1株枯草芽胞杆菌的鉴定及其弹性蛋白酶结构研究

鉴定弹性蛋白酶产生菌株EL32,确定该酶的基本结构。采用分子生物学、形态学及生理生化性质对菌株EL32进行鉴定;用硫酸铵沉淀、离子交换层析和分子筛层析纯化酶蛋白;借助肽指纹图谱及酶基因克隆技术研究该酶的一级结构;利用同源建模的方法研究该酶的空间结构。菌株EL32的16S rDNA与枯草芽胞杆菌16S rDNA的同源性达到99%,其菌落呈乳白色,其细胞革兰染色阳性,具有芽胞;发酵葡萄糖试验产酸不产气,明胶水解试验呈阳性,能够水解淀粉,V-P反应呈阳性,鉴定为枯草芽胞杆菌。从菌株BL32发酵液中纯化得到了弹性蛋白酶,SDS-PAGE分析显示其分子量为31 ku。用LTQ-MS测定肽指纹图谱表明该弹性蛋白酶是枯草芽胞杆菌蛋白酶subtilisin,该酶的基因和蛋白质序列与枯草芽胞杆菌蛋白酶subtilisin的同源性都高达99%。菌株EL32弹性蛋白酶的三维结构含有6个α-螺旋,7个扭曲的平行β-折叠以及2个反平行的β-折叠,His、Asp和Ser是其活性中心的关键基团。鉴定了1株产弹性蛋白酶的枯草芽胞杆菌,确定了其弹性蛋白酶是蛋白酶subtilisin,为该枯草芽胞杆菌蛋白酶的应用提供了基础。     

枯草芽孢杆菌SN-02发酵液的抑菌谱及稳定性研究

目的:研究枯草芽孢杆菌SN-02发酵液的抑菌谱及稳定性。方法:以28种植物病原菌为供试菌,杯碟法测定SN-02菌发酵液的抑菌谱;以烟草靶斑病菌为指示菌,杯碟法测定发酵液的热稳定性、酸碱稳定性及传代稳定性。结果:SN-02菌发酵液对28种供试菌株的抑菌圈直径在20mm以上。将发酵液于120℃处理2.5h,-20℃处理25d抑菌活性没有明显变化;发酵液在pH 4~9时抑菌活性无明显变化,在pH 1~3和pH 10条件下抑菌活性明显下降;连续培养10代,发酵液抑菌活性没有下降。结论:SN-02菌发酵液抑菌谱较广,耐高温和低温,传代稳定性好,但在强酸和强碱条件下稳定性较差。     

凝结芽胞杆菌G1及其抑菌物质的研究

目的获得具有良好性状的产细菌素菌株。方法测定菌株形态学和生理生化学特性以进行菌种鉴定;采用牛津杯法测定抑菌谱及细菌素的理化特性。结果经鉴定实验菌株为凝结芽胞杆菌,所产细菌素在100℃加热60min后,残余活力为90％;在pH5～9范围内,抗菌活力稳定。结论凝结芽胞杆菌G1可产性能良好的抑制革兰阳性菌生长的细菌素。     

静态强磁场对枯草芽胞杆菌的影响研究

目的:研究静态强磁场下微生物的生物学效应.方法:以超导磁体产生的静态强磁场为基础,枯草芽孢杆菌为模式生物,通过测定生长曲线、芽胞生成率、蛋白酶表达量、蛋白酶活力等研究静态强磁场条件下微生物性状的变化.结果:强磁场可以影响枯草芽胞杆菌的芽胞形成速率,抑制营养体的死亡;测定菌体生长过程中蛋白酶的含量以及碱性蛋白酶和中性蛋白酶的酶活力,发现磁场处理前后蛋白酶的含量没有发生显著性变化,处理组碱性蛋白酶的酶活力明显高于对照组,而中性蛋白酶酶活力则低于对照组.结论:强磁场可以延长枯草芽孢杆菌的世代周期,降低菌体死亡率,对细菌酶活性的影响因酶的种类不同而异.     

