基于序列和结构分析的酶热稳定性改造策略<sup>*</sup>

doi:10.13523/j.cb.2105021

中国生物工程杂志

2021, Vol. 41

Issue (10): 100-108 DOI: 10.13523/j.cb.2105021

综述

基于序列和结构分析的酶热稳定性改造策略^*

明玥¹,赵自通¹,王鸿磊²,梁志宏^1,^3,^**(

)

1 中国农业大学食品科学与营养工程学院北京 100083
2 中国农业大学烟台研究院烟台 264670
3 农业农村部转基因生物安全评价重点实验室北京 100083

Modification Strategy of Enzyme Thermal Stability Based on Sequence and Structure Analysis

MING Yue¹,ZHAO Zi-tong¹,WANG Hong-lei²,LIANG Zhi-hong^1,^3,^**(

)

1 College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083, China
2 Yantai Institute of China Agricultural University, Yantai 264670, China
3 Key Laboratory of Safety Assessment of Genetically Modified Organism, Ministry of Agriculture and Rural Affairs,Beijing 100083, China

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摘要：

功能酶被广泛应用于食品、化工、医药等领域,但却容易受高温环境限制,导致催化效率降低。以分子改造为目的的蛋白质工程技术是解决这一问题的关键环节,其能够对酶结构和功能进行改造,获得热稳定性好的工业酶。传统的定向进化方法只能依靠随机突变进行人工筛选,具有效率低、针对性差等缺点;理性设计作为酶热稳定性改造的主要方法,可借助各种计算机程序和软件预测潜在突变位点,但其要求对酶的催化机制、热稳定性机制有深入了解。对于大多数天然酶而言,酶的序列和晶体结构是最容易获取的信息,也是预测功能的重要基础。从酶的序列和晶体结构入手,重点介绍了共识突变、基于序列偏好性的突变、截短柔性区域、优化分子内相互作用力、刚化催化活性区域及计算机辅助筛选柔性位点等常用策略,这些策略具有筛选效率高、改造准确性高、实用性强等优点。结合多种酶的热稳定性改造案例进行分析,旨在为不同酶的改造策略选择提供有效参考,同时也为工业酶的耐热性研究提供理论支持。

关键词： 序列分析; 结构分析; 热稳定性; 改造策略

Abstract:

Functional enzymes have been widely used in the fields of food, chemicals, medicine, etc. However, high temperature reduces the catalytic efficiency of enzymes. The protein engineering technology can be used as a key link to modify enzymes’ structure and function, and to obtain industrial enzymes with thermostability. Traditional directed evolution methods can only rely on random mutations for manual screening with low efficiency. Rational design, as the main method of thermostability modification, can be used to predict potential mutation sites with various computer programs and software, but it requires deep understanding of the catalytic and thermal stability mechanism. For most natural enzymes, it is easy to obtain sequence and crystal structure, and is also an important basis for predicting function. This paper focuses on the modification strategies as follows: common mutation, the mutation based on amino acid preference, trunking of flexible regions, the optimization of intramolecular interactions, the modification of catalytic active regions and computer-aided design with the sequence and crystal structure analysis. These strategies have the advantages of high screening efficiency and modification accuracy, and strong practicability. And it also analyzes the thermostability modification cases of different enzymes, aiming to provide an effective reference for the selection of modification strategies, and also give theoretical support for the heat resistance research of industrial enzymes.

Key words: Sequence analysis Structural analysis Thermostability Modification strategies

收稿日期: 2021-05-12 出版日期: 2021-11-08

ZTFLH:

TQ033

基金资助: * 山东省重点研发计划(2019JZZY011014);国家自然科学基金(31671947)

通讯作者: 梁志宏 E-mail: lzh105@cau.edu.cn

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引用本文:

明玥,赵自通,王鸿磊,梁志宏. 基于序列和结构分析的酶热稳定性改造策略^*[J]. 中国生物工程杂志, 2021, 41(10): 100-108.

