分子伴侣共表达对嗜热环糊精葡萄糖基转移酶异源可溶性表达的影响

Effects of chaperone co-expression on heterologous solubility expression of thermophilic cyclodextrin glucosetransferase

摘要:

【目的】通过优化表达条件，提高嗜热环糊精葡萄糖基转移酶(CGTase)的可溶性表达和胞外酶活性。【方法】构建含cgt基因的重组表达质粒pET-28a(+)-ompA-cgt，筛选最适诱导温度，并构建5种分子伴侣共表达系统(pKJE8、pKJE7、pGro7、pTf16和pG-Tf2，5种分子伴侣质粒分别与重组表达质粒pET-28a(+)-ompA-cgt共表达)，筛选最适分子伴侣质粒，优化共表达条件。【结果】通过SDS-PAGE分析和测定胞外酶活，CGTase基因在大肠杆菌中实现表达，且具有一定量的重组CGTase分泌至胞外；25 °C诱导时CGTase的可溶性表达和在胞外上清中的酶活都最高；分子伴侣质粒pKJE8使酶的胞外活性提高了48.6%，效果最为显著；当L-阿拉伯糖浓度为0.5 g/L时，分子伴侣质粒pKJE8使酶的胞外活性提高了68.5%。【结论】通过优化表达条件及使用分子伴侣共表达系统提高了环糊精葡萄糖基转移酶的可溶性表达和胞外酶活，为该酶进一步相关研究奠定了基础。

Abstract:

[Objective] To improve the soluble expression level and enzyme activity of CGTase in prokaryotic expression system, the expression conditions were optimized. [Methods] The cgt gene was cloned from Geobacillus sp. B1 and cloned into expression vector pET-28a(+). The optimum induction temperature was selected by enzyme activity assay and SDS-PAGE. The molecular chaperone co-expression system was constructed and the optimum molecular chaperone vector was screened. [Results] The results was described as follows: the cgt gene was amplified and cloned into vector pET-28a(+) successfully, the enzyme activity and soluble expression level of CGTase was highest when induced at 25 °C; in the molecular chaperone co-expression system, the five vectors containing different molecular chaperones (pKJE8, pKJE7, pGro7, pTf16 and pG-Tf2) improved and the enzyme activity and soluble expression level of CGTase in varying degrees when co-expression with recombinant plasmid pET-28a(+)-ompA-cgt, and pKJE8 was confimed to contain the optimum molecular chaperones combination and the enzyme activity of CGTase improved 48.6%. When L-Arabinose concentration of 0.5 g/L, molecular chaperone plasmid pKJE8 made extracellular enzyme activity increased 68.5%. [Conclusion] These results can provide a potential value for further studies of CGTase.