益生枯草芽胞杆菌B15的性能检测及培养条件研究

对筛选得到的枯草芽胞杆菌B15进行了产酶及拮抗性能的检测,结果表明该菌株能够分泌淀粉酶、蛋白酶、纤维素酶,同时对常见病原体有一定的拮抗作用,具有作为益生菌进行发酵和研制微生态制剂的潜力。采用单因子试验,对B15菌株的培养液组成（碳源、氮源）及培养条件进行了研究,结果表明：以1.5%葡萄糖及0.5%酵母浸出物制成的培养液,在35℃、初始pH6.5左右,装液量为20 ml/250 ml,接种量2%的条件下培养B15,可将活菌数目从原始的1.15×109CFU/ml提高至1.61×109CFU/ml。     

枯草芽胞杆菌菌肥对有机冬瓜根区土壤微生态的影响

【背景】微生物肥料已广泛应用于我国有机作物的种植,其对有机种植土壤微生态的影响尚需科学评测。【目的】高通量测序技术可用于精确分析土壤微生物群落,从细菌、真菌群落结构和多样性的角度阐释枯草芽胞杆菌菌肥对有机农田根区土壤微生物群落的影响。【方法】在有机农田轮作种植条件下,施用枯草芽胞杆菌菌肥后提取冬瓜根区土壤基因组DNA,通过PCR扩增建立文库,利用IlluminaMiSeq高通量测序技术,并结合相关生物信息学方法分析土壤细菌16SrRNA基因V3-V4区和真菌ITS1区的多样性指数及群落结构;测定根区土壤化学性质及酶活性,分析有机冬瓜果实品质,并作相关分析。【结果】从6个有机冬瓜根区土壤样本中获得14199个细菌操作分类单元(OTU)和3378个真菌OTU,细菌和真菌文库测序覆盖率分别在98%、99%以上。枯草芽胞杆菌菌肥会在一定程度上提高土壤细菌种群多样性而降低真菌种群多样性,丰富了细菌群落结构,但显著降低了真菌群落丰富度(P0.05);并减少了根区土壤特有细菌和真菌物种。变形菌门、厚壁菌门和放线菌门是优势细菌,子囊菌门是优势真菌;枯草芽胞杆菌菌肥会提高绿弯菌门和子囊菌门的相对丰度,比例分别为46.23%、10.01%;降低变形菌门和担子菌门的相对丰度,比例分别为11.14%、74.72%。枯草芽胞杆菌菌肥显著降低了土壤pH,显著提高了有机冬瓜果实总氨基酸、可溶性固形物等营养成分含量(P0.05)。【结论】施用枯草芽胞杆菌菌肥改变有机冬瓜根区土壤细菌和真菌的丰富度和多样性,降低了土壤pH,提高了有机冬瓜果实品质。     

死谷芽胞杆菌B10发酵条件的研究与抗菌谱的测定

死谷芽胞杆菌B10可抑制多种食用菌病原木霉菌的生长,同时对其他多种食用菌病原真菌有拮抗作用,因此是1株很有潜力的生防菌,具有开发成生防产品的潜能。实验通过摇瓶培养,对死谷芽胞杆菌B10产生抑菌活性物质的发酵培养基和培养条件进行优化,并对其抑菌谱进行了测试。结果表明,实验产生抑菌活性物质的最佳培养基为NB液体培养基,最佳发酵条件为:菌株种龄18 h,发酵周期36 h,初始培养基pH为7.0,培养温度为30℃,摇瓶装液量为100 mL/250 mL,接种量为4%,摇床转速为170 r/min。B10发酵无菌滤液对多种食用菌病原真菌有明显抑制作用,其中对哈茨木霉T22抑制作用最强,抑菌率达90.56%。     

海洋短小枯草芽胞杆菌的鉴定及抑菌活性分析

对从连云港东西连岛海泥样品中分离得到的菌株Bacillus pumilus HX2-2的分类地位、生长条件和抑菌活性进行了研究。经过形态特征、生理生化性质及16S r DNA序列分析鉴定,该菌属于短小芽胞杆菌。不同温度、盐度、pH培养条件下测定菌液吸光度OD600值,表明该菌是一株轻度嗜盐菌,最适温度、盐度、pH分别为30℃、3%、7.0~8.0。在不同病原真菌的平板抑菌活性试验中,该菌对草莓尖胞镰刀菌、马铃薯炭疽病菌和水稻立枯丝核菌表现出显著的抑菌作用。菌株B.pumilus HX2-2是一株短小芽胞杆菌,具有广谱抑菌活性,具有进一步研究的价值。     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

