食品与发酵工业 >

2024 , Vol. 50 >Issue 5: 22 - 28

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035608

研究报告

宏基因组来源高活性酯酶的鉴定及其酶学性质表征

  • 张颖 ,
  • 董耀 ,
  • 吕云斌 ,
  • 王绍琛 ,
  • 冯治洋
展开
  • (南京农业大学 食品科技学院,江苏 南京,210000)
第一作者：硕士研究生(冯治洋教授为通信作者,E-mail:zfeng@njau.edu.cn)

收稿日期: 2023-03-24

  修回日期: 2023-04-21

  网络出版日期: 2024-04-09

基金资助

国家自然科学基金面上项目(31770049)

收起

Identification and characterization of a metagenome derivedesterase with high activity

  • ZHANG Ying ,
  • DONG Yao ,
  • LYU Yunbin ,
  • WANG Shaochen ,
  • FENG Zhiyang
Expand
  • (College of Science and Technology, Nanjing Agricultural University, Nanjing 210000, China)

Received date: 2023-03-24

  Revised date: 2023-04-21

  Online published: 2024-04-09

Fold

摘要

土壤中大量的非培养微生物是新酶发现的重要资源。该研究利用功能宏基因组学策略筛选获得高活性酯酶,并对其进行鉴定分析。采用三丁酸甘油酯平板法对土壤微生物宏基因组文库中具有酯酶活性的克隆进行筛选,获得高活性的阳性克隆,通过生物信息学分析和生化实验对酯酶的酶学性质进行研究表征。获得一个比活力高达(3 957±3) U/mg 的酯酶 AesZF899,该酯酶为不含信号肽的酯酶第四家族胞内酯酶,蛋白大小34 kDa,与来自Afipia sp.P52-10中未表征的α/β水解酶(GenBank登录号为WP_034470467.1)一致性达76.43%。AesZF899的最佳底物为对硝基苯乙酸酯,酶反应的最适pH值和最适温度分别是9.0和40 ℃。AesZF899在30 ℃温育8 h后仍可保持初始活性,在35、40 ℃分别温育7和4 h后仍可保持50%的活性,在反应温度大于80 ℃后酶活性丧失。终浓度10 mmol/L的Fe3+可以增强酶活性,而终浓度10 mmol/L的Na+、Li+和Mg2+有较强的抑制作用。体积分数为1%二甲基亚砜可以增强酶活性,异戊醇也对酶活性有一定的促进作用。

关键词： 酯酶; HSL家族; 宏基因组; 异源表达; 高活性

本文引用格式

张颖 , 董耀 , 吕云斌 , 王绍琛 , 冯治洋 . 宏基因组来源高活性酯酶的鉴定及其酶学性质表征[J]. 食品与发酵工业, 2024 , 50(5) : 22 -28 . DOI: 10.13995/j.cnki.11-1802/ts.035608

Abstract

The large amont of uncultured microorganisms in soil are important resources for the discovery of new enzymes.In this study, a novel esterase with high activity was identified by functional metagenomics strategy and its enzymatic properties was characterized.The clones from a soil metagenomic library were screened on triglyceride plates to obtain a positive clone with the highest activity.The properties of the esterase derived from the postive clone were studied by bioinformatics analysis and biochemical experiments.An esterase AesZF899 with specific activity of (3 957±3) U/mg was obtained.AesZF899 is an intracellular enzyme belonging to the Fourth family of esterase, and shared the highest identities of 76.43% with an uncharacterized α/β hydrolase (GenBank entry number WP_034470467.1) from Afipia sp.P52-10.AesZF899 has no signal peptide and its molecular weight is 34.0 kDa.AesZF899 exhibited the highest activity to p-nitrophenylacetate, and the optimal pH and temperature of the enzyme were 9.0 and 40 ℃, respectively.AesZF899 could still maintain the initial esterase activity after incubating the protein at 30 ℃ for 8 h, and maintained 50% activity after incubating at 35 or 40 ℃ for 7 or 4 h, respectively.The esterase was lost after incubating at 80 ℃.The esterase activity of AesZF899 could be improved in buffer containing 10 mmol/L of Fe3+, and partly inhibited in buffers containing 10 mmol/L of Fe3+, and partly inhibited in buffers containing 10 mmol/L of Na+, Ni+ and Mg2+.And the esterase activity of AesZF899 could be improved in buffer containing 1% DMSO or isoamyl alcohol.A normo-thermal stable esterase AesZF899 was obtained from a soil metagenomic library.

Key words： esterase; HSL family; metagenomic library; heterologous expression; high activity