MING Yue,ZHAO Zi-tong,WANG Hong-lei,LIANG Zhi-hong. Modification Strategy of Enzyme Thermal Stability Based on Sequence and Structure Analysis. China Biotechnology, 2021, 41(10): 100-108.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105021 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I10/100

表1 16个家族中嗜热蛋白与嗜温蛋白的残基取代偏好性

表2 不同氨基酸在β转角中的位置偏好性

表3 分子内相互作用对酶热稳定性的改善效果

图1 Escherichia coli转酮醇酶在不同温度(300 K,340 K,370 K)的RMSF值[18]

[1]	Yu H R, Huang H. Engineering proteins for thermostability through rigidifying flexible sites. Biotechnology Advances, 2014, 32(2): 308-315. doi: 10.1016/j.biotechadv.2013.10.012
[2]	Victorino da Silva Amatto I, Gonsales da Rosa-Garzon N, Antônio de Oliveira Simões F, et al. Enzyme engineering and its industrial applications. Biotechnology and Applied Biochemistry, 2021: 2117. DOI: 10.1002/bab.2117. doi: 10.1002/bab.2117
[3]	Eijsink V G H, Bjørk A, Gåseidnes S, et al. Rational engineering of enzyme stability. Journal of Biotechnology, 2004, 113(1-3): 105-120. doi: 10.1016/j.jbiotec.2004.03.026
[4]	Xu P, Ni Z F, Zong M H, et al. Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design. International Journal of Biological Macromolecules, 2020, 150: 9-15. doi: 10.1016/j.ijbiomac.2020.02.033
[5]	Dotsenko A S, Dotsenko G S, Rozhkova A M, et al. Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase. Biochimie, 2020, 176: 103-109. doi: S0300-9084(20)30139-5 pmid: 32621943
[6]	Jochens H, Aerts D, Bornscheuer U T. Thermostabilization of an esterase by alignment-guided focussed directed evolution. Protein Engineering, Design and Selection, 2010, 23(12): 903-909. doi: 10.1093/protein/gzq071 pmid: 20947674
[7]	Li S F, Xu J Y, Bao Y J, et al. Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability. Journal of Biotechnology, 2015, 210: 8-14. doi: 10.1016/j.jbiotec.2015.06.406
[8]	Pezeshgi Modarres H, Dorokhov B D, Popov V O, et al. Understanding and engineering thermostability in DNA ligase from Thermococcus sp. 1519. Biochemistry, 2015, 54(19): 3076-3085. pmid: 25900264
[9]	Steiner K, Schwab H. Recent advances in rational approaches for enzyme engineering. Computational and Structural Biotechnology Journal, 2012, 2(3): e201209010. doi: 10.5936/csbj.201209010
[10]	Modarres H P, Mofrad M R, Sanati-Nezhad A. Protein thermostability engineering. RSC Advances, 2016, 6(116): 115252-115270. doi: 10.1039/C6RA16992A
[11]	Montanucci L, Fariselli P, Martelli P L, et al. Predicting protein thermostability changes from sequence upon multiple mutations. Bioinformatics, 2008, 24(13): i190-i195. doi: 10.1093/bioinformatics/btn166
[12]	Böhm G, Jaenicke R. Relevance of sequence statistics for the properties of extremophilic proteins. International Journal of Peptide and Protein Research, 1994, 43(1): 97-106. pmid: 7908011
[13]	Gromiha M M, Oobatake M, Sarai A. Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins. Biophysical Chemistry, 1999, 82(1): 51-67. pmid: 10584295
[14]	Zhao W, Wang X Z, Deng R Q, et al. Discrimination of thermostable and thermophilic lipases using support vector machines. Protein and Peptide Letters, 2011, 18(7): 707-717. pmid: 21342095
[15]	Lopez-Camacho C, Salgado J, Lequerica J L, et al. Amino acid substitutions enhancing thermostability of Bacillus polymyxa β-glucosidase A. Biochemical Journal, 1996, 314(3): 833-838. doi: 10.1042/bj3140833
[16]	Liu Y H, Yuan D J, Zhao Z X, et al. A novel strategy to improve the thermostability of Penicillium camembertii mono-and di-acylglycerol lipase. Biochemical and Biophysical Research Communications, 2018, 500(3): 639-644. doi: 10.1016/j.bbrc.2018.04.123
