肽聚糖水解酶基因缺失对解淀粉芽胞杆菌活菌数量及产碱性蛋白酶的影响

为了探究肽聚糖水解酶对解淀粉芽胞杆菌(Bacillus amyloliquefaciens)活细胞数量以及碱性蛋白酶产量的影响,对解淀粉芽胞杆菌TCCC111018中的5个肽聚糖水解酶基因(lytC、lytD、lytE、lytF、lytG)分别进行敲除.通过分析对比基因缺失前后的生物量以及碱性蛋白酶酶活力发现,敲除菌株...     

土壤中可编码乌头酸异构酶的芽胞杆菌菌株筛选及鉴定

【目的】以芽胞杆菌(Bacillus)为筛选对象,分离土壤中可编码乌头酸异构酶(aconitate isomerase,AI)的革兰氏阳性(Gram positive,G+)菌株,以丰富对AI分布的科学认识,为其生物学功能研究奠定理论与材料基础。【方法】采用土样高温预处理法、含反式乌头酸(trans-aconitic acid,TAA)唯一碳源的ACO固体平板培养法,结合16S rDNA基因序列同源性分析,筛选能够编码AI的芽胞杆菌目的菌株。【结果】共分离得到22株能够利用TAA碳源的细菌菌株,成功鉴定了其中的16株,分别为巨大芽胞杆菌(Bacillus megaterium) 2株,阿氏芽胞杆菌(Bacillus aryabhattai) 7株,短小芽胞杆菌(Bacillus pumilus) 1株,未鉴定到种的芽胞杆菌(Bacillus sp.) 6株;且它们所含AI编码基因与已知AI基因在序列上存在差异。【结论】首次证明可编码AI的芽胞杆菌细菌种类具有多样性,暗示G+细菌广泛编码AI的可能性,更新了AI几乎只在G–细菌中分布的观点,为后续深入挖掘AI基因及其生物学功能研究提供更多可用微生物资源。     

Bacillus subtilis WHNB02植酸酶phyC基因的克隆及序列分析

采用PCR法获得产植酸酶芽孢杆菌(Bacillus subtilis)WHNB02株植酸酶的全长phyc基因,并将其克隆到pUC18-T载体。序列分析表明该基因全长1152bp,编码一个383个氨基酸的多肽,信号肽切割位点位于第26个氨基酸残基之后。系统进化树表明,来源于7株芽孢杆菌的植酸酶在遗传上分为两大类。将Bacillus subtilis WHNB02植酸酶phyC基因序列及其氨基酸序列在GenBank中登录,登录号分别为AF220075和AA043434．1。     

一株高产纳豆激酶菌株的鉴定与分析

鉴定高产纳豆激酶菌株Td,分析纳豆激酶的分子特征。利用菌体形态、生理生化特征、以及分子生物学方法对菌株Td进行鉴定;并采用MALDI-TOF质谱测定与分析、SDS-PAGE和纤溶活性测定等方法检测纳豆激酶特性,利用PCR方法扩增纳豆激酶的基因全长。结合菌体形态、生理生化特征和16S r DNA、gyr A基因序列、DNA-DNA杂交率等实验结果,鉴定菌株Td为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis);菌株Td发酵产生的纳豆激酶产量可达300 mg/L以上,占发酵液总蛋白的40%以上;纤溶活性达230 U/m L以上;氨基酸序列与subtilisin E的序列相似性最高;基因全长序列为1 143 bp。枯草芽孢杆菌枯草亚种Td是一株高产、高活性纳豆激酶的产生菌,具有优良的工业化开发价值。     

枯草芽胞杆菌fmbj SrfAC-A结构域的活性测定

[目的]在体外研究surfactin合成酶的A结构域,为获得新的surfactin类似物奠定基础.[方法]本文从枯草芽胞杆菌(Bacillus subtilis)fmbj中克隆出surfactin合成酶第7个模块的A结构域基因(SrfAC-A),与表达质粒pET-23a相连后在大肠杆菌表达系统中表达,用Ni-NTA亲和柱对重组蛋白SrfAC-A进行分离纯化后测定其活性.[结果]克隆所得的A结构域对Ile有选择活性,而对其他氨基酸基本无活性.[结论]Surfactin合成酶中的A结构域能在体外独立行使其选择底物氨基酸的功能.     

