Please wait a minute...
Food and Fermentation Industries    2022, Vol. 48 Issue (20) : 70-77     DOI: 10.13995/j.cnki.11-1802/ts.030432
Substrate affinity design for the improvement of nitrilase Nit6803 activity
LIU Xinyue, HAN Laichuang, LIU Zhongmei*
(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
Download: PDF(7325 KB)   HTML 
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
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.
Keywords nitrilase      enzyme-substrate affinity      rational design      enzyme activity     
Issue Date: 18 November 2022
URL:     OR
[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.
[1] ZHANG Weimiao, CHENG Zhongyi, ZHOU Li, ZHOU Zhemin. Modification of the key amino residues locating the substrate channel entrance of nitrile hydratase that regulate the enzyme activity[J]. Food and Fermentation Industries, 2022, 48(9): 8-13.
[2] CHEN Zhichao, WANG Jinduo, XU Qingyang. Effects of trace elements and growth factors on L-phenylalanine fermentation[J]. Food and Fermentation Industries, 2022, 48(8): 82-89.
[3] ZHU Liping, YANG Qiang, JIANG Wei, LI Qun, LIN Bin, TANG Jie, CHEN Shenxi. Mold communities and enzyme activity characteristics in light-flavor Xiaoqu Baijiu[J]. Food and Fermentation Industries, 2022, 48(7): 70-77.
[4] GONG Dachun, WANG Delin, WAN Li, LIU Run, LYU Yucai, LUO Huajun, SONG Ting. Stability of CpCR mutants from Candida parapsilosis ATCC 7330 carbonyl reductase[J]. Food and Fermentation Industries, 2022, 48(2): 59-64.
[5] FENG Chengcheng, CAI Zizhe, CHEN Qiong, OU Shiyi, XIE Xiaodong, WANG Yong, MARTIN J T REANEY, ZHANG Ning. Probiotic properties of β-glucosidase producing lactic acid bacteria[J]. Food and Fermentation Industries, 2022, 48(15): 85-90.
[6] LIU Xiaomin, XIE Xiangyun, LIN Liangcai, ZHANG Yuhang, XIAO Dongguang. Enzymatic properties of Aspergillus niger lipase tabI and its synthesis ability of ethyl lactate[J]. Food and Fermentation Industries, 2022, 48(10): 9-15.
[7] YANG Ju, MAO Yin, HUANG Xiaoqiang, ZHOU Shenghu, DENG Yu. Computational design of 5-carboxyl-2-pentenoyl-CoA reductase from Thermobifida fusca to enhance adipic acid production[J]. Food and Fermentation Industries, 2021, 47(7): 1-7.
[8] LIU Rui, TAO Leren, WAN Kang. The effect of microwave treatment on the storage quality of ‘Xindaping' potato[J]. Food and Fermentation Industries, 2021, 47(5): 168-173.
[9] ZHOU Ming, ZHU Xiaojuan, YAO Meixiang, LU Jianqing, CHEN Kaka, ZHU Fengni, CHEN Jinyin, SHEN Yonggen. Correlation between flavonoids content, related enzymes activity and antioxidant capacity during the maturation of ‘Xiushui Huahong’ sweet orange[J]. Food and Fermentation Industries, 2021, 47(4): 60-67.
[10] LI Jingzhu, HU Mengjun, ZHANG Jianhua. Recombinant expression of protein-glutaminase and optimization of fermentation conditions[J]. Food and Fermentation Industries, 2021, 47(3): 294-301.
[11] MEI Manli, SUN Pengjie, XU Qingyang. Increasing yield of adenosine by membrane dialysis fermentation technique[J]. Food and Fermentation Industries, 2021, 47(23): 30-37.
[12] MIAO Zhoudi, CHEN Xiwen, PENG Zheng, MAO Xinzhe, DU Guocheng, ZHANG Juan. Improving the thermostability of Bacillus subtilis keratinase by rational design[J]. Food and Fermentation Industries, 2021, 47(19): 50-56.
[13] SHANG Yuting, GONG Jinsong, WANG Shunzhi, LU Zhenming, LI Heng, SHI Jinsong, XU Zhenghong. Culture conditions and magnetic immobilization of nitrilase-producing strain[J]. Food and Fermentation Industries, 2021, 47(16): 128-134.
[14] DU Jianhui, LIU Song, LU Xinyao, CHEN Jian. Improving thermostability of transglutaminase by introducing intramolecular disulfide bonds[J]. Food and Fermentation Industries, 2021, 47(15): 1-8.
[15] ZHANG Chen, JIA Meng, MA Yaqin. Application of β-glucosidase activity stabilization technology in citrus flavor enhancement[J]. Food and Fermentation Industries, 2021, 47(11): 303-309.
Full text



Copyright © Food and Fermentation Industries, All Rights Reserved.
Powered by Beijing Magtech Co. Ltd