参考文献

[1] JOCHENS H, HESSELER M, STIBA K, et al.Protein engineering of α/β-hydrolase fold enzymes[J].ChemBioChem, 2011, 12(10):1508-1517.
[2] OLLIS D L, CHEAH E, CYGLER M, et al.The α/β hydrolase fold[J].Protein Engineering, Design and Selection, 1992, 5(3):197-211.
[3] BOURNE Y, MARCHOT P.Hot spots for protein partnerships at the surface of cholinesterases and related α/β hydrolase fold proteins or domains:A structural perspective[J].Molecules, 2017, 23(1):35.
[4] ARPIGNY J L, JAEGER K E.Bacterial lipolytic enzymes:Classification and properties[J].The Biochemical Journal, 1999, 343(1):177-183.
[5] RAUWERDINK A, KAZLAUSKAS R J.How the same core catalytic machinery catalyzes 17 different reactions:The serine-histidine-aspartate catalytic triad of α/β-hydrolase fold enzymes[J].ACS Catalysis, 2015, 5(10):6153-6176.
[6] MANDRICH L, PEZZULLO M, ROSSI M, et al.SSoNΔ andSsoNΔlong:Two thermostable esterases from the same ORF in the archae on Sulfolobus solfataricus?[J].Archaea, 2006, 2(2):109-115.
[7] BEN CHEIKH R, BERTICAT C, BERTHOMIEU A, et al.Characterization of a novel high-activity esterase in Tunisian populations of the mosquito Culex pipiens[J].Journal of Economic Entomology, 2008, 101(2):484-491.
[8] JEGANNATHAN K R, NIELSEN P H.Environmental assessment of enzyme use in industrial production:A literature review[J].Journal of Cleaner Production, 2013, 42:228-240.
[9] KIM T D.Bacterial hormone-sensitive lipases (bHSLs):Emerging enzymes for biotechnological applications[J].Journal of Microbiology and Biotechnology, 2017, 27(11):1907-1915.
[10] BERLEMONT R, SPEE O, DELSAUTE M, et al.Novel organic solvent-tolerant esterase isolated by metagenomics:Insights into the lipase/esterase classification[J].Revista Argentina De Microbiologia, 2013, 45(1):3-12.
[11] KONO Y, TOMITA T.A role of highly active carboxylesterase in an organophosphorus insecticide resistant strain of Culex pipiens molestus[J].Medical Entomology and Zoology, 1993, 44(1):15-21.
[12] 黄循柳, 黄仕杰, 郭丽琼, 等.宏基因组学研究进展[J].微生物学通报, 2009, 36(7):1058-1066.
HUANG X L, HUANG S J, GUO L Q, et al.Advances of metagenomics[J].Microbiology, 2009, 36(7):1058-1066.
[13] SANTILLAN J Y, DETTORRE L A, LEWKOWICZ E S, et al.New and highly active microbial phosphotriesterase sources[J].FEMS Microbiology Letters, 2016, 363(24):276.
[14] 时玉萍. 基于功能筛选的酯酶基因的克隆及其酶学性质的研究[D].上海:上海海洋大学, 2013.
SHI Y P.Gene cloning, expression, and characterization of two novel esterase:Screening based on functional method[D].Shanghai:Shanghai Ocean University, 2013.
[15] KANG C H, OH K H, LEE M H, et al.A novel family VII esterase with industrial potential from compost metagenomic library[J].Microbial Cell Factories, 2011, 10:41.
[16] WANG S C, GAO X, GAO Y J, et al.Tetracycline resistance genes identified from distinct soil environments in China by functional metagenomics[J].Frontiers in Microbiology, 2017, 8:1406.
[17] WATERHOUSE A, BERTONI M, BIENERT S, et al.SWISS-MODEL:Homology modelling of protein structures and complexes[J].Nucleic Acids Research, 2018, 46(W1):W296-W303.
[18] BIENERT S, WATERHOUSE A, DE BEER T A P, et al.The SWISS-MODEL Repository:New features and functionality[J].Nucleic Acids Research, 2017, 45(D1):D313-D319.
[19] GUEX N, PEITSCH M C, SCHWEDE T.Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer:A historical perspective[J].Electrophoresis, 2009, 30(Suppl 1):S162-S173.
[20] STUDER G, REMPFER C, WATERHOUSE A M, et al.QMEANDisCo:Distance constraints applied on model quality estimation[J].Bioinformatics, 2020, 36(6):1765-1771.
[21] BERTONI M, KIEFER F, BIASINI M, et al.Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology[J].Scientific Reports, 2017, 7:10480.
[22] SECUNDO F, CARREA G, TARABIONO C, et al.Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents[J].Biotechnology and Bioengineering, 2004, 86(2):236-240.
[23] 何平, 黄卓烈, 巫光宏, 等.二甲基亚砜对木瓜蛋白酶活性与构象的影响[J].华南农业大学学报, 2008, 29(3):37-41.
HE P, HUANG Z L, WU G H, et al.Effect of dimethyl sulfoxide on the activity and conformation of papain[J].Journal of South China Agricultural University, 2008, 29(3):37-41.
[24] 刘隽, 郑忠亮, 左振宇, 等.微水相中氨基酰化酶催化拆分反应的研究[J].食品工业科技, 2009, 30(8):143-145.
LIU J, ZHENG Z L, ZUO Z Y, et al.Study on the resolution catalysed by aminoacylase in micro-aqueous phase[J].Science and Technology of Food Industry, 2009, 30(8):143-145.
[25] 刘喆, 李家霖, 白利平.微生物酯酶研究进展[J].微生物学报, 2023, 63(2):451-464.
LIU Z, LI J L, BAI L P.Research advances in microbial esterases[J].Acta Microbiologica Sinica, 2023, 63(2):451-464.
[26] LI G L, XU L, ZHANG H J, et al.A de novo designed esterase with p-nitrophenyl acetate hydrolysis activity[J].Molecules, 2020, 25(20):4658.
[27] MA J, MA Y P, LI Y Q, et al.Characterization of feruloyl esterases from Pecoramyces sp.F1 and the synergistic effect in biomass degradation[J].World Journal of Microbiology and Biotechnology, 2022, 39(1):1-12.
[28] 马春辉. 酯酶在化学合成中的应用[J].化工设计通讯, 2018, 44(6):68.
MA C H.Application of esterase in chemical synthesis[J].Chemical Engineering Design Communications, 2018, 44(6):68.
[29] 黄嫒, 马志强, 王玮, 等.豌豆酯酶的纯化和酯酶活性表征及其对农药的敏感性[J].食品与发酵工业, 2019, 45(11):78-84.
HUANG A, MA Z Q, WANG W, et al.Purification, characterization and pesticides-sensitivity of plant-esterase from Pisum sativum Linn[J].Food and Fermentation Industries, 2019, 45(11):78-84.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司