研究报告

黑曲霉脂肪酶Lip A的异源表达与酶学性质分析

  • 王雅琪 ,
  • 凡林林 ,
  • 李文瑶 ,
  • 李弘轩 ,
  • 杜丽平 ,
  • 王洪 ,
  • 马立娟
展开
  • 1(天津科技大学 生物工程学院,天津,300457)
    2(工业发酵微生物教育部重点实验室,天津,300457)
    3(中国轻工业浓香型白酒固态发酵重点实验室,四川 宜宾,644000)
第一作者:硕士研究生(马立娟副研究员为通信作者,E-mail:malj@tust.edu.cn)

收稿日期: 2022-04-07

  修回日期: 2022-05-30

  网络出版日期: 2023-04-06

基金资助

中国轻工业浓香型白酒固态发酵重点实验室开放基金项目(2021zd012)

Heterologous expression and enzymatic characterization of Aspergillus niger lipase Lip A

  • WANG Yaqi ,
  • FAN Linlin ,
  • LI Wenyao ,
  • LI Hongxuan ,
  • DU Liping ,
  • WANG Hong ,
  • MA Lijuan
Expand
  • 1(College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China)
    2(Key Laboratory of Industrial Fermentation Microbiology Ministry of Education, Tianjin, 300457, China)
    3(Key Laboratory of Wuliangye-Flavor Liquor Solid-state Fermentation, China National Light Industrial, Yibin 644000, China)

Received date: 2022-04-07

  Revised date: 2022-05-30

  Online published: 2023-04-06

摘要

脂肪酶在油脂分解和脂质合成方面具有广泛应用,是一种很有前途的生物催化剂。该研究对来源于黑曲霉的脂肪酶Lip A进行了异源表达及催化特性研究。结果表明,该重组脂肪酶的最适温度为50 ℃,最适pH为8.0,并且在40~60 ℃和pH 5.0~8.0有较好的稳定性。该酶对有机试剂有较好的耐受性,金属离子Ca2+和Mg2+对脂肪酶水解活性有促进作用,Fe3+、Zn2+和Cu2+严重抑制该酶活性。反应动力学参数表明,对硝基苯酚乙酸酯是脂肪酶Lip A的最适底物,其反应Km为(0.228±0.05) mmol/L,催化常数Kcat为(0.023±0.001) s-1。生物信息学分析表明,脂肪酶Lip A是abH23同源家族Rhizomucor mihei脂肪酶中的一员。黑曲霉脂肪酶Lip A具有催化乳酸和乙醇生成乳酸乙酯特性,为其在酿造领域的应用奠定了基础。

本文引用格式

王雅琪 , 凡林林 , 李文瑶 , 李弘轩 , 杜丽平 , 王洪 , 马立娟 . 黑曲霉脂肪酶Lip A的异源表达与酶学性质分析[J]. 食品与发酵工业, 2023 , 49(5) : 25 -31 . DOI: 10.13995/j.cnki.11-1802/ts.031865

Abstract

Lipase has been widely used in the decomposition and synthesis of lipid,which is a promising biocatalyst. In this study, a putative lipase (Lip A) from Aspergillus niger was heterologous expressed in E. coli Rosetta(DE3) with success. The optimal reaction temperature and pH of lipase Lip A was at 50℃and pH 8.0, which means this lipase was a thermo-alkaline lipase. Lipase Lip A showed good stability in a range of temperature of 40-60℃ and pH of 5.0-8.0, and it had good tolerance to organic reagent. Lipase Lip A activity was enhanced by Ca2+ and inhibited seriously by Fe3+ and Cu2+. The kinetic parameters ofshowed that pNP-C2 was its optimal substrate, and the Km and Kcat towards pNP-C2 were (0.228±0.05) mmol/L and (0.023±0.001) s-1, respectively. Phylogenetic analysis showed that lipase Lip A was a member of the abH23 homologous family Rhizomucor mihei lipase. The lipase Lip A could catalyze the synthesis of the flavor ester ethyl lactate under aqueous phase, which showed a bright prospect for its application in the field of brewing.

