诱导Paracin1.7生成的刺激因子的分离与纯化

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

中国农学通报 ›› 2020, Vol. 36 ›› Issue (32): 17-22.doi: 10.11924/j.issn.1000-6850.casb2020-0150

诱导Paracin1.7生成的刺激因子的分离与纯化

田甜¹^,²(), 孙艳阳¹^,², 康杰¹^,², 宋刚¹^,², 高冬妮¹^,², 葛菁萍¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150500
²微生物黑龙江省高校重点实验室/黑龙江大学生命科学学院,哈尔滨 150080

收稿日期:2020-06-04 修回日期:2020-08-10 出版日期:2020-11-15 发布日期:2020-11-19
通讯作者: 葛菁萍
作者简介:田甜,女,1997年出生,黑龙江哈尔滨人,硕士研究生,研究方向:微生物资源挖掘与利用。通信地址:150080 黑龙江省哈尔滨市南岗区学府路74号,Tel:0451-86609016,E-mail: tiantianhlju@126.com。
基金资助:
国家自然科学基金“产细菌素乳酸菌对东北酸菜发酵体系微生物区系功能的影响及其分子机制的解析”(31470537);黑龙江省自然科学基金重点项目“乙酸代谢与副干酪乳杆菌群体感应互作探究及对细菌素产生的影响”(ZD2020C008)

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

Tian Tian¹^,²(), Sun Yanyang¹^,², Kang Jie¹^,², Song Gang¹^,², Gao Dongni¹^,², Ge Jingping¹^,²()

¹Agricultural Microorganisms Technology Education Engineering Research Center, Harbin 150500
²Key Laboratory of Microbiology of Heilongjiang Province, Life Science College, Heilongjiang University, Harbin 150080

Received:2020-06-04 Revised:2020-08-10 Online:2020-11-15 Published:2020-11-19
Contact: Ge Jingping

摘要/Abstract

摘要：

旨在分离纯化枯草芽孢杆菌(Bacillus subtilis)中能够诱导细菌素Paracin1.7分泌的刺激因子,并探明其对副干酪乳杆菌(Lactobacillus paracasei) HD1.7细菌素生成量及群体感应相关基因luxS表达的影响。本研究通过60%硫酸铵沉淀、CM Sepharose Fast Flow弱阳离子交换柱层析中度纯化及Superdex 75凝胶层析精度纯化,将获得的层析分离物与L. paracasei HD1.7共培养,检测共培养发酵液的抑菌活性及luxS基因的转录水平,并用SDS-PAGE与Native-PAGE检测分离物质。结果表明L. paracasei HD1.7抑菌活性为126.68%;luxS基因上调表达,为对照菌株的2.43倍;刺激因子的表观分子量约为30 kDa。本研究利用三步法初步分离纯化出诱导Paracin1.7生成的刺激因子,明确该刺激因子可以启动种间群体感应相关基因luxS,为L. paracasei HD1.7的群体感应研究奠定了理论和技术基础。

关键词: 枯草芽孢杆菌, 共培养, 细菌素, 刺激因子, 分离纯化

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

中图分类号:

S182

田甜, 孙艳阳, 康杰, 宋刚, 高冬妮, 葛菁萍. 诱导Paracin1.7生成的刺激因子的分离与纯化[J]. 中国农学通报, 2020, 36(32): 17-22.

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.

