琼胶酶AgaD在大肠杆菌中的高效表达

High expression of agarase AgaD in Escherichia coli

摘要:

摘要:【目的】构建琼胶酶AgaD的高效表达体系，优化发酵条件提高重组酶的表达量。【方法】首先根据大肠杆菌(E.coli)密码子偏好性，优化并合成AgaD的基因，使其适合E.coli表达系统;考察了不同的E.coli表达宿主;根据N端法则构建了突变体;评价了培养基中添加CaCl2和甘氨酸(Gly)对重组酶表达的影响。【结果】成功构建了琼胶酶AgaD 的高效表达体系，确定了E.coli AD494(DE3)为最适表达宿主;利用N端法则提高了重组酶的稳定性，缩短了发酵时间;通过在培养基中添加CaCl2和甘氨酸(Gly)进一步提高了胞外酶产量。最终，发酵上清中重组酶的活力由20 U/L提高至11300 U/L，比优化前提高了500余倍。【结论】构建了琼胶酶AgaD的高效表达体系，为GH96家族琼胶酶的深入研究奠定了基础。

Abstract:

Abstract:［Objective］ We constructed highly efficient expression systems for agarase AgaD and optimized its culture conditions．［Methods］First，the codon usage of AgaD was optimized to make it suitable for expression in E.coli． Then，the gene expression vector was transformed into different E．coli hosts． According to the“N-end rule”that is related to the in vivo half-life of a protein，a mutant was constructed． Finally，the effects of CaCl2 and glycine on enzyme production were evaluated．［Results］A highly efficient expression system of agarase AgaD was constructed，named pET-22b(+)-optagaDx-AD494 (DE3)．Replacing N-terminal second amino acid phenylalanine with alanine significantly improved agarase production and shortened the fermentation period．The extracellular enzyme activity was further up-regulated by CaCl2 and glycine． After optimization，the extracellular enzyme production raised from 20 U/L to 11300 U/L，more than 500 folds.［Conclusion］The high expression system of AgaD provides good basis for further studying agarases.