Food and Fermentation Industries >

2023 , Vol. 49 >Issue 19: 22 - 29

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034342

Expression of Aspergillus niger pelanase in Escherichia coli and enzymatic property characterization

  • ZHANG Haiyun ,
  • MA Jianing ,
  • LI Yangyang ,
  • YANG Penghui ,
  • SONG Weiyan ,
  • LIU Song
Expand
  • 1(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
    2(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    3(School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China)

Received date: 2022-11-22

  Revised date: 2022-12-23

  Online published: 2023-11-01

Fold

Abstract

Acid pectin lyase can break the a-1,4 glycosidic bond in pectin by b-trans-elimination to form unsaturated oligogalacturonic acid, which is important for the degradation of highly esterified pectin in food and other industries. In this study, acidic pectin lyase (pelA) from Aspergillus niger AG11 was expressed in Escherichia coli BL21 (DE3), and the protein tag optimization and enzymatic properties were investigated. It was finally determined that the highest intracellular enzyme activity of pelA was achieved at 3.25 U/mL with the N-terminal fusion of carbohydrate-binding structural domain (CBM), 4.12-fold higher than the initial condition. The enzymatic properties of pelA were determined before and after CBM resection. The results showed that the specific enzyme activity, optimum reaction temperature and optimum reaction pH of pelA were 15.45 U/mg, 40°C and 5.0, respectively. PelA maintained more than 80% activity at 30-50°C incubation for 2 h, which was the same as pelA+CBM. The relative enzyme activity was maintained above 60% by incubation at 40 ℃ and pH 4.0-5.0 for 2 h. At this time, pelA+CBM had more than 80% activity. Using citrus pectin as the substrate, the Km and kcat of pelA were 4.44 mmol/L and 31.44 s-1 , respectively. The results of the study laid the foundation for the molecular modification of pelA and its application in industrial production.

Key words： Aspergillus niger; acid pectin lyase; intracellular expression; optimization of melting labels; enzymatic properties

Cite this article

ZHANG Haiyun , MA Jianing , LI Yangyang , YANG Penghui , SONG Weiyan , LIU Song . Expression of Aspergillus niger pelanase in Escherichia coli and enzymatic property characterization[J]. Food and Fermentation Industries, 2023 , 49(19) : 22 -29 . DOI: 10.13995/j.cnki.11-1802/ts.034342

