产絮菌的差异表达蛋白质及其功能解析研究
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摘要
生物絮凝剂作为一种高效、无毒、无二次污染、具有生物可降解性和安全性的绿色水处理剂,代表了絮凝剂的重要研发方向之一,可广泛应用于给水和废水处理领域。复合型生物絮凝剂(CBF)是本实验室开发的一种高效生物絮凝剂,经过两段式发酵生产,产絮菌群由高效产絮菌F2和F6构建,产絮菌F2、F+(F2与F6混合培养)产生的生物絮凝剂主要活性成分为胞外分泌的多糖类物质。本研究从蛋白质组学角度研究产絮菌产絮过程差异表达蛋白质及其功能调控,从分子水平研究产絮菌的产絮机理,提出工业化生产生物絮凝剂的调控策略。
首先,比较在基本培养基和产絮培养基培养条件下产絮菌所产生的生物絮凝剂在产量、成分上的差别,与基本培养基培养条件下相比,在产絮培养基培养条件下,产絮菌F2、F6会形成一些差异表达蛋白质,这些差异表达蛋白质是导致在产絮培养基培养条件下,诱导和产生大量生物絮凝剂主要活性成分多糖的直接原因。
然后,利用SDS-PAGE、2-DE和质谱结合的蛋白质组学研究手段对产絮菌F2、F6在基本培养基和产絮培养基不同培养条件下差异表达蛋白质进行分离、鉴定和功能解析。通过SDS-PAGE,研究在基本培养基和产絮培养基培养条件下,产絮菌F2、F6总蛋白的变化情况,共鉴定出58个产絮菌F2产絮过程差异表达蛋白质,14个产絮菌F6产絮过程差异表达蛋白质。通过2-DE,研究在基本培养基和产絮培养基培养条件下,产絮菌F2可溶性总蛋白的变化情况,共有28个产絮菌F2产絮过程差异表达蛋白质点得到成功鉴定。通过对产絮菌产絮过程差异表达蛋白质的功能解析,从蛋白质组学层面揭示了产絮菌的产絮机理。
最后,利用多项摇瓶试验,进行产絮菌生产生物絮凝剂的相关调控规律研究,建立起发酵条件,差异表达蛋白质的功能和产絮变化三者之间的关系,对工业上生产生物絮凝剂提出底物水平调控、发酵过程参数、菌种复壮三个层面的调控策略。
本研究从蛋白质组学层面研究产絮菌的产絮机理,为全面解析和调控产絮菌产絮代谢途径奠定坚实的理论基础。基于产絮菌产絮过程差异表达蛋白质的功能解析,提出工业化生产生物絮凝剂的调控策略,为提高生物絮凝剂产量,调控生物絮凝剂工业化生产起到重要的指导作用。
As an efficient, nontoxic, non-secondary contaminative, biodegradable and environmental-friendly green wastewater disposal agent, bioflocculant is one of the most potential flocculants and has been widely used in treating various industrial and domestic sewages. The compound bioflocculant (CBF) was produced by our lab, which was a novel bioflocculant produced by two bioflocculant producing bacteria of F2 and F6 through two-step fermentation. The effective component of bioflocculant produced by F2 and F+ (mixed cultivation from F2 and F6) was determined as extracellular polysaccharide. In the paper, differentially expressed proteins related to flocculating metabolic pathway of bioflocculant producing bacteria and regulation of their functions were researched by proteomics methods, the flocculating mechanisms were revealed in molecular level, regulating strategies of producing bioflocculant in industry were proposed.
First, the difference of production and component of bioflocculant between in bioflocculant media and in minimal media were researched. F2 and F6 expressed differentially expressed proteins in bioflocculant media compared with minimal media. These differentially expressed proteins were the direct reason to induce and produce large amount of main and effective polysaccharide of bioflocculant in bioflocculant media.
Then, differentially expressed proteins of F2 and F6 in bioflocculant media compared with minimal media were separated, identified and analyzed in function by proteomics technologies——SDS-PAGE, two-dimensional electrophoresis (2-DE) and mass spectrum (MS). The changes of total proteins of F2 and F6 grown in bioflocculant media compared with minimal media were researched by SDS-PAGE. 58 of differentially expressed proteins of F2 and 14 of differentially expressed proteins of F6, which were related to flocculating metabolic pathway, were identified. The changes of cytosol proteins of F2 grown in bioflocculant media compared with minimal media were researched by 2-DE. 28 of differentially expressed proteins related to flocculating metabolic pathway of F2 were identified. By analyzing functions of differentially expressed proteins related to flocculating metabolic pathway of bioflocculant producing bacteria, the flocculating mechanisms of bioflocculant producing bacteria were revealed in proteomics level.
Finally, through a number of shake flask tests, the related regulation laws in production of bioflocculant were researched and the relationship between fermentation conditions, functions of differentially expressed proteins and changes in producing bioflocculant were established. Three dimentional regulating strategies, such as substrate, fermentation parameters and rejuvenation of bioflocculant producing bacteria, were brought up to regulate bioflocculant producing bacteria to produce bioflocculant by compound substrate in industry.