图/表 6

参考文献 27

[1]	葛菁萍, 平文祥, 宋刚, 等. 细菌素Paracin1.7的纯化与性质(英文)[J]. 微生物学报, 2009,49(5):610-617.
[2]	Ge J P, Sun Y Y, Xin X, et al. Purification and partial characterization of a novel bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice[J]. Scientific Reports, 2016,6(1):19366-19372.
[3]	雷虹, 李秀凉, 庄海霁, 等. 副干酪乳杆菌HD1.7产生抗菌物质的初步研究[J]. 工业微生物, 2007,37(4):16-19.
[4]	Antonio M B, Belén C G, Helena L P, et al. Induction of bacteriocin production by coculture is widespread among plantaricin-producing Lactobacillus plantarum strains with different regulatory operons[J]. Food Microbiology, 2013,33(1):40-47. URL pmid: 23122499
[5]	Jia F F, Pang X H, Zhu D Q, et al. Role of the luxS gene in bacteriocin biosynjournal by Lactobacillus plantarum KLDS1.0391: A proteomic analysis[J]. Scientific Reports, 2017,7(1):13871-13884. doi: 10.1038/s41598-017-13231-4 URL pmid: 29066774
[6]	Beatriz R B, Yolanda S, Laura N, et al. Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must[J]. Food Microbiology, 2007,24(5):482-491. doi: 10.1016/j.fm.2006.09.003 URL pmid: 17367681
[7]	Raquel T, Tomás G C, Carmen P, et al. Lactobacillus acidophilus La-5 increases lactacin B production when it senses live target bacteria[J]. International Journal of Food Microbiology, 2009,132(2-3):109-116. doi: 10.1016/j.ijfoodmicro.2009.04.004 URL pmid: 19411126
[8]	Svetoch E A, Eruslanov B V, Perelygin V V, et al. Inducer bacteria, unique signal peptides, and low-nutrient media stimulate in vitro bacteriocin production by Lactobacillus spp. and Enterococcus spp. strains[J]. Journal of Agricultural and Food Chemistry, 2010,58(10):6033-6038. doi: 10.1021/jf902802z URL pmid: 20429596
[9]	满丽莉, 孟祥晨, 王辉, 等. 群体感应系统在乳酸菌产细菌素中的作用[J]. 食品科学, 2011,32(13):360-364.
[10]	赵畅, 王宁, 王文昭, 等. 基于Lux型群体感应系统干预的生物被膜调控在污水处理中的研究进展与前景[J]. 生物工程学报, 2017,33(9):1596-1610.
[11]	Li Z, Nair S K. Quorum sensing: how bacteria can coordinate activity and synchronize their response to external signals?[J]. Protein Science, 2012,21(10):1403-1417. doi: 10.1002/pro.2132 URL pmid: 22825856
[12]	Williams P, Winzer K, Chan W C, et al. Look who's talking: communication and quorum sensing in the bacterial world[J]. Biological Sciences, 2007,362(1483):1119-1134. URL pmid: 17360280
[13]	杜艳芬, 廖璞. 微生物群体感应系统的研究进展[J]. 国际检验医学杂志, 2013,34(03):334-336.
[14]	Pereira C S, McAuley J R, Taga M E, et al. Sinorhizobium meliloti, a bacterium lacking the autoinducer-2 (AI-2) synthase, responds to AI-2 supplied by other bacteria[J]. Molecular Microbiology, 2008,70(05):1223-1235.
[15]	DeKeersmaecker S C J, Vanderleyden J. Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter[J]. Microbiology, 2003,149(08):1953-1956.
[16]	Ren Y W, Li S S, Wu Z X, et al. The influences of Bacillus subtilis on the virulence of Aeromonas hydrophila and expression of luxS gene of both bacteria under co-cultivation[J]. Current Microbiology, 2017,74(06):718-724.
[17]	Pereira C S, Thompson J A, Xavier K B. AI-2-mediated signalling in bacteria[J]. Fems Microbiology Reviews, 2013,37(02):156-181.
[18]	Marques J C, Lamosa P, Russell C, et al. Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein[J]. The Journal of Biological Chemistry, 2011,286(20):18331-18343. doi: 10.1074/jbc.M111.230227 URL pmid: 21454635
[19]	Ge J P, Fang B Z, Wang Y, et al. Bacillus subtilis enhances production of Paracin1.7, a bacteriocin produced by Lactobacillus paracasei HD1-7, isolated from Chinese fermented cabbage[J]. Annals of Microbiology, 2014,64(4):1735-1743.
[20]	果马. 枯草芽孢杆菌对副干酪乳杆菌细菌素生成和LuxS基因表达的影响. 哈尔滨:黑龙江大学, 2014: 34-36.
[21]	纪晓磊, 李昆达, 高冬妮, 等. 枯草芽胞杆菌信号分子的分离纯化[J]. 中国农学通报, 2018,34(26):42-47.
[22]	王子佳, 李红梅, 弓爱君, 等. 蛋白质分离纯化方法研究进展[J]. 化学与生物工程, 2009,26(8):8-11.
[23]	Chandra M R S, Lee Y S, Park I H, et al. Isolation, purification and characterization of a thermostable β-mannanase from Paenibacillus sp. DZ₃[J]. Journal of the Korean Society for Applied Biological Chemistry, 2011,54(3):325-331.
[24]	Lü X, Hu P, Dang Y, et al. Purification and partial characterization of a novel bacteriocin produced by Lactobacillus casei TN-2 isolated from fermented camel milk (Shubat) of Xinjiang Uygur Autonomous region, China[J]. Food Control, 2014,43(05):276-283.
[25]	Abdel-Fattah A F, Hashem A M, Ismail A M S, et al. Purification and some properties of β-mannanase from Aspergillus Oryzae NRRL 3448[J]. Journal of Applied Sciences Research, 2009,5(12):2067-2073.
[26]	Sawa N, Zendo T, Kiyofuji J, et al. Identification and characterization of lactocyclicin Q, a novel cyclic bacteriocin produced by Lactococcus sp. strain QU 12[J]. Applied and Environmental Microbiology, 2009,75(06):1552-1558.
[27]	Saloomeh M J, Kvist V F, Lene J. The quorum sensing luxS gene is induced in Lactobacillus acidophilus NCFM in response to Listeria monocytogenes[J]. International Journal of Food Microbiology, 2011,149(03):269-273.

