枯草芽孢杆菌蛋白质分泌机制研究进展

综述了枯草芽孢杆菌不同蛋白质分泌机制,重点讨论了大多数细菌蛋白分泌的Sec途径,包括Sec途径的信号肽,信号肽酶,SecYEG通道,与分泌有关的各种细胞因子以及Sec途径的限制因素,此外还简要讨论了Tat途径,该途径能够转运折叠迅速或归密的蛋白质。     

枯草芽孢杆菌用于外源蛋白质生产和分泌的可能性

成功地利用枯草芽孢杆菌分泌外源基因产物,需要一个缺失胞外蛋白酶的寄主,需要创造遗传调控外源基因在生长期间表达的因子和营养条件,还需要进一步了解寄主的分泌机制以提高其分泌效率。     

信号肽对酵母外源蛋白质分泌效率的影响

根据酵母表达系统在表达外源蛋白方面的独特优势,利用酵母表达系统高效分泌并纯化具有生物学活性的蛋白质,已受到广泛关注。信号肽在蛋白质的分泌中起着重要作用,可引导蛋白分泌至胞外,大大提高蛋白的表达量,在工业化生产外源蛋白的纯化工艺方面具有重要意义。该文将从酵母表达系统中对信号肽的选择、改造、偏爱密码子和增强子的应用等几个方面进行优化的探讨,以提高蛋白质在酵母系统中的分泌效率。     

外源蛋白在芽孢杆菌中分泌表达的研究进展

芽孢杆菌是很有潜力的分泌型基因工程宿主菌。本文概述了利用芽孢杆菌分泌表达外源基因时,影响目的蛋白产率的一些主要因素,如蛋白酶水解作用、缺乏适宜的分子伴侣、信号肽的选择不当等,并讨论了相应的解决对策。     

过表达TatAdCd转位酶对枯草芽孢杆菌脂肪酶分泌的影响

【目的】研究过表达枯草芽孢杆菌Tat运输途径的Tat Ad Cd转位酶对促进脂肪酶分泌的影响。【方法】用cdd基因的串联启动子和前导区,替换tat AD-CD操纵子的启动子和前导区,并在染色体sac B基因位点整合表达;采用q RT-PCR方法表征tat AD-CD操纵子的表达水平;用脂肪酶表达质粒p HP13L转化Tat Ad Cd转位酶过表达菌株,构建产脂肪酶重组菌。通过测定脂肪酶活性,以及聚丙烯酰胺凝胶电泳,考察Tat Ad Cd转位酶过表达对脂肪酶分泌的影响。【结果】tat AD-CD操纵子被过表达,其胞内m RNA相对水平提高了185倍。Tat Ad Cd转位酶的过表达,使脂肪酶发酵单位提高了40%。【结论】使用cdd基因的串联启动子和前导区,能够有效地过表达目的基因;枯草芽孢杆菌脂肪酶可以同时经由Sec途径和Tat途径分泌;过表达Tat Ad Cd转位酶,能够显著提高脂肪酶的分泌量。     

外源蛋白在芽孢杆菌中分泌表达的研究进展

芽孢杆菌是很有潜力的分泌型基因工程宿主菌。本文概述了利用芽孢杆菌分泌表达外源基因时,影响目的蛋白产率的一些主要因素,如蛋白酶水解作用、缺乏适宜的分子伴侣、信号肽的选择不当等,并讨论了相应的解决对策。     

乳酸菌蛋白质分泌表达研究进展

食品级乳酸菌不仅是食品或消化道中传递异源蛋白质的合适的候选菌,在工业发酵中还可用于生产蛋白质。在过去20多年中,人们设计了许多乳酸菌蛋白质表达和标记系统,这些系统已用在乳酸菌工程菌的细胞内或细胞外生产各种细菌、病毒和真核生物来源的蛋白质。在目的蛋白生产和发酵中,分泌表达由于可持续培养和简化纯化步骤并使目的蛋白与其靶位相互作用而优于细胞质表达。目前只有少数研究报道了目的蛋白在乳酸菌细胞内或分泌表达产量的比较,研究表明分泌表达比细胞质表达更优越。     

信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响

枯草芽孢杆菌具有良好的安全性和优异的外分泌能力,是目前常用的脂肪酶异源分泌表达体系。在枯草体系中如何提高脂肪酶分泌量是目前的研究热点。该体系中信号肽与目的基因不匹配和目标蛋白总表达量不高是导致脂肪酶LipS分泌表达效果较差的重要原因。针对这些问题,依据信号肽结构特征的不同(正电荷与疏水氨基酸个数的差异)对枯草芽孢杆菌体系中Sec和Tat这两个主要分泌途径的信号肽进行了筛选,结果显示Tat途径中的phoD信号肽分泌效果明显优于其他信号肽。在此基础上,通过替换使用诱导型表达载体、突变优化phoD信号肽,并考察了表达条件的影响,显著提高了脂肪酶LipS的分泌表达量,发酵液中转酯酶活达到62.07U/L,占总酶活的62.30%,较原始分泌表达体系的分泌量提高了13.7倍。     

基于信号肽策略提高外源蛋白在枯草芽孢杆菌中的表达

枯草芽孢杆菌(Bacillus subtilis)作为一种重要的原核表达宿主菌,具有蛋白分泌能力强、遗传背景清晰、无密码子偏好性、生长迅速和非致病性等优点,一直都是外源蛋白表达优选的模式菌株.信号肽(signal peptide)是一段存在于前体蛋白N-端的短肽链,其功能在于引导和调节前体蛋白的折叠,在蛋白转移和分泌过...     

