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食品与发酵工业  2022, Vol. 48 Issue (20): 70-77    DOI: 10.13995/j.cnki.11-1802/ts.030432
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
刘欣悦, 韩来闯, 刘中美*
(江南大学 生物工程学院,江苏 无锡,214122)
Substrate affinity design for the improvement of nitrilase Nit6803 activity
LIU Xinyue, HAN Laichuang, LIU Zhongmei*
(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
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摘要 腈水解酶(Nitrilase,EC,是一类可以将腈类物质一步水解为羧酸的酶,是多种重要大宗化工品、药物中间体的理想生物催化剂。但是天然酶活性低、热稳定性差限制了其在工业上的应用。近年来通过蛋白质改造来解决酶活性与稳定性间的“trade-off”效应以提升催化性能的研究较多。该研究提出一种酶-底物亲和设计改造策略,以来源于Syechocystis sp.PCC6803的腈水解酶Nit6803作为改造对象,结合基于Rosetta的Cartesian_ddG方法和基于自由能微扰的酶-底物亲和力计算,对Nit6803的催化口袋进行单点突变及组合突变设计。基于此获得了活性显著提升的单点突变体F64Y、W170G,及组合突变体F64Y/W170G。其中,F64Y/W170G的比酶活力达到(22.48±0.64) U/mg,为野生型的4.56倍,且该突变体的热稳定性不低于野生型。通过分批补加3-氰基吡啶进行全细胞催化表明F64Y/W170G催化能力强于野生型,在达到相同转化率情况下极大的缩短了催化时间。结果表明,该研究提出的设计策略可以有效提升酶的活性而不影响其稳定性,为酶的理性设计改造提供了新的思路。
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关键词:  腈水解酶  酶-底物亲和  理性设计  酶活力    
Abstract: Nitrilase (EC is an ideal biocatalyst for a variety of essential bulk chemicals and pharmaceutical intermediates, due to its capability to catalyze nitrile to carboxylic acid with high stereoselectivity under mild reaction conditions. However, the low activity and poor thermal stability of natural enzymes still limit its industrial application. In recent years, it has been a research hotspot to break through the bottleneck of ‘trade-off' between activity and stability through protein engineering. This study proposes a novel enzyme-substrate affinity design strategy. The nitrilase Nit6803 derived from Syechocystis sp. PCC6803 was improved in activity through the rational design combining the Cartesian_ddG method in Rosetta suite and the enzyme-substrate affinity calculation based on free energy perturbation. The single-point mutants F64Y, W170G, and combination mutant F64Y/W170G with significantly improved activity were obtained. Among them, the specific enzyme activity of F64Y/W170G reached (22.48±0.64) U/mg, which was 4.56 times that of the wild type, and the thermal stability maintained. Whole-cell catalysis by adding 3-cyanopyridine in batches showed that F64Y/W170G had stronger catalytic ability than wild type, and greatly shortened the catalysis time when reaching the same conversion rate. The results demonstrated that the engineering strategy proposed in this study can effectively enhance the enzyme activity without decreasing its stability, which provides a new idea for the rational design of enzymes.
Key words:  nitrilase    enzyme-substrate affinity    rational design    enzyme activity
收稿日期:  2021-12-13      修回日期:  2022-01-26           出版日期:  2022-10-25      发布日期:  2022-11-18      期的出版日期:  2022-10-25
基金资助: 江苏省自然科学基金(BK20210470);中国博士后科学基金(2021M701461)
作者简介:  硕士研究生(刘中美教授为通信作者,
刘欣悦,韩来闯,刘中美. 底物亲和设计提高腈水解酶Nit6803活性[J]. 食品与发酵工业, 2022, 48(20): 70-77.
刘欣悦,韩来闯,刘中美. Substrate affinity design for the improvement of nitrilase Nit6803 activity[J]. Food and Fermentation Industries, 2022, 48(20): 70-77.
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[1] CHHIBA-GOVINDJEE V P, VAN DER WESTHUYZEN C W, BODE M L, et al.Bacterial nitrilases and their regulation[J].Applied Microbiology and Biotechnology, 2019, 103(12):4 679-4 692.
[2] JEZ J M.Plant nitrilase:A new job for an old enzyme[J].The Biochemical Journal, 2019, 476(7):1 105-1 107.
[3] ATALAH J, CÁCERES-MORENO P,ESPINA G,et al.Thermophiles and the applications of their enzymes as new biocatalysts[J].Bioresource Technology, 2019, 280:478-488.
[4] SHEN J D, CAI X, LIU Z Q, et al.Nitrilase:A promising biocatalyst in industrial applications for green chemistry[J].Critical Reviews in Biotechnology, 2021, 41(1):72-93.
