摘要
【目的】有机溶剂对微生物有强烈的毒害作用致使绝大多数微生物不能在较高的有机溶剂浓度下生长。为了探究微生物的耐溶剂性机制,由野生型假单胞菌Pseudomonas putida JUCS驯化获得一株能够在60%(V/V)的环己烷中生长的菌株P.putidaJUCT1。【方法】采用蛋白质二维电泳对P.putida JUCT1在不同溶剂条件下的蛋白组分表达量的差异进行分析比对。【结果】从总共22个表达量差异均超过50%的蛋白质中,选取了3个高丰度蛋白质,通过MALDI-TOF/TOF鉴定为:3-羟基异丁酸水解酶、蛋白质延伸因子EF-Ts、异分支酸水解酶超家族(编码基因分别为mmsB、tsf、PSEEN0851)。将这3个基因在大肠杆菌中重组表达,3个蛋白均能不同程度地提高E.coli JM109的耐溶剂性,其中3-羟基异丁酸脱氢酶(编码基因mmsB)对菌株的溶剂耐受性影响最为显著。【结论】证明了运用蛋白组学的方法研究微生物的耐溶剂性的可行性,并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供理论依据。
[Objective] Most microorganisms can not grow under organic solvents of high con-centration due to their toxicity.To elucidate the mechanisms of organic-solvent-tolerant（OST） of microorganism,an OST mutant P.putida JUCT1 capable of growing in the presence of 60%（V/V） cyclohexane was obtained by gradient adaptation in cyclohexane.[Methods] Two-dimensional gel electrophoresis（2-DE） was used to compare and analyze the total cellular protein of JUCT1 when growing in the presence of 60%（V/V） cyclohexane or not.[Results] From 22 proteins whose intensity values show over 50% discrepancies under different solvent conditions,3 high abundance protein spots were identified by MALDI-TOF/TOF spectra as 3-hydroxyisobutyrate dehydrogenase,protein chain elongation factor EF-Ts,and isochorismatase superfamily hy-drolase,and their corresponding genes mmsB,tsf,and PSEEN0851 were expressed in the E.coli respectively.These the solvent tolerance of JM109 was significantly increase by these three pro-teins,particularly 3-hydroxyisobutyrate dehydrogenase.[Conclusion] In this work,proteomics analysis was proven to be an effective strategy for exploring OST mechanism of microbial cells.Importantly,this work provides molecular basis for constructing OST whole-cell catalyst for in-dustrial applications.
出处
《微生物学通报》
CAS
CSCD
北大核心
2012年第4期468-476,共9页
Microbiology China
基金
国家973计划课题(No.2011CB710800)
江苏省自然科学基金课题(No.BK2011150)
教育部新世纪优秀人才支持计划(No.NCET-11-0658)
江苏省高校优势学科建设工程资助项目
关键词
耐溶剂性
二维电泳分析
恶臭假单胞菌
菌株驯化
环己烷
Organic-solvent-tolerant（OST）
Proteomic analysis
Pseudomonas putida
Adaptation
Cyclohexane