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

诱导Paracin1.7生成的刺激因子的分离与纯化

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 27

相关文章 15

编辑推荐

Metrics

本文评价

关于本刊

作者中心

审者中心

在线期刊

联系我们

[1]	濮永瑜, 包玲凤, 何翔, 刘芮, 张庆, 施竹凤, 何永宏, 杨佩文. 烟草青枯病和黑胫病拮抗细菌的筛选、鉴定及防效研究[J]. 中国农学通报, 2022, 38(7): 116-123.
[2]	蔡永占, 王瑞宝, 白涛, 刘有才, 韩小女, 王斌, 华小兵, 李诚, 毛明林, 字文霖. 3株枯草芽孢杆菌对烟草白粉病的防效[J]. 中国农学通报, 2022, 38(25): 125-129.
[3]	房佳敏, 马成瑶, 张彦龙. 黑木耳凝集素对RAW264.7细胞免疫调节作用的研究[J]. 中国农学通报, 2021, 37(9): 125-130.
[4]	刘明, 唐佳文, 沙爽, 孟剑侠, 程丽, 李冲伟. 基于分子蒸馏技术紫苏油α-亚麻酸纯化效果的研究[J]. 中国农学通报, 2021, 37(33): 135-141.
[5]	潘月, 杨志馨, 杨晓丽, 王旭, 宋刚. 细菌素Paracin 1.7在乳酸乳球菌NZ9000中的异源表达及抑菌性质的研究[J]. 中国农学通报, 2021, 37(26): 15-23.
[6]	姜雪雍, 岳元春, 孙养存, 高冬妮, 平文祥, 葛菁萍. 副干酪乳杆菌与芽孢杆菌属共培养种间关系对产细菌素的影响[J]. 中国农学通报, 2021, 37(24): 124-132.
[7]	万璐, 闫佳佳, 郑春英. 内生真菌发酵对刺五加异嗪皮啶合成的影响[J]. 中国农学通报, 2021, 37(13): 122-126.
[8]	梁艳琼, 李锐, 吴伟怀, 谭施北, 习金根, 郑金龙, 陆英, 贺春萍, 易克贤. 基于HS-SPME-GC-MS的Bacillus subtilis Czk1挥发性物质的萃取条件优化[J]. 中国农学通报, 2021, 37(11): 24-31.
[9]	孙莹, 王海曼, 宋刚, 葛菁萍. 利用氦氖激光诱变提高枯草芽孢杆菌纤溶酶活力的研究[J]. 中国农学通报, 2020, 36(35): 28-36.
[10]	夏树立, 韩静, 王丽学, 杨春蕾, 刘长悦, 王海亮, 吴贶, 史慧玲. 一种高产L-赖氨酸枯草芽孢杆菌的构建研究[J]. 中国农学通报, 2020, 36(30): 98-105.
[11]	张心青, 张萧萧, 杨传伦, 郭南南, 马娜娜, 蔡倩倩, 王秀芝. 一株对番茄枯萎病有拮抗作用的枯草芽孢杆菌的ARTP诱变与筛选[J]. 中国农学通报, 2020, 36(26): 44-49.
[12]	张炎, 房保柱, 平文祥, 葛菁萍. 副干酪乳杆菌HD1.7高密度培养产细菌素Paracin1.7条件优化[J]. 中国农学通报, 2020, 36(24): 116-124.
[13]	邹鸿飞, 舒晓燕, 张月琴, 刘媛, 杨文玉, 赵桂英. D-101大孔树脂对香椿叶中黄酮类成分的分离纯化及抗氧化活性研究[J]. 中国农学通报, 2020, 36(24): 159-164.
[14]	刘维维, 金晓, 辛寒晓, 史庆华, 姚强, 刘丽英, 孙中涛. 强化型EM菌剂对金针菇菌糠堆肥的影响[J]. 中国农学通报, 2020, 36(22): 78-85.
[15]	许蓉,孙健,高冬妮,黄守锋,宋刚. 东北酸菜发酵过程菌群变化偶联酸菜品质的初步探究[J]. 中国农学通报, 2018, 34(33): 152-159.