[17]	Guruprasad K, Rajkumar S. Gβ- and gγ-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials. Journal of Biosciences, 2000, 25(2): 143-156. pmid: 10878855
[18]	Yu H R, Yan Y H, Zhang C, et al. Two strategies to engineer flexible loops for improved enzyme thermostability. Scientific Reports, 2017, 7: 41212. doi: 10.1038/srep41212
[19]	于浩然. 理性设计改造荧光素酶的热稳定性. 天津: 天津大学, 2014.
	Yu H R. Engineering thermostability of luciferase by rational design. Tianjin: Tianjin University, 2014.
[20]	Mamonova T B, Glyakina A V, Galzitskaya O V, et al. Stability and rigidity/flexibility-two sides of the same coin. Biochimica et Biophysica Acta, 2013, 1834(5): 854-866. doi: 10.1016/j.bbapap.2013.02.011 pmid: 23416444
[21]	Wang X Y, Ma R, Xie X M, et al. Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering. Scientific Reports, 2017, 7: 15287. doi: 10.1038/s41598-017-12659-y
[22]	Nisha M, Satyanarayana T. Characterization of recombinant amylopullulanase (gt-apu) and truncated amylopullulanase (gt-apuT) of the extreme thermophile Geobacillus thermoleovorans NP33 and their action in starch saccharification. Applied Microbiology and Biotechnology, 2013, 97(14): 6279-6292. doi: 10.1007/s00253-012-4538-6 pmid: 23132347
[23]	Damnjanović J, Nakano H, Iwasaki Y. Deletion of a dynamic surface loop improves stability and changes kinetic behavior of phosphatidylinositol-synthesizing Streptomyces phospholipase D. Biotechnology and Bioengineering, 2014, 111(4): 674-682. doi: 10.1002/bit.25149 pmid: 24222582
[24]	Liu L, Yu H R, Du K, et al. Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region. Microbial Cell Factories, 2018, 17(1): 165. doi: 10.1186/s12934-018-1012-x
[25]	Yang T C, Legault S, Kayiranga E A, et al. The N-terminal β-sheet of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability. Applied and Environmental Microbiology, 2012, 78(9): 3059-3067. doi: 10.1128/AEM.07576-11
[26]	Chen J, Yu H M, Liu C C, et al. Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions. Journal of Biotechnology, 2013, 164(2): 354-362. doi: 10.1016/j.jbiotec.2013.01.021
[27]	Liu L, Deng Z M, Yang H Q, et al. In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica to improve thermostability. Applied and Environmental Microbiology, 2014, 80(3): 798-807. doi: 10.1128/AEM.03045-13
[28]	Niu C T, Zhu L J, Xu X, et al. Rational design of disulfide bonds increases thermostability of a mesophilic 1, 3-1, 4-β-glucanase from Bacillus terquilensis. PLoS One, 2016, 11(4): e0154036. doi: 10.1371/journal.pone.0154036
[29]	Bi J H, Chen S H, Zhao X H, et al. Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability. Applied Microbiology and Biotechnology, 2020, 104(17): 7551-7562. doi: 10.1007/s00253-020-10764-z
[30]	Wang R, Wang S, Xu Y, et al. Enhancing the thermostability of Rhizopus chinensis lipase by rational design and MD simulations. International Journal of Biological Macromolecules, 2020, 160: 1189-1200. doi: S0141-8130(20)33412-7 pmid: 32485250
[31]	Jiang X, Wang Y R, Wang Y, et al. Exploiting the activity-stability trade-off of glucose oxidase from Aspergillus niger using a simple approach to calculate thermostability of mutants. Food Chemistry, 2021, 342: 128270. doi: 10.1016/j.foodchem.2020.128270
[32]	Sakabe M, Asanuma D, Kamiya M, et al. Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization. Journal of the American Chemical Society, 2013, 135(1): 409-414. doi: 10.1021/ja309688m
[33]	Pikkemaat M G, Linssen A B M, Berendsen H J C, et al. Molecular dynamics simulations as a tool for improving protein stability. Protein Engineering, Design and Selection, 2002, 15(3): 185-192. doi: 10.1093/protein/15.3.185
