定性,在催化反应过程中受到温度、产物浓度等影响而逐渐失活,这严重影响了NHase的工业化应用。近年来,针对这个问题,研究者通过酶分子改造,在NHase的稳定性提高方面取得了一定进展。本文中,笔者主要对近年来提高NHase稳定性的分子改造方面取得的进展进行总结,包括基于生物信息学解析的盐桥增设、自由能降低、同源片段重组和基因融合等策略。这些成果不仅推动了生物催化法生产酰胺类化合物的发展,还将为NHase蛋白质改造工程提供理论支持。
Nitrile hydratase(NHase,EC a key metalloenzyme in the hydration of nitriles to their corresponding amides that are widely used in the industrial production of highly value amides.However,the poor thermostability of NHase is the major factor preventing its extensive industrial application.We summarize here the molecular modification methods to improve the stability of NHase,including salt bridge insertion,free energy reduction,homologous fragment recombination,and gene fusion.These achievements not only improve the development of biocatalytic production of amide compounds,but also provide theoretical support for NHase protein engineering.


[1] PELT S V,QUIGNARD S,KUBAC D,et al.Nitrile hydratase CLEAs:the immobilization and stabilization of an industrially important enzyme[J].Green Chem,2008,10(4):395-400.
[2] NOGUCHI T,NOJIRI M,TAKEI K,et al.Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy[J].Biochemistry,2003,42(40):11642-11650.
[3] KOMEDA H,KOBAYASHI M,SHIMIZU S.A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase(L-NHase)induced by its reaction product[J].J Biol Chem,1996,271(26):15796-15802.
[4] PRASAD S,BHALLA T C.Nitrile hydratases(NHases):at the interface of academia and industry[J].Biotechnol Adv,2010,28(6):725-741.
[5] FOERSTNER K U,DOERKS T,MULLER J,et al.Anitrile hydratase in the eukaryote Monosiga brevicollis[J].PLoS ONE,2008,3(12):e3976.
[6] ROSE M J,BETTERLEY N M,MASCHARAK P K.Thiolate S-oxygenation controls nitric oxide(NO)photolability of a synthetic iron nitrile hydratase(Fe-NHase)model derived from mixed carboxamide/thiolate ligand[J].J Am Chem Soc,2009,131(24):8340-8341.
[7] NELP M T,SONG Y,WYSOCKI V H,et al.A protein-derived oxygen is the source of the amide oxygen of nitrile hydratases[J].J Biol Chem,2016,291(15):7822-7829.
[8] CHENG Z,CUI W,XIA Y,et al.Modulation of NHase regioselectivity towards dinitriles by tailoring the substrate binding pocket residues[J].ChemCatChem,2018,10:449-458.
[9] NOJIRI M,YOHDA M,ODAKA M,et al.Functional expression of nitrile hydratase in Escherichia coli:requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine[J].J Biochem,1999,125(4):696-704.
[10] LU J,ZHENG Y,YAMAGISHI H,et al.Motif CXCC in nitrile hydratase activator is critical for NHase biogenesis in vivo[J].FEMS Lett,2003,553(3):391-396.
[11] GUMATAOTAO N,LANKATHILAKA K P,BENNETT B,et al.The iron-type nitrile hydratase activator protein is a GTPase[J].Biochem J,2017,474(2):247-258.
[12] LIU Y,CUI W,FANG Y,et al.Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli[J].BMC Biotechnol,2013,13(1):48.
[13] ZHOU Z,HASHIMOTO Y,SHIRAKI K,et al.Discovery of post-translational maturation by self-subunit swapping[J].Proc Natl Acad Sci USA,2008,105(39):14849-14854
[14] XIA Y,PEPLOWSKI L,CHENG Z,et al.Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase[J].Biotechnol Bioeng,2019,116(3):481-489.
[15] KUHNM L,MARTINEZ S,GUMATAOTAO N,et al.The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli[J].Biochem Biophys Res Commun,2012,424(3):365-370.
[16] HOPMANN K H,GUO J D,HIMO F.Theoretical investigation of the first-shell mechanism of nitrile hydratase[J].Inorg Chem,2007,46(12):4850-4856.
[17] HASHIMOTO K,SUZUKI H,TANIGUCHI K,et al.Catalytic mechanism of nitrile hydratase proposed by time-resolved X-ray crystallography using a novel substrate,tert-butylisonitrile[J].J Biol Chem,2008,283(52):36617-36623.
[18] HOPMANN K H,HIMO F.Theoretical investigation of the second-shell mechanism of nitrile hydratase[J].Eur J Inorg Chem,2008,2008(9):1406-1412.
[19] HOPMANNK H.Full reaction mechanism of nitrile hydratase:acyclic intermediate and an unexpected disulfide switch[J].Inorg Chem,2014,53(6):2760-2762.
