信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响

Effects of different signal peptides and chemical penetrators on extracellular production of recombinant cyclodextrin glycosyltransferase

摘要:

【目的】研究不同的信号肽和化学通透剂对重组环糊精葡萄糖基转移酶(CGTase)胞外分泌的影响，提高CGTase的胞外分泌量。【方法】扩增地芽孢杆菌CHB1 (Geobacillus sp. CHB1)的CGTase基因，构建带有地芽孢杆菌CHB1自身信号肽、OmpA、PelB信号肽和不带信号肽的4种重组质粒；比较4种重组质粒对重组CGTase胞外分泌的影响，筛选最优的信号肽；考察甘氨酸、Triton X-100、SDS和Tween 80四种化学通透剂对重组CGTase胞外分泌的影响，确定最佳的化学通透剂及其浓度。【结果】OmpA信号肽介导的分泌效果最好，胞外酶活达到7.44 U/mL，分别是PelB、CHB1信号肽的2.04倍和11.27倍，不带信号肽的重组质粒菌胞外检测不到酶活；携带OmpA信号肽的重组质粒菌发酵48 h，同时添加浓度为0.6%的甘氨酸和0.3%的Triton X-100，胞外酶活达最大到14.27 U/mL；SDS和Tween 80对该酶的胞外分泌具有明显的抑制作用。【结论】OmpA信号肽的介导效果最佳，同时添加浓度为0.6%和0.3%的甘氨酸和Triton X-100可以有效促进胞外分泌，为该重组酶的高效胞外分泌提供了一种有效的方法。

Abstract:

[Objective] Effects of signal peptides and chemical penetrators on extracellular secretion of cyclodextrin glycosytransferase (CGTase) in recombinant Escherichia coli strain BL21(DE3) was observed in this study in order to lay foundation for fermentation technology of CGTase. [Methods] The CGTase gene was cloned by PCR with genomic DNA of Geobacillus sp. CHB1 as template. Four recombinant plasmids were constructed which included the signal peptide of Geobacillus sp. CHB1, OmpA, PelB and no signal peptide. The recombinant plasmids were respectively transformed into E. coli BL21(DE3) and then induced to express. The extracellular enzyme activities of the target proteins were analyzed and measured to screen the best signal peptide. Chemical penetrators like glycine, SDS, Triton X-100 and Tween 80 were added into the culture to determine the effect of chemical additives on extracellular secretion of recombinant CGTase. [Results] The results showed that these four recombinant plasmids could successfully express CGTase in E. coli BL21(DE3) and OmpA was the optimal signal peptide. Extracellular enzyme activity induced by OmpA could reach 7.44 U/mL which were 2.04- and 11.27-folds of PelB and CHB1, respectively. No extracellular CGTase activity was detected in E. coli BL21(DE3) with the no signal peptide. Extracellular enzyme activities induced by OmpA were 9.27 U/mL and 9.75 U/mL by addition of 0.6% glycine and 0.3% Triton X-100, respectively, and it could reach 14.27 U/mL by synergistic action of glycine and Triton X-100 after 48 h. However, SDS and Tween 80 had obvious inhibition on extracellular CGTase activity. [Conclusion] The extracellular CGTase activity in E. coli BL21(DE3) induced by OmpA could reach the highest by addition of 0.6% glycine and 0.3% Triton X-100.