References

[1] 许均华, 李高阳, 李志坚.高产果胶酶菌株的选育及其发酵生产的研究进展[J].食品与机械, 2011, 27(1):146-150.
XU J H, LI G Y, LI Z J.Research progress in pectinase high-yield strains and the fermentation of pectinase[J].Food & Machinery, 2011, 27(1):146-150.
[2] FU Y J, LIU W, ZU Y G, et al.Enzyme assisted extraction of luteolin and apigenin from pigeonpea[Cajanus cajan (L.) Mill sp.]leaves[J].Food Chemistry, 2008, 111(2):508-512.
[3] 张海燕, 吴天祥.微生物果胶酶的研究进展[J].酿酒科技, 2006(9):82-85.
ZHANG H Y, WU T X.Research progress in pectinase[J].Liquor-Making Science & Technology, 2006(9):82-85.
[4] KASHYAP D R, VOHRA P K, CHOPRA S, et al.Applications of pectinases in the commercial sector:A review[J].Bioresource Technology, 2001, 77(3):215-227.
[5] HE Y L, PAN L, WANG B.Efficient over-expression and application of high-performance pectin lyase by screening Aspergillus niger pectin lyase gene family[J].Biotechnology and Bioprocess Engineering, 2018, 23(6):662-669.
[6] YıLDıZ S, MATA-GÓMEZ M A, TARı C, et al.Partial purification of a polygalacturonase from a new Aspergillus sojae mutant and its application in grape mash maceration[J].International Journal of Food Science & Technology, 2017, 52(3):834-842.
[7] ANAND G, YADAV S, YADAV D.Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 7589[J].Biocatalysis and Agricultural Biotechnology, 2017, 10:264-269.
[8] ATANASOVA L, DUBEY M, GRUJIĆ M, et al.Evolution and functional characterization of pectate lyase PEL12, a member of a highly expanded Clonostachys rosea polysaccharide lyase 1 family[J].BMC Microbiology, 2018, 18(1):178.
[9] JOHN J, SURENDRANATHAN KAIMAL K K, SMITH M L, et al.Advances in upstream and downstream strategies of pectinase bioprocessing:A review[J].International Journal of Biological Macromolecules, 2020, 162:1086-1099.
[10] AHMED I, ZIA M A, HUSSAIN M A, et al.Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger;its purification and characterization[J].Journal of Radiation Research and Applied Sciences, 2016, 9(2):148-154.
[11] SIDDIQUI M A, PANDE V, ARIF M.Production, purification, and characterization of polygalacturonase from Rhizomucor pusillus isolated from decomposting orange peels[J].Enzyme Research, 2012, 2012:138634.
[12] 吴倩, 周峻沛, 唐湘华, 等.Penicillium sp.JF09酸性果胶酶的发酵条件、酶学性质及其在苹果汁澄清中的应用潜力研究[J].食品科技, 2013, 38(8):42-47.
WU Q, ZHOU J P, TANG X H, et al.The acid pectinase from Penicillium sp.JF09:Fermentation optimization, enzymatic characterization and potential for application in apple juice clarification[J].Food Science and Technology, 2013, 38(8):42-47.
[13] EL ENSHASY H A, ELSAYED E A, SUHAIMI N, et al.Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system[J].BMC Biotechnology, 2018, 18(1):71.
[14] AMIN F, BHATTI H N, BILAL M.Recent advances in the production strategies of microbial pectinases:A review[J].International Journal of Biological Macromolecules, 2019, 122:1017-1026.
[15] PÉREZ-FUENTES C, CRISTINA RAVANAL M, EYZAGUIRRE J.Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization[J].Fungal Biology, 2014, 118(5-6):507-515.
[16] 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.
[17] 彭小波. 果胶裂解酶在毕赤酵母中高效表达及酶学性质分析[D].武汉:武汉轻工大学, 2018.
PENG X B.High-level expression pectate lyase in Pichia pastoris and enzymatic determination[D].Wuhan:Wuhan Polytechnic University, 2018.
[18] DAMAK N, ABDELJALIL S, KOUBAA A, et al.Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli[J].International Journal of Biological Macromolecules, 2013, 62:549-556.
[19] 谢茂芳, 薛保国, 吴坤.黑曲霉果胶裂解酶基因的克隆与原核表达[J].河南农业科学, 2013, 42(1):82-85.
XIE M F, XUE B G, WU K.Cloning and prokaryotic expression of pectin lyase gene from Aspergillus niger[J].Journal of Henan Agricultural Sciences, 2013, 42(1):82-85.
[20] 杨欣伟. 黑曲霉EIM-6果胶裂解酶基因的克隆与表达[D].福州:福建师范大学, 2009.
YANG X W.Cloning and expression of pectin lyase gene from Aspergillus niger EIM-6[D].Fuzhou:Fujian Normal University, 2009.
[21] HADJ SASSI A, TRIGUI-LAHIANI H, ABDELJALIL S, et al.Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli[J].International Journal of Biological Macromolecules, 2017, 95:256-262.
[22] 王颖, 董晓燕, 孙彦.蛋白质复性技术研究进展[J].生物工程进展, 2002, 22(2):61-65.
WANG Y, DONG X Y, SUN Y.Research advances in protein renaturation technology[J].Progress in Biotechnology, 2002, 22(2):61-65.
[23] KAY T.Overview of bacterial expression systems for heterologous protein production:From molecular and biochemical fundamentals to commercial systems[J].Applied Microbiology and Biotechnology, 2006, 72(2):211-222.
[24] 周俊杰. 融合标签酰基载体蛋白对外源蛋白热稳定性的影响研究[D].广州:华南农业大学, 2019.
ZHOU J J.Effect of fusion label acyl carrier protein on thermal stability of exogenous proteins[D].Guangzhou:South China Agricultural University, 2019.
[25] ECHE S, GORDON M L.Recombinant expression of HIV-1 protease using soluble fusion tags in Escherichia coli:A vital tool for functional characterization of HIV-1 protease[J].Virus Research, 2021, 295:198289.
[26] ZONDAGH J, WILLIAMS A, ACHILONU I, et al.Overexpression, purification and functional characterisation of wild-type HIV-1 subtype C protease and two variants using a thioredoxin and his-tag protein fusion system[J].The Protein Journal, 2018, 37(4):369-379.
[27] MAHALIK S, SHARMA A K, MUKHERJEE K J.Genome engineering for improved recombinantprotein expression in Escherichia coli[J].Microbial Cell Factories, 2014, 13(1):177.
[28] HOFFMAM Z B, ZANPHORLIN L M, COTA J, et al.Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase[J].New Biotechnology, 2016, 33(4):467-472.
[29] MALGAS S, PLETSCHKE B I.The effect of an oligosaccharide reducing-end xylanase, BhRex8A, on the synergistic degradation of xylan backbones by an optimised xylanolytic enzyme cocktail[J].Enzyme and Microbial Technology, 2019, 122:74-81.
[30] TEO S C, LIEW K J, SHAMSIR M S, et al.Characterizing a halo-tolerant GH10 xylanase from Roseithermus sacchariphilus strain RA and its CBM-truncated variant[J].International Journal of Molecular Sciences, 2019, 20(9):2284.
Options
Outlines

/

〈 〉

Powered by Beijing Magtech Co. Ltd,.