纳豆激酶的原核表达、纯化及其特性分析（英文）

纳豆激酶(Nattokinase)作为一种新型溶解血栓的纤维蛋白溶解酶,有着广阔的市场前景和巨大的产业化潜力。本研究从枯草芽胞杆菌(Bacillus subtilis natto)发酵液中提取、纯化纳豆激酶,依次通过硫酸铵盐析、凝胶过滤层析和疏水层析方法纯化纳豆激酶,纯化倍数为5.2,纯化率为46.3%。纯化后的纳豆激酶经SDS-PAGE电泳显示相对分子量约为28 kD a,纤维蛋白平板法显示活性为4 580 IU/mg。但纳豆激酶在枯草芽胞杆菌发酵液中的含量较低,因此在原核表达载体大肠埃希菌(Escherichia coli)BL21(DE3)中克隆并高效表达了纳豆激酶基因,其纳豆激酶产量显著提高,但酶活相对降低。     

枯草芽胞杆菌BSD-2中ybfB基因敲除及生物信息学分析

在前期突变体库研究中发现,ybf B缺失的枯草芽胞杆菌Bacillus subtilis BSD-2丧失了其抑真菌活性。本研究通过敲除并恢复BSD-2中ybf B基因,进一步确认其缺失对菌株生物活性的影响,并利用生物信息学手段对其基因序列和编码蛋白质序列进行了预测分析。本研究成功敲除BSD-2菌株的ybf B基因,构建突变株B-Y-k,并成功构建其回复突变株B-Y,平板对峙实验结果显示突变其ybf B基因该菌株失去其抗真菌活性,回复突变株抗真菌活性恢复,表明该基因与其抗真菌相关。生物信息学分析显示该基因全长1 251 bp,编码416个氨基酸,具有12个跨膜螺旋区,为疏水性蛋白,推测该蛋白为疏水性跨膜转运蛋白,可能参与活性物质的转运。     

海洋中分离的抗白念珠菌芽胞杆菌性状特征及系统鉴定

对筛选自中国南海、黄海、渤海4个地区近海海水样品的7株有抗白念珠菌活性,且稳定性较强的芽胞杆菌的形态特征、培养特征及生理生化试验等进行系统分析比较,结果表明,LU-B02为凝结芽胞杆菌(Bacillus coagulans),LU-B13为蜡样芽胞杆菌(Bacillus cereus),其他各菌株为短小芽胞杆菌(Bacillius pumilus),除LU-B02耐盐度5%外,其他耐盐度达10%以上。它们均属于芽胞杆菌的第一群。16S r DNA基因同源性序列比较进一步证实LU-B02为凝结芽胞杆菌,它与凝结芽胞杆菌标准菌株ATCC15950在鉴定特征上虽然相同,但与后者相比,最高生长温度较低,耐盐性较强,可以利用阿拉伯糖、木糖、甘露醇等碳源,可水解酪素,并可稳定地产生抗白念珠菌活性物质,将其命名为Bacillus coagulans subsp.heishijiaosis。尚未见抗白念珠菌的凝结芽胞杆菌菌株的有关报道。     

蛋白激发子PeaT1在枯草芽胞杆菌中的分泌表达及重组菌株提高小麦抗旱和促生的作用

PeaT1是从极细链格孢菌Alternaria tenuissima中分离的一种蛋白激发子,具有促进植物生长和诱导植物产生系统获得抗性的功能,为了实现peaT1基因在枯草芽胞杆菌Bacillus subtilis中的分泌表达,增加其应用途径,从枯草芽胞杆菌基因组DNA中分别扩增获得P43启动子和nprB基因的信号肽序列,并用SOE (Splicing by over lapping extension) 方法与peaT1基因连接,将连接产物克隆到大肠杆菌-枯草芽胞杆菌穿梭表达载体pHY300-PLK上,构建了重组表达载体pHY43N-peaT1。将重组载体转化枯草芽胞杆菌WB800菌株,SDS-PAGE和Western blotting分析证实,在NprB信号肽的引导下,枯草芽胞杆菌成功分泌表达了PeaT1蛋白。构建的重组菌株能够显著增强幼苗抗旱性,提高小麦株高。     

Bt新型基因sip的克隆、表达和生物信息学分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白对农业害虫有特异的杀虫活性,对环境无污染、对人畜无害,成为目前为止研究和应用最为广泛的生物杀虫剂.其中内毒素(insecticidal crystal proteins,ICPs)和营养期杀虫蛋白(vegetative insecticidal protein,VIP)研究范围广、技术成熟,而分泌期杀虫蛋白(Secreted insecticidal protein,Sip)相关报道较少.为进一步发掘我国苏云金芽胞杆菌的菌株资源和新型基因资源,从不同生态环境中分离出400株Bt野生菌株进行sip基因的克隆鉴定,其中Bt野生菌株QZL26扩增得到1 038 bp的碱基的序列,编码345个氨基酸,与Sip1A蛋白氨基酸序列同源性为91.83％.测序结果提交GenBank注册,登录号为JQ965994.     