[20] MACDONALD C A,BOYD R J.Competing nitrile hydratase catalytic mechanisms:is cysteine-sulfenic acid acting as a nucleophile[J].Comput Theor Chem,2015,1070:48-54.
[21] GUO L,FANG W,GUO L,et al.Biodegradation of the neonicotinoid insecticide acetamiprid by actinomycetes Streptomyces canus CGMCC 13662 and characterization of the novel nitrile hydratase involved[J].J Agric Food Chem,2019,67(21):5922-5931.
[22] PEI X,WANG J,GUO W,et al.Efficient biodegradation of dihalogenated benzonitrile herbicides by recombinant Escherichia coli harboring nitrile hydratase-amidase pathway[J].Biochem Eng J,2017,125:88-96.
[23] HANN E C,EISENBERG A,FAGER S K,et al.5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23[J].Bioorg Med Chem,1999,7(10):2239-2245.
[24] RYUNO K,NAKAMURA T.Biocatalyst process:enzymatic transformation of nitrile compounds and the application[J].ChemInform,2003,34(43):517-522.
[25] WIESER M,TAKEUCHI K,WADA Y,et al.Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1:purification,substrate specificity and comparison with the analogous high-molecular-mass enzyme[J].FEMS Microbiol Lett,1998,169(1):17-22.
[26] TEILUM K,OLSEN J G,KRAGELUND B B.Protein stability,flexibility and function[J].Biochim Biophys Acta,2011,1814(8):969-976.
[27] NESTL B M,NEBEL B A,HAUER B.Recent progress in industrial biocatalysis[J].Curr Opin Chem Biol,2011,15(2):187-193.
[28] CHEN J,YU H,LIU C,et al.Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions[J].J Biotechnol,2013,164(2):354-362.
[29] XIA Y,CUI W,CHENG Z,et al.Improving thermostability and catalytic efficiency of the subunit-fused nitrile hydratase by semi-rational engineering[J].ChemCatChem,2018(10):1370-1375.
[30] CUI Y,CUI W,LIU Z,et al.Improvement of stability of nitrile hydratase via protein fragment swapping[J].Biochem Biophys Res Commun,2014,450(1):401-408.
[31] XIA Y,CUI W,LIU Z,et al.Construction of a subunit-fusion nitrile hydratase and discovery of an innovative metal ion transfer pattern[J].Sci Rep,2016,6(1):19183.
[32] SUN Z,LIU Q,QU G,et al.Utility of B-factors in protein science:interpreting rigidity,flexibility,and internal motion and engineering thermostability[J].Chem Rev,2019,119:1626-1665.
[33] PEI X,ZHANG H,MENG L,et al.Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy[J].Process Biochem,2013,48(12):1921-1927.
[34] SINGH R,PANDEY D,DHARIWAL S,et al.Bioconversion of acrylonitrile using nitrile hydratase activity of Bacillus sp.APB-6[J].Biotech,2018,8(5):225.
[35] ZHOU L,MOU H,GAO J,et al.Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E.coli by macromolecular cross-linking agent[J].Chin J Chem Eng,2017,25(4):487-492.
[36] YOKOT K,SATOU K,OHKI S Y.Comparative analysis of protein thermostability:differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins[J].Sci Technol Adv Materials,2006,7(3):255-262.
[37] YIP K,STILLMAN T J,BRITTON K L,et al.The structure of Pyrococcusfuriosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures[J].Structure,1995,3(11):1147-1158.
[38] GURRY T,NERENBERG P S,STULTZ C M.The contribution of interchain salt bridges to triple-helical stability in collagen[J].Biophys J,2010,98(11):2634-2643.
[39] LIU J,YU H,SHEN Z.Insights into thermal stability of thermophilic nitrile hydratases by molecular dynamics simulation[J].J Mol Graphics Modelling,2008,27(4):529-535.
[40] ZHAO H,CHOCKALINGAM K,CHEN Z.Directed evolution of enzymes and pathways for industrial biocatalysis[J].Curr Opin Biotechnol,2002,13(2):104-110.
[41] PACKER M S,LIU D R.Methods for the directed evolution of proteins[J].Nat Rev Genet,2015,16(7):379-394.
[42] 何玉财,周琼,林春昕,等.一种腈水解酶的高通量筛选方法[J].高校化学工程学报,2012(5):853-857.
[43] 郑裕国,林志坚,郑仁朝,等.一种腈转化酶的高通量筛选方法:101838681[P].,2010-09-22.
[44] CARBONE M N,ARNOLD F H.Engineering by homologous recombination:exploring sequence and function within a conserved fold[J].Curr Opin Struct Biol,2007,17(4):454-459.
