研究报告

大肠杆菌分泌表达裂解性多糖单加氧酶发酵条件的优化

  • 郭宵 ,
  • 安亚静 ,
  • 柴成程 ,
  • 路福平 ,
  • 刘夫锋
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  • (工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,工业酶国家工程实验室,天津科技大学 生物工程学院,天津,300457)
博士研究生(刘夫锋教授为通讯作者,E-mail:fufengliu@tust.edu.cn)

收稿日期: 2019-10-08

  网络出版日期: 2020-04-10

基金资助

国家重点研发专项(2018YFA0901700)

Fermentation condition optimization of recombinant lytic polysaccharidemonooxygenase extracellularly expressed in Escherichia coli

  • GUO Xiao ,
  • AN Yajing ,
  • CHAI Chengcheng ,
  • LU Fuping ,
  • LIU Fufeng
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  • (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education; Tianjin KeyLaboratory of Industrial Microbiology; National Engineering Laboratory for Industrial Enzymes;College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)

Received date: 2019-10-08

  Online published: 2020-04-10

摘要

裂解性多糖单加氧酶(Lytic polysaccharide monooxygenases, LPMOs)是一种铜离子依赖型的氧化酶,可以降解生物质中的顽固多糖。因此,LPMOs在木质纤维素转化为生物燃料或其他有价值的寡糖方面得到了广泛的研究。该研究对来源嗜热毁丝霉的MtC1LPMO在大肠杆菌中进行了异源表达,首先研究了不同信号肽对重组蛋白表达的影响,结果表明PelB信号肽可以成功将MtC1LPMO分泌至细胞外,提高了MtC1LPMO的总可溶性蛋白含量。为进一步提高MtC1LPMO胞外表达量,对诱导时期、诱导剂浓度、诱导温度、发酵时间和乙醇浓度进行了优化。实验结果表明,在培养基OD600值达到0.9时,添加终浓度为0.5 mmol/L的IPTG和终体积分数为2%的乙醇,然后于30 ℃发酵16 h后重组MtC1LPMO的表达量最高,可溶性蛋白总含量为12.65 mg/L,为优化前的2.05倍。其中细胞外蛋白含量也达到了最高,为8.51 mg/L,细胞外比率为67.30%,以2,6-二甲氧基苯酚为底物在标准条件下测定的比活力为10.3 U/g。

本文引用格式

郭宵 , 安亚静 , 柴成程 , 路福平 , 刘夫锋 . 大肠杆菌分泌表达裂解性多糖单加氧酶发酵条件的优化[J]. 食品与发酵工业, 2020 , 46(5) : 31 -37 . DOI: 10.13995/j.cnki.11-1802/ts.022822

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent oxidases that are involved in the degradation of recalcitrant polysaccharides in biomass. Consequently, LPMOs have been widely used in the production of fuels and other value-added oligosaccharides from lignocellulose. In this study, a gene encoding LPMO from Myceliophthora thermophila C1 (MtC1LPMO) was expressed in Escherichia coli BL21(DE3). Firstly, the effect of signal peptides on the expression level of MtC1LPMO was studied. It was found that the fused MtC1LPMO with PelB signal peptide was secreted into the culture medium, increasing greatly the total content of soluble MtC1LPMO. To further improve the expression level of MtC1LPMO, the effects of induction time, inducer concentration, ethanol stress, induction incubation time and temperature on the productivity of the recombinant MtC1LPMO in E. coli BL21 were investigated. The highest yield of soluble MtC1LPMO was obtained by adding 0.5 mmol/L IPTG and 2% ethanol at a final concentration to the medium when OD600 reached 0.9 and further incubated at 30 ℃ for 16 h. The protein concentration of total soluble MtC1LPMO was 12.65 mg/L, which was 2.05 times of that before optimization. The extracellular ratio was 67.30% with the extracellular concentration of 8.51 mg/L. The specific activity of MtC1LPMO was 10.3 U/g using 2,6-dimethoxyphenol as the substrate at the standard condition.

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