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.     

枯草芽胞杆菌乙酰木聚糖酯酶的鉴定及诱导剂对酯酶活力的影响

以枯草芽胞杆菌CICC 20034为研究对象,对其分泌的高相对分子质量酯酶进行鉴定,并考察诱导剂对其活力的影响。结果表明:枯草芽胞杆菌CICC 20034可分泌一种相对分子质量为1.07×105的酯酶,经蛋白质质谱鉴定为乙酰木聚糖酯酶,单体分相对子质量为3.56×104。在发酵培养基中添加乙酸乙酯和木糖可以显著的促进乙酰木聚糖酯酶的活力,而三丁酸甘油酯和大分子诱导剂——木聚糖、玉米芯粉和壳聚糖对酯酶的活力几乎无促进作用。枯草芽胞杆菌CICC 20034以乙酸乙酯为诱导剂时最高比酶活为0.62 U/mL,为已知报道的野生细菌乙酰木聚糖酯酶的最高酯酶活力。     

A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1 % sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.     

应用多重PCR鉴定微生物肥料常用芽孢杆菌

[目的]枯草群芽孢杆菌中枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(B.amyloliq-wefaciens)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus)是微生物肥料中常用菌种,用传统方法鉴定费时费力,有必要建立检测和鉴定这些芽孢杆菌的种特异性PCR方法.[方法]利用已登录的gyrA、rpoA和16s rRNA基因序列分别设计和筛选上述菌种的特异引物并建立多重PCR反应体系.[结果]以基因组DNA为模板,扩增芽孢杆菌、类芽孢杆菌和短芽孢杆菌3属15种的标准菌株(共33株),4个目标种分别产生了大小不同的唯一的产物,除个别种与短小芽孢杆菌引物有交叉反应外,其余参考菌株均为阴性.从23株枯草群菌株的基因组DNA扩增发现,PCR鉴定与常规鉴定结果一致.[结论]本文建立的多重PCR方法具有较好的特异性,可快速准确鉴定枯草群的4个种,在微生物肥料检测方面有良好的实用前景.     

YesT: a new rhamnogalacturonan acetyl esterase from Bacillus subtilis

YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichiacoli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.     

Characterization and mapping of the Bacillus subtilis prtR gene.

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.     

The crystal structure of the cephalosporin deacetylating enzyme acetyl xylan esterase bound to paraoxon explains the low sensitivity of this serine hydrolase to organophosphate inactivation

Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for β-lactam antibiotic synthesis) AXE/CAH (acetyl xylan esterase/cephalosporin acetyl hydrolase) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon (diethyl-p-nitrophenyl phosphate, also called paraoxon-ethyl), reflected in a high K(i) for it (~5 mM) and in a slow formation (t(?)~1 min) of the covalent adduct of the enzyme and for DEP (E-DEP, enzyme-diethyl phosphate, i.e. enzyme-paraoxon). The crystal structure of the E-DEP complex determined at 2.7 ? resolution (1 ?=0.1 nm) reveals strain in the active Ser1?1-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active-site-size limitation imposed by bulky conserved aromatic residues that may exclude as substrates esters having acyl groups larger than acetate. Interestingly, in the doughnut-like homohexamer of the enzyme, the six active sites are confined within a central chamber formed between two 60°-staggered trimers. The exclusive access to this chamber through a hole around the three-fold axis possibly limits the size of the xylan natural substrates. The enzyme provides a rigid scaffold for catalysis, as reflected in the lack of movement associated with paraoxon adduct formation, as revealed by comparing this adduct structure with that also determined in the present study at 1.9 ? resolution for the paraoxon-free enzyme.     

Carbohydrate esterase family 4 enzymes: substrate specificity

The substrate specificity of selected enzymes classified under Carbohydrate Esterase family 4 (CE4) has been examined. Chitin deacetylase from Mucor rouxii and both a native and a truncated form of acetyl xylan esterase from Streptomyces lividans were found to be active on both xylan and several soluble chitinous substrates. Furthermore, the activities of all enzymes examined were significantly increased in the presence of Co(2+) when chitinous substrates were employed. However, the presence of this metal ion did not result in enhancing the activities of the enzymes when xylan was used as substrate. An acetyl xylan esterase from Bacillus pumilus, classified under Carbohydrate Esterase family 7, was found to be inactive towards all chitinous substrates tested. Finally, all enzymes examined were inactive towards cell wall peptidoglycan.     

Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.