The Stimulating Factor Inducing the Production of Paracin1.7: Separation and Purification

doi:10.11924/j.issn.1000-6850.casb2020-0150

Abstract

Abstract:

The purpose is to separate and purify the stimulating factors in Bacillus subtilis that can induce the secretion of bacteriocin Paracin1.7, and to explore its influence on the production of Lactobacillus paracasei HD1.7 bacteriocin and the expression of quorum sensing related gene luxS. After 60% ammonium sulfate precipitation, moderate purification by CM Sepharose Fast Flow weak cation-exchange chromatography and accurate purification by Superdex 75 gel filtration, the chromatography isolates were obtained, and they were co-cultured with Lactobacillus paracasei HD1.7. The antimicrobial activity of the co-culture fermentation broth and the transcription level of luxS gene were detected. The SDS-PAGE and Native-PAGE were used to detect the isolated substances. The results showed that the antimicrobial activity of Lactobacillus paracasei HD1.7 was 126.68%. The expression of luxS gene was up-regulated, which was 2.43 folds of that of the original strain. The apparent relative molecular weight of the stimulating factor was about 30 kDa. In this study, the stimulating factor that induces the production of Paracin 1.7 was preliminarily separated and purified by the three-step method, and it was confirmed that the stimulating factor could activate the interspecies quorum sensing related gene luxS. The study could lay a theoretical and technical foundation for further exploration of the quorum sensing of Lactobacillus paracasei HD1.7.

Key words: Bacillus subtilis, co-culture, bacteriocin, stimulating factor, separation and purification

CLC Number:

S182

Tian Tian, Sun Yanyang, Kang Jie, Song Gang, Gao Dongni, Ge Jingping. The Stimulating Factor Inducing the Production of Paracin1.7: Separation and Purification[J]. Chinese Agricultural Science Bulletin, 2020, 36(32): 17-22.

Figures/Tables 6

References 27

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引物名称	引物序列(5′→3′)
luxS-up	ATGGCAAAAGTTGAAAGTTTCAC
luxS-down	CTAGACAACCTGACGCGTGTACG
ldh-up	GACACATAAGAAAGGATG
ldh-down	TACTGACGAGTTTCGATGTC

引物名称	引物序列(5′→3′)
luxS-up	ATGGCAAAAGTTGAAAGTTTCAC
luxS-down	CTAGACAACCTGACGCGTGTACG
ldh-up	GACACATAAGAAAGGATG
ldh-down	TACTGACGAGTTTCGATGTC

样品	体积/mL	总蛋白/mg	总活力/AU	比活力/(AU/mg)	纯化倍数	得率/%
无细胞浓缩发酵上清液	2000	8371.84	132291.50	15.80	1	100.00
硫酸铵沉淀法粗提	200	679.37	104200.50	153.37	9.71	78.76
CM Sepharose FF	10	37.76	46850.00	1240.73	78.53	35.41
Superdex 75	5	2.74	18123.00	6641.23	420.33	13.70

样品	体积/mL	总蛋白/mg	总活力/AU	比活力/(AU/mg)	纯化倍数	得率/%
无细胞浓缩发酵上清液	2000	8371.84	132291.50	15.80	1	100.00
硫酸铵沉淀法粗提	200	679.37	104200.50	153.37	9.71	78.76
CM Sepharose FF	10	37.76	46850.00	1240.73	78.53	35.41
Superdex 75	5	2.74	18123.00	6641.23	420.33	13.70