[34]	Zhang L J, Tang X M, Cui D B, et al. A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107. Protein Science, 2014, 23(1): 110-116. doi: 10.1002/pro.2388
[35]	Tu T, Luo H Y, Meng K, et al. Improvement in thermostability of an Achaetomium sp. strain Xz8 endopolygalacturonase via the optimization of charge-charge interactions. Applied and Environmental Microbiology, 2015, 81(19): 6938-6944. doi: 10.1128/AEM.01363-15
[36]	You S, Tu T, Zhang L J, et al. Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions. Biotechnology for Biofuels, 2016, 9: 124. doi: 10.1186/s13068-016-0544-8
[37]	Yang M, Yang S X, Liu Z M, et al. Rational design of alginate lyase from Microbulbifer sp. Q7 to improve thermal stability. Marine Drugs, 2019, 17(6): 378. doi: 10.3390/md17060378
[38]	Ren Y X, Luo H Y, Huang H Q, et al. Improving the catalytic performance of Proteinase K from Parengyodontium album for use in feather degradation. International Journal of Biological Macromolecules, 2020, 154: 1586-1595. doi: 10.1016/j.ijbiomac.2019.11.043
[39]	Guo Y J, Tu T, Zheng J, et al. Improvement of BsAPA aspartic protease thermostability via autocatalysis-resistant mutation. Journal of Agricultural and Food Chemistry, 2019, 67(37): 10505-10512. doi: 10.1021/acs.jafc.9b03959
[40]	Matsumiya Y, Nishikawa K, Aoshima H, et al. Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site. Journal of Biochemistry, 2004, 135(4): 547-553. pmid: 15115781
[41]	Matsumiya Y, Murata N, Inouye K, et al. Further stabilization of Leu^155 mutant thermolysins by mutation of an autodegradation site. Applied Biochemistry and Biotechnology, 2012, 166(3): 735-743. doi: 10.1007/s12010-011-9462-1 pmid: 22139731
[42]	Niu C F, Yang P L, Luo H Y, et al. Engineering of Yersinia phytases to improve pepsin and trypsin resistance and thermostability and application potential in the food and feed industry. Journal of Agricultural and Food Chemistry, 2017, 65(34): 7337-7344. doi: 10.1021/acs.jafc.7b02116
[43]	Sun Z T, Liu Q, Qu G, et al. Utility of B-factors in protein science: interpreting rigidity, flexibility, and internal motion and engineering thermostability. Chemical Reviews, 2019, 119(3): 1626-1665. doi: 10.1021/acs.chemrev.8b00290
[44]	Li Y F, Hu F J, Wang X M, et al. A rational design for trypsin-resistant improvement of armillariella tabescens β-mannanase MAN47 based on molecular structure evaluation. Journal of Biotechnology, 2013, 163(4): 401-407. doi: 10.1016/j.jbiotec.2012.12.018
[45]	Zhang S B, Wu Z L. Identification of amino acid residues responsible for increased thermostability of feruloyl esterase a from Aspergillus niger using the PoPMuSiC algorithm. Bioresource Technology, 2011, 102(2): 2093-2096. doi: 10.1016/j.biortech.2010.08.019
[46]	Kellogg E H, Leaver-Fay A, Baker D. Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins, 2011, 79(3): 830-838. doi: 10.1002/prot.22921
[47]	Capriotti E, Fariselli P, Casadio R. I-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Research, 2005, 33(suppl_2): W306-W310. doi: 10.1093/nar/gki375
[48]	Guerois R, Nielsen J E, Serrano L. Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. Journal of Molecular Biology, 2002, 320(2): 369-387. doi: 10.1016/S0022-2836(02)00442-4
[49]	李冠霖. 脂肪酶/酯酶的理性设计和改造研究. 武汉: 华中科技大学, 2018.
	Li G L. Rational design and modification of lipase/esterase. Wuhan: Huazhong University of Science and Technology, 2018.
[50]	You S, Xie C, Ma R, et al. Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase. Biotechnology for Biofuels, 2019, 12: 278. doi: 10.1186/s13068-019-1620-7

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