食品与发酵工业 >

2022 , Vol. 48 >Issue 13: 16 - 24

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.029933

研究报告

重组大肠杆菌产卤代烷脱卤酶的发酵条件优化

  • 名晓东 ,
  • 郭自涛 ,
  • 陈剑雄 ,
  • 顾正华 ,
  • 辛瑜 ,
  • 孙海彦 ,
  • 关彦明 ,
  • 张梁
展开
  • 1(江南大学 粮食发酵工艺与技术国家工程实验室,江苏 无锡,214122)
    2(中国热带农业科学院热带生物技术研究所,海南 海口,571101)
    3(中国食品发酵工业研究院有限公司,北京,100015)
第一作者：硕士研究生(张梁教授为通信作者,E-mail:zhangl@jiangnan.edu.cn)

收稿日期: 2021-11-02

  修回日期: 2021-11-28

  网络出版日期: 2022-08-03

基金资助

国家重点研发计划绿色生物制造专项(2021YFC2100300);国家现代农业产业技术体系项目(CARS-11-HNSHY);中国热带农业科学院基本科研业务费项目(1630052021012,1630052020023,1630052020024)

收起

Optimization of fermentation conditions of recombinant Escherichia coli expressing haloalkane dehalogenase

  • MING Xiaodong ,
  • GUO Zitao ,
  • CHEN Jianxiong ,
  • GU Zhenghua ,
  • XIN Yu ,
  • SUN Haiyan ,
  • GUAN Yanming ,
  • ZHANG Liang
Expand
  • 1(National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China)
    2(Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China)
    3(China National Research Institute of Food and Fermentation Industries, Beijing, 100015, China)

Received date: 2021-11-02

  Revised date: 2021-11-28

  Online published: 2022-08-03

Fold

摘要

对产卤代烷脱卤酶的重组大肠杆菌BL21(DE3)/pET28a-DhaA进行发酵优化以提高其表达量。该研究从TB培养基出发,通过单因素实验与响应面实验在摇瓶水平上对培养基各成分进行优化;其次,在最优培养基的基础上对发酵的工艺条件进行优化;最后,在5 L发酵罐中进行了初步放大验证。结果表明,最优的培养基组成为:甘油10 g/L,酵母粉23 g/L,蛋白胨14 g/L,MgSO4·7H2O 1.3 g/L,ZnSO4·7H2O 0.1 g/L,酶活力可达到(1 182.94±10.86) U/L,较初始培养基提高了94%;最优的发酵工艺条件为:接种量5%,装液量40 mL/250 mL,37 ℃培养至OD600为1.8时加入终浓度为0.4 mmol/L的异丙基-β-D-硫代半乳糖苷于20 ℃诱导22 h,酶活力可达到(3 585.83±15.02) U/L,提高至未进行优化前的5.87倍;5 L发酵罐初步放大验证实验中,酶活力最高达到(9 682.62±191.16) U/L,提高至摇瓶水平的2.7倍。通过在摇瓶水平对发酵培养基与工艺进行优化,以及在5 L发酵罐中进行放大验证,极大地提高了卤代烷脱卤酶的酶活力,为放大生产奠定了基础。

关键词： 卤代烷脱卤酶; 大肠杆菌; 发酵优化; 响应面法

本文引用格式

名晓东 , 郭自涛 , 陈剑雄 , 顾正华 , 辛瑜 , 孙海彦 , 关彦明 , 张梁 . 重组大肠杆菌产卤代烷脱卤酶的发酵条件优化[J]. 食品与发酵工业, 2022 , 48(13) : 16 -24 . DOI: 10.13995/j.cnki.11-1802/ts.029933

