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文章摘要
β-N-乙酰葡萄糖苷内切酶Endo S异源表达及发酵优化<sub>*</sub>
Heterologous Expression and Fermentation Optimization of Endo Sfrom Streptococcus pyogenes
投稿时间:2018-06-28  修订日期:2018-09-28
DOI:
中文关键词: β-N-乙酰葡萄糖苷内切酶endo S  重组大肠杆菌  异源表达  发酵优化
英文关键词: endo  S, recombinant  E.coli, heterologous  expression, fermentation  optimization
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
杨敏 糖化学与生物技术教育部重点实验室生物工程学院江南大学 1033231434@qq.com 
赵恺 江苏省无锡市江南大学生物工程学院  
濮晶晶 糖化学与生物技术教育部重点实验室  
洪皓飞 糖化学与生物技术教育部重点实验室  
赵鑫锐 糖化学与生物技术教育部重点实验室  
吴志猛 糖化学与生物技术教育部重点实验室 zwu@jiangnan.edu.cn 
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中文摘要:
      Endo S(EC 3.2.1.96)是一种来源于化脓链球菌的β-N-乙酰葡萄糖苷内切酶,可以特异性水解免疫球蛋白G(IgG)可结晶区(Fc片段)N糖链。为实现其高效表达,本研究构建endo S重组表达大肠杆菌E.coli BL21(DE3)/pET28a-endo S,并验证表达蛋白糖苷水解活性,在摇瓶水平单因素优化蛋白表达,考察了基础培养基种类、碳源、氮源、无机盐、培养基初始pH、发酵温度和时间等因素对产酶的影响,确定最优发酵培养基组成为(g/L):酵母粉10.0,甘油4.0,牛肉浸膏25.4,NaCl 5.0,pH 8.0,最优培养条件为:20oC诱导表达24 h,蛋白表达量为225 mg/L发酵液,是优化前的4.5倍,同时也是目前报道最高表达量的5.6倍。
英文摘要:
      Endo S (EC 3.2.1.96) is a β -N-acetylglucoside endonuclease from Streptococcus pyogenes, which can specifically hydrolyze the N-sugar chain of immunoglobulin G (IgG) crystallizable region (FC fragment). To achieve high efficient expression of Endo S, a heterologous expression strain E.coli BL21(DE3)/pET28a-endo S was constructed and the glycoside hydrolase activity of protein expressed was conformed. Single-factor optimization of Endo S fermentation medium components and conditions was performed at shake flask level,including screening of basal mediums,nitrogen sources,carbon sources,mineral salts,expression temperature and time,initial pH of cultural medium and so on. The optimized medium is as following (g/L): beef extract 25.4, yeast powder 10.0, NaCl 5.0, glycerol 4.0, pH 8.0. Protein expression is induced by 0.25 mM IPTG at 20°C for 24 h with a final level of 225 mg/L fermentation broth, which increases 3.5-fold compared to that before optimization and it is 5.6 times higher than the highest yield reported.
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