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

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(Ig G)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。

Endo S(EC 3.2.1.96)is aβ-N-acetylglucosaminidases from Streptococcus pyogenes,which can specifically hydrolyze the Fc N-glycan of immunoglobulin G(Ig G).In this study,recombinant plasmid BL21(DE3)/pET28 a-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 p H 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 at20℃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.