Abstract

In this study, the fermentation of a recombinant Escherichia coli BL21(DE3)/pET28a-DhaA was optimized to improve its production of haloalkane dehalogenase. Firstly, based on TB medium, the components of culture medium were optimized at shaking flask level by single factor experiment and response surface experiment. Secondly, the fermentation conditions were optimized base on the optimal culture medium. Finally, the preliminary amplification was verified in a 5 L fermentation tank. The results showed that the optimal medium composition was glycerol 10 g/L, yeast powder 23 g/L, peptone 14 g/L, MgSO4·7H2O 1.3 g/L, ZnSO4·7H2O 0.1 g/L. The enzyme activity can reach (1 182.94±10.86) U/L, which was 94% higher than that of the initial medium. The optimal fermentation conditions were: the inoculum volume 5%, the loading volume 40 mL/250 mL, 0.4 mmol/L IPTG was added to induce 22 h at 20 ℃, when the OD600 reached 1.8 at 37 ℃. The enzyme activity was achieved at (3 585.83±15.02) U/L, which was 5.87 times higher than the initial conditions. At last, the enzyme activity reached (9 682.62±191.16) U/L in a 5 L fermentation tank,which was 2.7 times higher than that of a shaking flask. After optimization, the enzyme activity of haloalkane dehalogenase was greatly improved, and our study provides a foundation for scale-up production.

Key words： haloalkane dehalogenase; Escherichia coli; fermentation optimization; response surface method

