食品与发酵工业 >

2022 , Vol. 48 >Issue 12: 138 - 144

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.030830

研究报告

链霉菌S-pMS02发酵制备磷脂酶D的关键技术研究

  • 王鋆坦 ,
  • 朱海华 ,
  • 张梦雪 ,
  • 郭碧珊 ,
  • 许君 ,
  • 刘红伟 ,
  • 王法云 ,
  • 王慧
展开
  • 1(河南省商业科学研究所有限责任公司,河南 郑州,450002)
    2(河南省食品质量安全控制工程技术研究中心,河南 郑州,450002)
    3(河南农业大学 生命科学学院,河南 郑州,450002)
    4(上海交通大学 公共卫生学院,上海,200025)
第一作者:双学士、工程师(朱海华高级工程师和王慧教授为共同通信作者,E-mail:94672834@qq.com;huiwang@shsmu.edu.cn)

收稿日期: 2022-01-15

  修回日期: 2022-03-02

  网络出版日期: 2022-07-15

基金资助

中央引导地方科技发展专项(YDZX20204100004126);河南省科学院科研开发基本科研费(200611115;220611032);河南省省级特聘研究员项目(200511003)

收起

Key technology of producing phospholipase D by fermentation of Streptomyces S-pMS02

  • WANG Juntan ,
  • ZHU Haihua ,
  • ZHANG Mengxue ,
  • GUO Bishan ,
  • XU Jun ,
  • LIU Hongwei ,
  • WANG Fayun ,
  • WANG Hui
Expand
  • 1(Henan Commercial Science Research Institute Co.Ltd., Zhengzhou 450002,China)
    2(Food Quality and Safety Control Engineering Technology Research Center of Henan Province, Zhengzhou 450002,China)
    3(School of Life Science, Henan Agricultural University, Zhengzhou 450002,China)
    4(School of Public Health, Shanghai Jiao Tong University, Shanghai 200025,China)

Received date: 2022-01-15

  Revised date: 2022-03-02

  Online published: 2022-07-15

Fold

摘要

实验室前期构建了重组链霉菌菌株S-pMS02用于高效分泌表达磷脂酶D(phospholipase D, PLD),但是其PLD产量依然难以满足大规模生产需求。为了进一步提高PLD产量,该研究对S-pMS02的最佳产酶条件进行了探索,并改进了制备工艺。结果显示,该菌株最适发酵产酶碳源为可溶性淀粉、氮源为硫酸铵酪蛋白氨基酸复合物,此外1.5 g/L的Mg2+能显著提高PLD的产量。在接种孢子浓度为1×105个/mL,发酵温度为32 ℃,发酵6 d时PLD酶活力达到最高,为25.56 U/mL。通过在发酵制备过程中使用剪切搅散和保温装置,进一步将PLD产量提升至33 U/mL,为最初的2倍。该研究的结果为促进PLD的生产以及工业化应用奠定了基础。

关键词： 磷脂酶D; 重组链霉菌; 发酵优化; 单因素试验

本文引用格式

王鋆坦 , 朱海华 , 张梦雪 , 郭碧珊 , 许君 , 刘红伟 , 王法云 , 王慧 . 链霉菌S-pMS02发酵制备磷脂酶D的关键技术研究[J]. 食品与发酵工业, 2022 , 48(12) : 138 -144 . DOI: 10.13995/j.cnki.11-1802/ts.030830

Abstract

Recombinant strain Streptomyces S-pMS02 was constructed in previous study for efficient secretion and expression of phospholipase D (PLD). However, the yield of PLD is still insufficient, which limits its large-scale production. In order to improve the yield of PLD, the fermentation conditions of recombinant S-pMS02 were investigated, also, the preparation process was improved. By single-factor experiments, the carbon source and nitrogen source for PLD production were optimized, which were soluble starch and the combination of ammonium sulfate/casein amino acid, respectively. Furthermore, the addition of Mg2+(1.5 g/L) significantly improved the yield of PLD (by 40%) in fermentation broth. The optimum inoculative concentration and fermentation temperature was 1×105 spores/mL and 32 ℃, respectively, and a maximum PLD activity of 25.56 U/mL was detected after 6 days. Moreover, the yield of PLD was further improved to 33 U/mL(2-fold versus initial)by using shear agitation and thermal insulation device in the preparation process. The preliminary results obtained in this work are expected to promote the large-scale industrial production of PLD.

Key words： phospholipase D; recombinant Streptomyces; optimization of fermentation conditions; single-factor experiments

