苏云金芽孢杆菌aiiA基因在枯草芽孢杆菌中的表达

根据NCBI上已公布的枯草芽孢杆菌BS168菌株的β-1,4-glucanase(eglS)基因以及苏云金芽孢杆菌的aiiA基因序列,设计引物并扩增得到eglS基因的启动子、信号肽序列和aiiA基因.构建重组表达载体Ps4-eaiiA,转化枯草芽孢杆菌BS168,得到枯草芽孢杆菌工程菌BS168/Ps4-eaiiA.该工程菌诱导发酵后经SDS-PAGE和Western blotting分析表明:aiiA基因实现了在枯草芽孢杆菌中的表达.对胡萝卜软腐欧文氏菌进行马铃薯的抗病性实验,结果表明aiiA基因在枯草芽孢杆菌中表达的AiiA蛋白对胡萝卜欧文氏杆菌表现出一定的抗病性.     

短小芽孢杆菌质粒pCJ转化枯草杆菌的研究

本文采用我国自己分离的短小芽孢杆菌抗四环素质粒pCJ3转化枯草杆菌的各种突变体.质粒DNA经氯化铯—溴化乙锭密度梯度离心纯化后转化枯草杆菌的感受态细胞.结果表明枯草杆菌BR151,SB202,168,QB1130及QB1133都可作为质粒pCJ3的受体,转化效率在10~3左右,其中以SB202这株突变体为最高,从各种转化体中提取的质粒及经BamHI消化后的质粒的电泳图均与原质粒pCJ3及其BamHI酶切片段相同,并具有pCJ3的抗四环素转化活性.将pCJ3DNA经BamHI酶切后重新用T_4-DNA连接酶连接再转化枯草杆菌细胞,其转化效率比原质粒高1—2个数量级.研究了二价金属离子、pH及培养基成分对转化的影响,结果证明:Ca~(++)对枯草杆菌转化有促进作用,Cu~(++),Zn~(++)则有抑制作用,转化的最适pH在7.2—7.5之间;营养丰富培养能提高转化效率.     

枯草芽孢杆菌启动子的克隆及功能验证

以pHT01穿梭质粒为骨架,构建以卡那霉素抗性基因为报告基因的启动子探针载体pHT-kan.利用该探针载体在大肠杆菌中克隆枯草芽孢杆菌168的启动子活性片段,挑取得到100个重组子.通过卡那霉素浓度梯度筛选出2个抗性最强的片段进行序列测定和分析,将启动子片段命名为BSP25、BSP31.将抗性最高的两个载体转入枯草芽孢杆菌168菌株,结果表明,它们可以在枯草芽孢杆菌中启动卡那霉素抗性基因的表达,重组菌株表现出卡那霉素抗性.     

一种改进的分离纯化枯草芽胞杆菌产脂肽抗生素的方法

对枯草芽胞杆菌B6-1的去细胞发酵液进行超滤(分子量截留6-8kD),取截留液用6 N HCI酸沉淀,沉淀再经氯仿/甲醇(2:1)和甲醇依次抽提,ODS-C18柱固相萃取,用不同浓度的甲醇水溶液依次梯度洗脱,活性最强的70%、80%、100%甲醇溶液合并.经LC-MS分析,得出这些物质属于脂肽类表面活性剂抗生素,部分分子量(1450、1464、1478、1506)与已经报道的C15、C16、C17、C19脂肽fengycins相吻合.     

枯草芽孢杆菌谷氨酰胺转氨酶的异源表达

以野生型枯草芽孢杆菌基因组DNA为模板,PCR扩增获得带有SD序列及谷氨酸棒状杆菌信号肽ΔS0949的BTG基因.将其与大肠杆菌-谷氨酸棒状杆菌穿梭表达载体pXMJ19连接,构建重组质粒pXMJ19-Sbtg转化谷氨酸棒状杆菌ATCC13032.经IPTG诱导后该重组菌发酵液具有交联酪蛋白的能力,表明该重组菌能够实现分泌表达.     

Purification and characterization of keratinase from a new Bacillus subtilis strain

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.     

枯草芽孢杆菌寡聚-1,6-葡萄糖苷酶基因在毕赤酵母中的表达研究

根据枯草芽孢杆菌（Bacillus subtilis）寡聚-1,6-葡萄糖苷酶基因序列设计引物,以pHBM003为模板,扩增得到寡聚-1,6-葡萄糖苷酶基因,克隆至毕氏酵母（Pichia pastoris）表达载体pHBM905上,获得重组毕氏酵母表达载体pHBM9053．将此质粒分别转化毕氏酵母GS115、KM71和SMD1168菌株,筛选获得重组毕赤酵母GS115（pHBM9053）、KM71（pHBM9053）和SMD1168（pHBM9053）;然后进行摇瓶诱导培养,这3株毕氏酵母分别在诱导培养60h、48h和24h后,酶活力达到最高,对应为2．233u／mL、0．34u／mL和1．235u／mL;GS115（pHBM9053）所产寡聚-1,6-葡萄糖苷酶的最适反应温度为75℃,最适反应pH值为6,在30～75℃、pH8～9范围内较稳定．     

荧光蛋白在枯草芽孢杆菌中的表达

枯草芽孢杆菌是生物工业用的重要宿主体系。为实现荧光蛋白从分子水平定量追踪枯草芽孢杆菌培养过程的目的,构建了一系列整合型表达载体pX-GFPmut1和游离型表达载体pSW1-GFPmut1,pSW1-CFP,pSW1-YFP,研究了不同颜色荧光蛋白在枯草芽孢杆菌中整合型表达和游离型表达的差异。最适合的荧光蛋白表达体系是用游离型质粒pSW1-GFPmut1在枯草芽孢杆菌中表达绿色荧光蛋白GFPmut1。含pSW1-GFPmut1枯草芽孢杆菌通过木糖诱导表达GFPmut1后,细胞破碎后的荧光强度和细胞浓度呈线性关系。结果表明,利用枯草芽孢杆菌表达荧光蛋白可以实现利用荧光蛋白快速定量枯草芽孢杆菌培养过程。     

Formation of proinsulin by immobilized Bacillus subtilis

There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol. Some of these compounds are now produced on an industrial scale whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as "clogging' of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.     

荧光蛋白在枯草芽孢杆菌中的表达

枯草芽孢杆菌是生物工业用的重要宿主体系。为实现荧光蛋白从分子水平定量追踪枯草芽孢杆菌培养过程的目的,构建了一系列整合型表达载体pX-GFPmut1和游离型表达载体pSW1-GFPmut1,pSW1-CFP,pSW1-YFP,研究了不同颜色荧光蛋白在枯草芽孢杆菌中整合型表达和游离型表达的差异。最适合的荧光蛋白表达体系是用游离型质粒pSW1-GFPmut1在枯草芽孢杆菌中表达绿色荧光蛋白GFPmut1。含pSW1-GFPmut1枯草芽孢杆菌通过木糖诱导表达GFPmut1后,细胞破碎后的荧光强度和细胞浓度呈线性关系。结果表明,利用枯草芽孢杆菌表达荧光蛋白可以实现利用荧光蛋白快速定量枯草芽孢杆菌培养过程。     

Modification of the rib operon derived from Bacillus subtilis and its expression in Escherichia coli

A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.