In the paper, the flocculating mechanisms of bioflocculant producing bacteria were researched in proteomics level, which can establish solid theory foundations to fully analyze and regulate flocculating metabolic pathway of bioflocculant producing bacteria. Based on analyzing functions of differentially expressed proteins related to flocculating metabolic pathway, regulating strategies were brought up, which can play important directing roles to raise the output of bioflocculant, to regulate bioflocculant producing bacteria to produce bioflocculant in industry.
首先,比较在基本培养基和产絮培养基培养条件下产絮菌所产生的生物絮凝剂在产量、成分上的差别,与基本培养基培养条件下相比,在产絮培养基培养条件下,产絮菌F2、F6会形成一些差异表达蛋白质,这些差异表达蛋白质是导致在产絮培养基培养条件下,诱导和产生大量生物絮凝剂主要活性成分多糖的直接原因。
然后,利用SDS-PAGE、2-DE和质谱结合的蛋白质组学研究手段对产絮菌F2、F6在基本培养基和产絮培养基不同培养条件下差异表达蛋白质进行分离、鉴定和功能解析。通过SDS-PAGE,研究在基本培养基和产絮培养基培养条件下,产絮菌F2、F6总蛋白的变化情况,共鉴定出58个产絮菌F2产絮过程差异表达蛋白质,14个产絮菌F6产絮过程差异表达蛋白质。通过2-DE,研究在基本培养基和产絮培养基培养条件下,产絮菌F2可溶性总蛋白的变化情况,共有28个产絮菌F2产絮过程差异表达蛋白质点得到成功鉴定。通过对产絮菌产絮过程差异表达蛋白质的功能解析,从蛋白质组学层面揭示了产絮菌的产絮机理。
最后,利用多项摇瓶试验,进行产絮菌生产生物絮凝剂的相关调控规律研究,建立起发酵条件,差异表达蛋白质的功能和产絮变化三者之间的关系,对工业上生产生物絮凝剂提出底物水平调控、发酵过程参数、菌种复壮三个层面的调控策略。
本研究从蛋白质组学层面研究产絮菌的产絮机理,为全面解析和调控产絮菌产絮代谢途径奠定坚实的理论基础。基于产絮菌产絮过程差异表达蛋白质的功能解析,提出工业化生产生物絮凝剂的调控策略,为提高生物絮凝剂产量,调控生物絮凝剂工业化生产起到重要的指导作用。
As an efficient, nontoxic, non-secondary contaminative, biodegradable and environmental-friendly green wastewater disposal agent, bioflocculant is one of the most potential flocculants and has been widely used in treating various industrial and domestic sewages. The compound bioflocculant (CBF) was produced by our lab, which was a novel bioflocculant produced by two bioflocculant producing bacteria of F2 and F6 through two-step fermentation. The effective component of bioflocculant produced by F2 and F+ (mixed cultivation from F2 and F6) was determined as extracellular polysaccharide. In the paper, differentially expressed proteins related to flocculating metabolic pathway of bioflocculant producing bacteria and regulation of their functions were researched by proteomics methods, the flocculating mechanisms were revealed in molecular level, regulating strategies of producing bioflocculant in industry were proposed.
First, the difference of production and component of bioflocculant between in bioflocculant media and in minimal media were researched. F2 and F6 expressed differentially expressed proteins in bioflocculant media compared with minimal media. These differentially expressed proteins were the direct reason to induce and produce large amount of main and effective polysaccharide of bioflocculant in bioflocculant media.
Then, differentially expressed proteins of F2 and F6 in bioflocculant media compared with minimal media were separated, identified and analyzed in function by proteomics technologies——SDS-PAGE, two-dimensional electrophoresis (2-DE) and mass spectrum (MS). The changes of total proteins of F2 and F6 grown in bioflocculant media compared with minimal media were researched by SDS-PAGE. 58 of differentially expressed proteins of F2 and 14 of differentially expressed proteins of F6, which were related to flocculating metabolic pathway, were identified. The changes of cytosol proteins of F2 grown in bioflocculant media compared with minimal media were researched by 2-DE. 28 of differentially expressed proteins related to flocculating metabolic pathway of F2 were identified. By analyzing functions of differentially expressed proteins related to flocculating metabolic pathway of bioflocculant producing bacteria, the flocculating mechanisms of bioflocculant producing bacteria were revealed in proteomics level.
Finally, through a number of shake flask tests, the related regulation laws in production of bioflocculant were researched and the relationship between fermentation conditions, functions of differentially expressed proteins and changes in producing bioflocculant were established. Three dimentional regulating strategies, such as substrate, fermentation parameters and rejuvenation of bioflocculant producing bacteria, were brought up to regulate bioflocculant producing bacteria to produce bioflocculant by compound substrate in industry.
In the paper, the flocculating mechanisms of bioflocculant producing bacteria were researched in proteomics level, which can establish solid theory foundations to fully analyze and regulate flocculating metabolic pathway of bioflocculant producing bacteria. Based on analyzing functions of differentially expressed proteins related to flocculating metabolic pathway, regulating strategies were brought up, which can play important directing roles to raise the output of bioflocculant, to regulate bioflocculant producing bacteria to produce bioflocculant in industry.
引文
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