参考文献

[1] QUINTERO J C, MOREIRA M T, FEIJOO G, et al.Anaerobic degradation of hexachlorocyclohexane isomers in liquid and soil slurry systems[J].Chemosphere, 2005, 61(4):528-536.
[2] 李安章, 邵宗泽.微生物卤代烷烃脱卤酶研究进展[J].微生物学报, 2015, 55(4):381-388.
LI A Z, SHAO Z Z.Research progress of microbial haloalkane dehalogenase-A review[J].Acta Microbiologica Sinica, 2015, 55(4):381-388.
[3] PROKOP Z, OPLUSTIL F, DEFRANK J, et al.Enzymes fight chemical weapons[J].Biotechnology Journal, 2006, 1(12):1 370-1 380.
[4] KOUDELAKOVA T, BIDMANOVA S, DVORAK P, et al.Haloalkane dehalogenases:Biotechnological applications[J].Biotechnology Journal, 2013, 8(1):32-45.
[5] KOUDELAKOVA T, CHOVANCOVA E, BREZOVSKY J, et al.Substrate specificity of haloalkane dehalogenases[J].The Biochemical Journal, 2011, 435(2):345-354.
[6] PROKOP Z, SATO Y, BREZOVSKY J, et al.Enantioselectivity of haloalkane dehalogenases and its modulation by surface loop engineering[J].Angewandte Chemie International Edition, 2010, 49(35):6 111-6 115.
[7] BIDMANOVA S, CHALOUPKOVA R, DAMBORSKY J, et al.Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons[J].Analytical and Bioanalytical Chemistry, 2010, 398(5):1 891-1 898.
[8] LOS G V, ENCELL L P, MCDOUGALL M G, et al.HaloTag:A novel protein labeling technology for cell imaging and protein analysis[J].ACS Chemical Biology, 2008, 3(6):373-382.
[9] KULAKOVA A N, LARKIN M J, KULAKOV L A.The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064[J].Microbiology (Reading, England), 1997, 143 (Pt 1):109-115.
[10] NAGATA Y, OHTSUBO Y, TSUDA M.Properties and biotechnological applications of natural and engineered haloalkane dehalogenases[J].Applied Microbiology and Biotechnology, 2015, 99(23):9 865-9 881.
[11] 赵渊中, 钟近艺, 郭楠.芥子气酶法洗消及烷基卤脱卤酶分子改造进展[J].环境科学与技术, 2017, 40(2):83-90.
ZHAO Y Z, ZHONG J Y, GUO N.Recent progress in enzymatic decontamination of sulfur mustard and HLDs modifications by protein engineering[J].Environmental Science & Technology, 2017, 40(2):83-90.
[12] NEWMAN J, PEAT T S, RICHARD R, et al.Haloalkane dehalogenases:Structure of a Rhodococcus enzyme[J].Biochemistry, 1999, 38(49):16 105-16 114.
[13] 赵渊中, 钟近艺, 郭楠, 等.多点突变提高DhaA对芥子气的活性和热稳定性[J].应用与环境生物学报, 2017, 23(4):714-718.
ZHAO Y Z, ZHONG J Y, GUO N, et al.Improvement in the thermostability and activity of DhaA against sulfur mustard by multipoint mutagenesis[J].Chinese Journal of Applied and Environmental Biology, 2017, 23(4):714-718.
[14] KOUDELAKOVA T, CHALOUPKOVA R, BREZOVSKY J, et al.Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel[J].Angewandte Chemie International Edition, 2013, 52(7):1 959-1 963.
[15] BEDNAR D, BEERENS K, SEBESTOVA E, et al.FireProt:energy- and evolution-based computational design of thermostable multiple-point mutants[J].PLoS Computational Biology, 2015, 11(11):e1004556.
[16] ZHAO Y Z, YU W L, ZHENG H, et al.PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA[J].Journal of Biotechnology, 2017, 254:25-33.
[17] 郑禾. 基于介孔泡沫吸附的DhaA稳定化方法与机理研究[D].北京:军事科学院, 2019.
ZHENG H.Study on the stabilization method and mechanism of DhaA adsorbed in mesocellular foam[D].Beijing:Academy of Military Sciences, 2019.
[18] WANG F L, SONG T Y, JIANG H, et al.Bacillus subtilis spore surface display of haloalkane dehalogenase DhaA[J].Current Microbiology, 2019, 76(10):1 161-1 167.
[19] 李安章. 食烷菌属中的卤代烷烃脱卤酶的生化鉴定和系统发育分析[D].厦门:厦门大学, 2013.
LI A Z.Biochemical identification and phylogenetic analysis of haloalkane dehalogenases in Alcanivorax[D].Xiamen:Xiamen University, 2013.
[20] 赵金星, 张梁, 顾正华, 等.大肠杆菌重组表达磷脂酶C的发酵工艺优化[J].食品科学, 2013, 34(9):143-149.
ZHAO J X, ZHANG L, GU Z H, et al.Optimization of fermentation process for recombinant Escherichia coli expression of phospholipase C[J].Food Science, 2013, 34(9):143-149.
[21] SHI X J, XIE J C, LIAO S Y, et al.High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid[J].Bioresource Technology, 2017, 241:795-801.
[22] HARRISON J S, KESHAVARZ-MOORE E, DUNNILL P, et al.Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli[J].Biotechnology and Bioengineering, 1997, 53(6):611-622.
[23] 吕欣, 段作营, 毛忠贵.氮源与无机盐对高浓度酒精发酵的影响[J].西北农林科技大学学报(自然科学版), 2003, 31(4):159-162.
LYU X, DUAN Z Y, MAO Z G.Study on the effects of nitrogen sources and inorganic salts on ethanol fermentation[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry, 2003, 31(4):159-162.
[24] 李建华, 李啸, 张娅, 等.金属离子对卤醇脱卤酶发酵生产的影响[J].中国酿造, 2012, 31(3):127-131.
LI J H, LI X, ZHANG Y, et al.Effects of metal ions on production of the halogen alcohol dehalogenase[J].China Brewing, 2012, 31(3):127-131.
[25] DUAN X G, HU S B, QI X H, et al.Optimal extracellular production of recombinant Bacillus circulans β-galactosidase in Escherichia coli BL21(DE3)[J].Process Biochemistry, 2017, 53:17-24.
[26] 胡耀辉, 王丹, 朴春红, 等.重组麦芽糖转葡萄糖基酶工程菌发酵条件的优化[J].食品科学, 2011, 32(19):141-146.
HU Y H, WANG D, PIAO C H, et al.Optimization of fermentation conditions for genetically engineered recombinant bacterium with amylomaltase gene[J].Food Science, 2011, 32(19):141-146.
[27] WANG H L, LI X M, MA Y H, et al.Process optimization of high-level extracellular production of alkaline pectate lyase in recombinant Escherichia coli BL21 (DE3)[J].Biochemical Engineering Journal, 2015, 93:38-46.
[28] ZOU C, DUAN X G, WU J.Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy[J].Bioresource Technology, 2014, 172:174-179.
[29] 谢颂洋, 顾正华, 郭自涛, 等.重组表达D-泛解酸内酯水解酶乳酸克鲁维酵母的发酵工艺优化[J].食品与发酵工业, 2021, 47(23):38-45.
XIE S Y, GU Z H, GUO Z T, et al.Optimized fermentation condition for production of D-lactonohydrolase in a Kluyveromyces lactis recombinant[J].Food and Fermentation Industries, 2021, 47(23):38-45.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司