• 站内检索
    高级检索
  • 最新录用
    更多
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
Heterologous Expression and Fermentation Optimization of Endo S from -Streptococcus pyogenes
  
DOI:10.3969/j.issn.1673-1689.2020.05.007
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:
作者单位
杨敏 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
赵恺 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
濮晶晶 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
洪皓飞 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
赵鑫锐 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
吴志猛 江南大学 糖化学与生物技术教育部重点实验室江苏 无锡 214122 
摘要点击次数: 74
全文下载次数: 54
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质表达,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:20 ℃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-acetylglucosaminidases from Streptococcus pyogenes, which can specifically hydrolyze the Fc N-glycan of immunoglobulin G(IgG). In this study, recombinant plasmid BL21(DE3)/pET28a-endo S was constructed and the protein was successfully expressed in E. coli BL21(DE3) cells with confirmed enzymatic activity. Then, single-factor optimization, including screening of basal mediums, nitrogen and carbon sources, mineral salts, expression temperature and time, and initial pH of cultural medium, was performed at shake flask level to express the Endo S in a high level. Under the optimized medium(beef extract 25.4 g/L, yeast powder 10.0 g/L, NaCl 5.0 g/L, glycerol 4.0 g/L, pH 8.0)and condition(induced by 0.25 mM IPTG at 20 ℃ for 24 h), the yield of recombinant enzyme was 225 mg/L after purification by Ni-resin affinity chromatography, which was 3.5 times higher than that before optimization and 5.6 fold higher than the highest yield reported.
查看全文   查看/发表评论  下载PDF阅读器