参考文献

[1] PARK J J, CHOI Y, JUNG E, et al.Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses[J].The EMBO Journal, 2021, 40(15):e107121.
[2] BRUTON A, NAUMAN J, HANES D, et al.Phosphatidylserine for the treatment of pediatric attention-deficit/hyperactivity disorder:A systematic review and meta-analysis[J].The Journal of Alternative and Complementary Medicine, 2021, 27(4):312-322.
[3] MOZZI R, BURATTA S, GORACCI G.Metabolism and functions of phosphatidylserine in mammalian brain[J].Neurochemical Research, 2003, 28(2):195-214.
[4] BIRGE R B, BOELTZ S, KUMAR S, et al.Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer[J].Cell Death and Differentiation, 2016, 23(6):962-978.
[5] HIRAYAMA S, TERASAWA K, RABELER R, et al.The effect of phosphatidylserine administration on memory and symptoms of attention-deficit hyperactivity disorder:A randomised, double-blind, placebo-controlled clinical trial[J].Journal of Human Nutrition and Dietetics, 2013, 27:284-291.
[6] HUANG T T, LYU X Q, LI J H, et al.Combinatorial fine-tuning of phospholipase D expression by Bacillus subtilis WB600 for the production of phosphatidylserine[J].Journal of Microbiology and Biotechnology, 2018, 28(12):2 046-2 056.
[7] 徐银凤, 赵雨, 孙迪, 等.蜡状芽孢杆菌磷脂酶D的活性形式及HKD活性区域的分子改造对其活性的影响[J].中国生物化学与分子生物学报, 2017, 33(11):1 161-1 167.
XU Y F, ZHAO Y, SUN D, et al.The active form of phospholipase D from Bacillus cereus and the effect of molecular modification of HKD active region on its activity[J].Chinese Journal of Biochemistry and Molecular Biology, 2017, 33(11):1 161-1 167.
[8] LIU Y H, HUANG L, FU Y, et al.A novel process for phosphatidylserine production using a Pichia pastoris whole-cell biocatalyst with overexpression of phospholipase D from Streptomyces halstedii in a purely aqueous system[J].Food Chemistry, 2019, 274:535-542.
[9] CHEN S, XU L, LI Y, et al.Bioconversion of phosphatidylserine by phospholipase D from Streptomyces racemochromogenes in a microaqueous water-immiscible organic solvent[J].Bioscience, Biotechnology, and Biochemistry, 2013,77(9):1 939-1 941.
[10] 代书玲, 张江, 商军.磷脂酶D高效产生菌的筛选及鉴定[J].江苏农业科学, 2013, 41(3):309-311.
DAI S L, ZHANG J, SHANG J.Screening and identification of the high phospholipase D-producing strains[J].Jiangsu Agricultural Sciences, 2013, 41(3):309-311.
[11] 朱海华, 王鋆坦, 王小瑞, 等.一种在链霉菌中高效表达磷脂酶D的方法及重组链霉菌:中国, CN112575023A[P].2021-03-30.
ZHU H H, WANG J T, WANG X R, et al.Method for high expression of phospholipase D in Streptomyces and recombinant Streptomyces:China, CN112575023A[P].2021-03-30.
[12] 黄琳, 马杰莹, 王爽, 等.Streptomyces septatus磷脂酶D在毕赤酵母中的重组表达及酶学性质分析[J].食品科学, 2020, 41(8):100-107.
HUANG L, MA J Y, WANG S, et al.Recombinant expression of phospholipase D from Streptomyces septatus in Pichia pastoris and analysis of its enzymatic properties[J].Food Science, 2020, 41(8):100-107.
[13] 梁丽敏, 刘春凤, 杜阳吉.链霉菌产磷脂酶D发酵培养基的优化[J].中国食品添加剂, 2014(9):114-120.
LIANG L M, LIU C F, DU Y J.Optimization of cultural medium for phospholipase D production by Streptomyces sp.[J].China Food Additives, 2014(9):114-120.
[14] 朱海华, 王鋆坦, 刘红伟, 等.一种利用基因工程链霉菌发酵获取磷脂酶D的制备方法:中国, CN112831446A[P].2021-05-25.
ZHU H H, WANG J T, LIU H W, et al.A preparation method for obtaining phospholipase D by fermentation of genetically engineered Streptomyces:China, CN112831446A[P].2021-05-25.
[15] 赵紫薇, 杨天奎, 牟英.色褐链霉菌产磷脂酶D的发酵条件研究[J].中国油脂, 2010, 35(11):52-57.
ZHAO Z W, YANG T K, MU Y.Fermentation conditions of phospholipase D production by Streptomyces chromofuscus[J].China Oils and Fats, 2010, 35(11):52-57.
[16] 韩海霞, 曹栋, 史苏佳, 等.链霉菌CA-1产磷脂酶D发酵条件的优化[J].食品工业科技, 2014, 35(13):176-180.
HAN H X, CAO D, SHI S J, et al.Optimization of fermentation conditions of phospholipase D from Streptomyces sp.CA-1[J].Science and Technology of Food Industry, 2014, 35(13):176-180.
[17] 相启森, 岳田利, 袁亚宏, 等.金属离子对褐黄孢链霉菌生长和纳他霉素生物合成的影响[J].西北农林科技大学学报(自然科学版), 2010, 38(1):209-215.
XIANG Q S, YUE T L, YUAN Y H, et al.Effects of metal ions on growth of Streptomyces gilvosporeus and biosynthesis of natamycin [J].Journal of Northwest A & F University (Natural Science Edition), 2010, 38(1):209-215.
[18] 钱娟, 庞洋, 王昕.磷脂酶D的制备及催化磷脂酰胆碱合成磷脂酰丝氨酸[J].中国油脂, 2017, 42(9):66-71.
QIAN J, PANG Y, WANG X.Preparation of phospholipase D and catalytic synthesis of phosphatidylserine from phosphatidylcholine[J].China Oils and Fats, 2017, 42(9):66-71.
[19] OGINO C, YASUDA Y, KONDO A, et al.Improvement of transphosphatidylation reaction model of phospholipase D from Streptovericilium cinnamoneum [J].Biochemical Engineering Journal, 2002, 10(2):115-121.
[20] 张莹. 在不同宿主中表达链霉菌磷脂酶D的研究[D].上海:华东理工大学, 2013.
ZHANG Y.Phospholipase D safe and efficient expression in different hosts[D].Shanghai:East China University of Science and Technology, 2013.
[21] 张业尼, 路福平, 李玉, 等.磷脂酰丝氨酸合成酶基因的克隆及在枯草芽孢杆菌中的表达[J].中国生物工程杂志, 2008,28(9):56-60.
ZHANG Y N, LU F P, LI Y, et al.Cloning of the phosphatidylserine synthase gene and its secretive expression in Bacillus subtilis[J].China Biotechnology, 2008, 28(9):56-60.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司