戊糖乳杆菌群体感应信号肽AIP的纯化及鉴定

Purification and Identification of Quorum Sensing Signal Peptide AIP of Lactobacillus pentosus

目的:对戊糖乳杆菌31-1的群体感应信号肽进行提取、纯化和鉴定。方法:经硫酸铵盐析、超滤、疏水层析、凝胶层析和RP-HPLC步骤纯化戊糖乳杆菌31-1的群体感应信号肽。用MALDI-TOF质谱测定纯化后的小肽AIP的分子质量,并用EDMAN降解法对其N端氨基酸测序。用组氨酸蛋白激酶抑制剂——氯氰碘柳胺验证其群体感应信号肽分子功能。结果:纯化的信号肽AIP的比活力达160 000 IU/m L,纯度提高了800倍。该小肽的分子质量为2 985.16 u。其N端前15个氨基酸序列为NH2-KSSAYSLQMGATAIK。氯氰碘柳胺所造成的戊糖乳杆菌31-1细菌素合成功能的缺失,不能通过添加小肽AIP而得以恢复,证实了小肽AIP的群体感应信号肽分子功能。结论:群体感应信号肽AIP的获得为戊糖乳杆菌31-1细菌素的发酵调控奠定了基础。

Objective： To purify and identify quorum sensing signal peptide of Lactobacillus pentosus 31-1. Methods： The active principle was purified to homogeneity by ammonium sulfate precipitation, ultrafilteration, hydrophobic interac- tion chromatography, gel filtration chromatography and reverse phase high-performance liquid chromatography （RP-HPLC）. MALDI-TOF mass spectrometer was used for determination molecular weight of the purified peptide AIP, and its N-ter- minal sequences was analyzed by Edman degradation. Closantel, a histidine protein kidnase inhibitor was used to verify the role of the purified peptide. Results： The purification resulted in an 800-fold increase in specific activity with a yield of 160 000 IU/mL of the induction activity. The mass of the purified peptide AIP was 2 985.16 u, its partial N-ter- minal sequence was： NH2-KSSAYSLQMGATAIK. Bacteriocin production inhibited by closantel could not be restored by peptide AlP, which further confirmed its quorum sensing signal molecule role. Conclusion： The obtaining of the quorum sensing peptide AIP laid the fundation for the regulation of bacteriocin fermentation in L. pentosus 31-1.