枯草芽孢杆菌芽孢中的芽孢衣

芽孢衣是赋予芽孢对有机溶剂和溶菌酶的抗性,以及对外界出芽诱导物的感应能力的保护性结构。该主要介绍由枯草芽孢杆菌(bacillus subtilis)所形成的芽孢的芽孢衣中已鉴定的主要蛋白质．及其基因表达调控和应用等方面的研究进展。     

中性蛋白酶基因诱导型表达分泌载体的构建

利用PCR方法分别扩增出sacB基因的启动子-信号肽序列（sacR）和枯草芽孢杆菌中性蛋白酶的前肽-成熟肽序列,将两者连接后克隆入载体pHP13中,构建了含有中性蛋白酶基因的诱导型表达分泌载体pHP13SN,再将其转化入枯草杆菌DB104,获得基因工程菌DB104(pHP13SN)。中性蛋白酶基因在蔗糖的诱导和sacR的调控下实现了分泌表达,并获得了具有生物学活性的中性蛋白酶。     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

Inducible resistance to zinc ions in Bacillus subtilis 168

Abstract Chromosomally determined resistance to Zn²⁺ ions is a property of Bacillus subtilis 168. Resistance is inducible and is not connected with Cd²⁺ resistance, characteristics of plasmid determined resistance in bacteria. A few proteins were shown to respond to inducible levels of the cation.     

Restriction on Conjugational Transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.     

途径优化强化枯草芽胞杆菌合成肝素前体

肝素前体是化学酶法合成肝素的起点,肝素前体的微生物高效合成具有重要意义。在已构建的产肝素前体的枯草芽胞杆菌((1.71±0.08)g/L)中,分析了UDP-葡萄糖醛酸(UDP-GlcUA)途径中关键酶基因(pgcA、gtaB、tuaD)以及UDP-乙酰氨基葡糖(UDP-GlcNAc)途径中关键酶基因(glmS、glmM、glmU)的过量表达对肝素前体产量及其分子量的影响。在此基础上,通过共表达tuaD、gtaB、glmU、glmM和glmS基因,摇瓶中肝素前体产量提高至(2.89±0.11)g/L,分子量为(75.90±1.18)kDa。通过在3 L发酵罐中进行补料分批发酵,肝素前体的产量最终积累到(7.25±0.36)g/L,分子量为(46.66±2.71)kDa,为工业化生产肝素奠定了基础。     

血红蛋白基因在枯草芽孢杆菌中的表达及其作用的研究

革兰氏阴性菌Ｖｉｔｒｅｏｓｃｉｌｌａ的血红蛋白与氧结合力强,能降低该菌需氧量。将该血红蛋白的结构基因（Ｖｇｂ）连接到枯草杆菌质粒ｐＡＫ４的β－内酰胺酶基因（ｂｌａ）启动子下（框架正确）,构建了重组质粒ｐＡＶ,并将此质粒先转化至枯草杆菌ＤＢ１０４,继而又转化到枯草杆菌碱性蛋白酶工程菌Ｇ３３１和产木聚糖酶枯草杆菌Ｂ５３。经酶切和电泳分析显示转化的质粒ＤＮＡ含有大小与Ｖｇｂ相同的电泳带,又经非放射性同位     

Emergence and development of chlorhexidine resistance during sporulation of Bacillus subtilis 168

Abstract During sporulation of Bacillus subtilis strain 168 initiated by step-down conditions, resistance to chlorhexidine diacetate (CHA) developed at about t _3.5, before heat but after toluene resistance. Mutants blocked at stage IV of sporulation were sensitive to all three treatments. Stage V mutants were toluene resistant but moderately sensitive to heat and CHA. A stage VI mutant was resistant to all three treatments. Thus, chlorhexidine resistance is likely to be a result of spore coat, rather than of cortex, development.     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168

Lipopeptides have a widespread role in different pathways of Bacillus subtilis; they can act as antagonists, spreader and immunostimulators. Plipastatin, an antifungal antibiotic, is one of the most important lipopeptide nonribosomly produced by Bacillus subtilis. Plipastatin has strong fungitoxic activity and involve in inhibition of phospholipase A2 and biofilm formation. For better understanding of the molecule and pathway by which lipopeptide plipastatin is synthesized, we present a computationally predicted structure of plipastatin using homology modeling. Primary and secondary structure analysis suggested that ppsD is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. The tertiary structure of protein (plipastatin synthase subunit D) was predicted by homology modeling. The results suggest a flexible structure which is also an important characteristic of active enzymes enabling them to bind various cofactors and substrates for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and QMEAN score.This predicted information will help in better understanding of mechanisms underlying plipastatin synthase subunit D synthesis. Plipastatin can be used as an inhibitor of various fungal diseases in plants.     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。