参考文献

[1] HILLS G.Industrial use of lipases to produce fatty acid esters[J].European Journal of Lipid Science and Technology, 2003, 105(10):601-607.
[2] STERGIOU P Y, FOUKIS A, FILIPPOU M, et al.Advances in lipase-catalyzed esterification reactions[J].Biotechnology Advances, 2013, 31(8):1 846-1 859.
[3] ANGAJALA G, PAVAN P, SUBASHINI R.Lipases:An overview of its current challenges and prospectives in the revolution of biocatalysis[J].Biocatalysis and Agricultural Biotechnology, 2016, 7:257-270.
[4] FILHO D G,SILVA A G, GUIDINI C Z..Lipases:Sources, immobilization methods, and industrial applications[J].Applied Microbiology and Biotechnology, 2019, 103(18):7 399-7 423.
[5] 王丹丹, 林萌莉, 周晓宏.脂肪酶对白酒四大酯的可逆合成与分解[J].食品科技, 2014, 39(12):74-78;83.
WANG D D, LIN M L, ZHOU X H.Reversible synthesis and hydrolysis of four main esters in Chinese liquor catalyzed by lipase[J].Food Science and Technology, 2014, 39(12):74-78;83.
[6] LI C, ZHOU J W, DU G C, et al.Developing Aspergillus niger as a cell factory for food enzyme production[J].Biotechnology Advances, 2020, 44:107630.
[7] PEL H J, DE WINDE J D, ARCHER D B, et al.Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88[J].Nature Biotechnology, 2007, 25(2):221-231.
[8] SHU Z Y, YAN Y J, YANG J K, et al.Aspergillus niger lipase:Gene cloning, over-expression in Escherichia coli and in vitro refolding[J].Biotechnology Letters, 2007, 29(12):1 875-1 879.
[9] SHU Z Y, DUAN M J, YANG J K, et al.Aspergillus niger lipase:Heterologous expression in Pichia pastoris, molecular modeling prediction and the importance of the hinge domains at both sides of the lid domain to interfacial activation[J].Biotechnology Progress, 2009, 25(2):409-416.
[10] XING S Q, ZHU R N, LI C Q, et al.Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from Aspergillus niger GZUF36[J].Journal of Food Science and Technology, 2020, 57(7):2 669-2 680.
[11] FERNÁNDEZ-LORENTE G, ORTIZ C, SEGURA R L, et al.Purification of different lipases from Aspergillus niger by using a highly selective adsorption on hydrophobic supports[J].Biotechnology and Bioengineering, 2005, 92(6):773-779.
[12] MHETRAS N C, BASTAWDE K B, GOKHALE D V.Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207[J].Bioresource Technology, 2009, 100(3):1 486-1 490.
[13] HARIDASAN NAMBOODIRI V M, CHATTOPADHYAYA R.Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger[J].Lipids, 2000, 35(5):495-502.
[14] XU Y Q, ZHAO J R, LIU X, et al.Flavor mystery of Chinese traditional fermented Baijiu:The great contribution of ester compounds[J].Food Chemistry, 2022, 369(1):130920.
[15] LEVISSON M, VAN DER OOST J, KENGEN S W M.Carboxylic ester hydrolases from hyperthermophiles[J].Extremophiles, 2009, 13(4):567-581.
[16] KRUIS A J, BOHNENKAMP A C, PATINIOS C, et al.Microbial production of short and medium chain esters:Enzymes, pathways, and applications[J].Biotechnology Advances, 2019, 37(7):107407.
[17] SUN J C, YU B, CURRAN P, et al.Optimisation of flavour ester biosynthesis in an aqueous system of coconut cream and fusel oil catalysed by lipase[J].Food Chemistry, 2012, 135(4):2 714-2 720.
[18] XU Y Q, WANG X C, LIU X, et al.Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu[J].Food Chemistry, 2021, 338:128025.
[19] YAO J, GUI L, YIN S C.A novel esterase from a soil metagenomic library displaying a broad substrate range[J].AMB Express, 2021, 11(1):38.
[20] KARI J, ANDERSEN M, BORCH K, et al.An inverse Michaelis-Menten approach for interfacial enzyme kinetics[J].ACS Catalysis, 2017, 7(7):4 904-4 914.
[21] 赵江, 胡松青.响应曲面法优化非水相酶催化合成乳酸乙酯[J].现代食品科技, 2009, 25(11):1 320-1 322;1 319.
ZHAO J, HU S Q.Optimization of lipase-catalyzed synthesis of ethyl lactate in non-aqueous media by response surface methodology[J].Modern Food Science and Technology, 2009, 25(11):1 320-1 322;1 319.
[22] XU Y Q, HUANG H Q, LU H Y, et al.Characterization of an Aspergillus niger for efficient fatty acid ethyl ester synthesis in aqueous phase and the molecular mechanism[J].Frontiers in Microbiology, 2022, 12:820380.
[23] ZHANG X, LI X Q, XIA L M.Expression of a thermo-alkaline lipase gene from Talaromyces thermophilus in recombinant Pichia pastoris[J].Biochemical Engineering Journal, 2015, 103:263-269.
[24] SCHWEIKER K L, MAKHATADZE G I.Protein stabilization by the rational design of surface charge-charge interactions[J].Methods in Molecular Biology (Clifton, N.J.), 2009, 490:261-283.
[25] SINCHAIKUL S, SOOKKHEO B, PHUTRAKUL S, et al.Optimization of a thermostable lipase from Bacillus stearothermophilus P1:Overexpression, purification, and characterization[J].Protein Expression and Purification, 2001, 22(3):388-398.
[26] KOUTINAS M, YIANGOU C, OSÓRIO N M, et al.Application of commercial and non-commercial immobilized lipases for biocatalytic production of ethyl lactate in organic solvents[J].Bioresource Technology, 2018, 247:496-503.
[27] BORNSCHEUER U T.Microbial carboxyl esterases:Classification, properties and application in biocatalysis[J].FEMS Microbiology Reviews, 2002, 26(1):73-81.
[28] PETROVSKAYA L E, NOVOTOTSKAYA-VLASOVA K A, SPIRINA E V, et al.Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library[J].FEMS Microbiology Ecology, 2016, 92(5):fiw046.
[29] FISCHER M, PLEISS J.The Lipase Engineering Database:A navigation and analysis tool for protein families[J].Nucleic Acids Research, 2003, 31(1):319-321.
[30] ZHANG Y P, CHEN P C, GAO Y, et al.DBP-PSSM:Combination of evolutionary profiles with the XGBoost algorithm to improve the identification of DNA-binding proteins[J].Combinatorial Chemistry & High Throughput Screening, 2022, 25(1):3-12.
文章导航

/