[45] SUN W,ZHU L,CHEN X,et al.The stability enhancement of nitrile hydratase from Bordetella petrii by swapping the C-terminal domain of β subunit[J].Appl Biochem Biotechnol,2016,178(8):1481-1487.
[46] PEI X,WANG J,WU Y,et al.Evidence for the participation of an extra α-helix at β-subunit surface in the thermal stability of Co-type nitrile hydratase[J].Appl Microbiol Biotechnol,2018,102(18):7891-7900.
[47] NAIEL A,RINAT B S,FUAD F.Conversion of TSH heterodimer to a single polypeptide chain increases bioactivity and longevity[J].Endocrinology,2012,153(2):954-60.
[48] MARSH J A,HERNANDEZ H,HALL Z,et al.Protein complexes are under evolutionary selection to assemble via ordered pathways[J].Cell,2013,153(2):461-470.
[49] LIU Y,CUI W,LIU Z,et al.Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide[J].J Biosci Bioeng,2014,118(3):249-252.
[50] CHEN Y,Song J,WANG M,et al.A novel molecular chaperone GroEL2 from Rhodococcus ruber,and its fusion chimera with nitrile hydratase for co-enhanced activity and stability[J].Chem Eng Sci,2018,192:235-243.


 ZHENG Yu-guo,JIN Xiao-feng,ZHENG Ren-chao,et al.A class of biocatalyst: cyanide-resistant nitrile hydratase[J].Chinese Journal of Bioprocess Engineering,2010,8(03):73.[doi:10.3969/j.issn.1672-3678.2010.03.015]
 ZHENG Yuguo,SHEN Yinchu.Biocatalysis and green process for chiral pharmaceutical chemicals[J].Chinese Journal of Bioprocess Engineering,2013,11(03):24.[doi:10.3969/j.issn.1672-3678.2013.02.004]
 YU Huilei,HUANG Lei,NI Yan,et al.Advances in synthesis of chiral alcohols by carbonyl bioreduction[J].Chinese Journal of Bioprocess Engineering,2013,11(03):71.[doi:10.3969/j.issn.1672-3678.2013.03.013]
 ZHAN Yijing,XU Zheng,XU Lu,et al.Research progress in production of L-ribose[J].Chinese Journal of Bioprocess Engineering,2013,11(03):74.[doi:10.3969/j.issn.1672-3678.2013.05.015]
 CAI Qian,WU Ming-huo,ZHENG Yu-guo,et al.Isolation of strains highly yielding p-hydroxyphenylacetamide nitrile hydratase[J].Chinese Journal of Bioprocess Engineering,2005,3(03):38.[doi:10.3969/j.issn.1672-3678.2005.03.008]
 WU Ming-huo,CAI Qian,ZHENG Yu-guo,et al.Screening for p-hydroxyphenylcyanide nitrilase and study of enzyme production conditions[J].Chinese Journal of Bioprocess Engineering,2005,3(03):32.[doi:10.3969/j.issn.1672-3678.2005.04.007]
 WEI Erman,MA Hongmin,QU Xudong,et al.Research progress of cytochrome P450 in asymmetric cyclopropane[J].Chinese Journal of Bioprocess Engineering,2017,15(03):28.[doi:10.3969/j.issn.1672-3678.2017.01.005]
 HE Qiaofei,WU Hui.Advances in biosynthesis of hydroxy fatty acids by Escherichia coli[J].Chinese Journal of Bioprocess Engineering,2017,15(03):71.[doi:10.3969/j.issn.1672-3678.2017.05.009]
 LI Ning.Advances in biocatalytic upgrading of 5-hydroxymethylfurfural[J].Chinese Journal of Bioprocess Engineering,2017,15(03):34.[doi:10.3969/j.issn.1672-3678.2017.06.005]
 WU Zheming,JIN Jianqiang,ZHENG Renchao.Advances in exploitation and application of amidase in organic synthesis[J].Chinese Journal of Bioprocess Engineering,2018,16(03):38.[doi:10.3969/j.issn.1672-3678.2018.01.005]
 ZHANG Jun,CUI Wenjing,LIU Yi,et al.βAla97 of nitrile hydratase from Pseudomonas putida affects regioselectivity towards adiponitrile[J].Chinese Journal of Bioprocess Engineering,2015,13(03):41.[doi:10.3969/j.issn.1672-3678.2015.03.008]


作者简介:沈瑞华(1970—),男,江苏如皋人,工程师,研究方向:发酵工程; 周哲敏(联系人),教授,E-mail:zhmzhou@jiangnan.edu.cn
SHEN Ruihua,GUO Junling,ZHOU Zhemin.Recent advances in stability reconstruction of nitrile hydratase[J].Chin J Bioprocess Eng,2020,18(3):269-276..
更新日期/Last Update: 2020-05-30