[5] GONG J S, SHI J S, LU Z H M, et al.Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis:Recent insights and promises[J].Critical Reviews in Biotechnology, 2017, 37(1):69-81.
[6] DESANTIS G, ZHU Z L, GREENBERG W A, et al.An enzyme library approach to biocatalysis:Development of nitrilases for enantioselective production of carboxylic acid derivatives[J].Journal of the American Chemical Society, 2002, 124(31):9 024-9 025.
[7] LI G Y, MARIA-SOLANO M A, ROMERO-RIVERA A, et al.Inducing high activity of a thermophilic enzyme at ambient temperatures by directed evolution[J].Chemical Communications, 2017, 53(68):9 454-9 457.
[8] YU H, DALBY P A.Exploiting correlated molecular-dynamics networks to counteract enzyme activity-stability trade-off[J].Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(52):E12 192-E12 200.
[9] 焦标. 腈水解酶分子改造及在R-扁桃酸生产中的应用[D].杭州:浙江工业大学, 2016.JIAO B.Engineering of nitrilase and its application in biosynthesis of R-mandelic acid[D].Hangzhou:Zhejiang University of Technology, 2016.
[10] SCHREINER U, HECHER B,OBROWSKY S,et al.Directed evolution of Alcaligenes faecalis nitrilase[J].Enzyme and Microbial Technology, 2010, 47(4):140-146.
[11] XUE Y P, YANG Y K, LYU S Z, et al.High-throughput screening methods for nitrilases[J].Applied Microbiology and Biotechnology, 2016, 100(8):3 421-3 432.
[12] JUMPER J, EVANS R, PRITZEL A, et al.Highly accurate protein structure prediction with AlphaFold[J].Nature, 2021, 596(7 873):583-589.
[13] LEMAN J K, WEITZNER B D, LEWIS S M, et al.Macromolecular modeling and design in Rosetta:Recent methods and frameworks[J].Nature Methods, 2020, 17(7):665-680.
[14] BENDER B J, GAHBAUER S, LUTTENS A, et al.A practical guide to large-scale docking[J].Nature Protocols, 2021, 16(10):4 799-4 832.
[15] OLLITRAULT P J, MIESSEN A, TAVERNELLI I.Molecular quantum dynamics:A quantum computing perspective[J].Accounts of Chemical Research, 2021, 54(23):4 229-4 238.
[16] HILDEBRAND P W, ROSE A S, TIEMANN J K S.Bringing molecular dynamics simulation data into view[J].Trends in Biochemical Sciences, 2019, 44(11):902-913.
[17] 汤晓芒. 通过优化蛋白表面的电荷分布来对腈水解酶的热稳定性进行理性设计[D].上海:华东理工大学, 2012.TANG X M.Rational stabilization of a nitrilase through optimization of protein's surface charge distribution[D].Shanghai:East China University of Science and Technology, 2012.
[18] YU S S, LI J L, YAO P Y, et al.Inverting the enantiopreference of Nitrilase-catalyzed desymmetric hydrolysis of prochiral dinitriles by reshaping the binding pocket with a mirror-image strategy[J].Angewandte Chemie, 2021,60(7):3 679-3 684.
[19] ZHANG Q, LU X, ZHANG Y, et al.Development of a robust nitrilase by fragment swapping and semi-rational design for efficient biosynthesis of pregabalin precursor[J].Biotechnology and Bioengineering, 2020, 117(2):318-329.
[20] JINDAL G, SLANSKA K, KOLEV V,et al.Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase[J].Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(2):389-394.
[21] YU S, YAO P, LI J, et al.Improving the catalytic efficiency and stereoselectivity of a nitrilase from Synechocystis sp.PCC6803 by semi-rational engineering en route to chiral γ-amino acids[J].Catalysis Science & Technology, 2019, 9(6):1 504-1 510.
[22] ZHANG L J, YIN B, WANG C H, et al.Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803[J].Journal of Structural Biology, 2014, 188(2):93-101.
[23] PARK H, BRADLEY P, GREISEN P Jr, et al.Simultaneous optimization of biomolecular energy functions on features from small molecules and macromolecules[J].Journal of Chemical Theory and Computation, 2016, 12(12):6 201-6 212.
[24] LIU Y, HAN L, CHENG Z H, et al.Enzymatic biosynthesis of L-2-aminobutyric acid by glutamate mutase coupled with L-aspartate-β-decarboxylase using L-glutamate as the sole substrate[J].ACS Catalysis, 2020, 10(23):13 913-13 917.
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[7] . [J]. Food and Fermentation Industries, 2002, 28(4): 10 .
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