基于麦芽糖诱导型启动子在枯草芽胞杆菌中异源表达麦芽四糖淀粉酶及其酶学性质的研究

麦芽四糖淀粉酶(Maltotetraose amylase, Mta)可以从淀粉的非还原末端特异性依次切割第4个α-1,4糖苷键形成麦芽四糖,目前在食品、医疗保健和造纸等领域具有重要应用。构建安全、高效的表达系统强化麦芽四糖淀粉酶的重组表达,进而降低以其为核心酶的麦芽四糖生物转化过程的生产成本具有迫切的现实需求。本研究将源自Pseudomonas saccharophila(DSM 654)的麦芽四糖淀粉酶基因mta在枯草芽胞杆菌Bacillus subtilis中重组表达,利用麦芽糖诱导型启动子实现其安全高效表达,之后对重组酶进行分离纯化和酶学性质表征。结果显示,将携带麦芽糖诱导型启动子P_glv的表达载体转入B.subtilis WB800N中,成功构建工程菌后进行诱导表达,并且利用金属离子螯合层析技术成功获得了Mta纯酶。酶学性质研究结果显示其最适反应温度为55℃,最适反应pH为7.5。动力学常数K_m为(1.26±0.17) g/L、k_cat/K_m为(2 275.07±32.83) L/s·g,...     

枯草芽孢杆菌抗菌肽生物合成的研究进展

革兰氏阳性菌模式生物--枯草芽孢杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径不同,可分为核糖体肽和非核糖体肽。其中,非核糖体肽分子量较小,一般为3000Da以下,其生物合成是通过多功能复合酶系--非核糖体肽链合成酶来完成的,多发生在菌体生长停止之后;而核糖体肽分子量较大,其合成多于菌体快速生长时期。非核糖体肽链合成酶和核糖体肽的合成及其调控均需基因参与,而这一系列基因就构成了各种抗菌肽生物合成的基因簇。对核糖体肽和非核糖体肽的生物合成及其相关调控机制进行了综述。     

Purification and Properties of l-Arginase from Bacillus subtilis

l-Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) was purified in a crystalline form from cells of Bacillus subtilis KY 3281 with an overall yield of 23.2%. The crystalline enzyme had a specific activity of 858 i.u./mg-protein and was ultracentrifugally homogeneous. It was estimated to have a molecular weight of 115,000±5000 by the method of Yphantis.

The enzyme highly specific for l-arginine showed the maximum activity at pH 10 with Mn²⁺ ion. The Km for l-arginine was 1.35 × 10^?2 m The activity was competitively inhibited by l-lysine, but not by l-ornithine and increased by the addition of Mn²⁺ or Co²⁺ ions. The stable pH and temperature ranges became wider in the presence of Mn²⁺ ion and l-threonine.     

枯草芽孢杆菌JA抗菌物特性的研究及抗菌肽的分离纯化

枯草芽孢杆菌JA分泌物中具有抑菌作用的粗提液通过DEAESepharose FF、SOURCE 15 PHE、Sephacry1 S200HR和反相HPLC多步柱层析分离纯化后,获得3种对水稻纹枯和小麦赤霉病菌均有抑菌作用的抗菌肽AFP1、AFP2和AFP3。MALDITOF质谱法测得其分子量分别为1462.645D、1476.390D和1490.530D。氨基酸组成分析结果表明,AFP1和AFP3由苏氨酸、异亮氨酸、酪氨酸、脯氨酸等多种氨基酸组成。目标产物热稳定性好、对蛋白酶有一定的耐受性,推断很可能是低分子量的环状脂肽。     

Long-Term Adaptation of Bacillus subtilis 168 to Extreme pH Affects Chemical and Physical Properties of the Cellular Membrane

We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.     

枯草芽胞杆菌甲壳素脱乙酰酶的筛选及酶学性质*

从海洋泥土中分离出产甲壳素脱乙酰酶菌株,确定该菌株为产碱属芽孢杆菌,其产酶适宜培养条件为：pH4．0,添加金属离子Ca^2 ,培养时间为80h,温度为350℃。所得甲壳素脱乙酰酶作用的最适温度为40℃～50℃,最适pH为4．5-5．0之间。     

Properties of glutamate dehydrogenase from Bacillus subtilis.

$\text{Bacillus subtilis}$ strain 168 possesses an NAD-dependent glutamate dehydrogenase. The level of this enzyme is influenced by the stage of growth, the source of nitrogen, and a high rate of tryptophan biosynthesis. The enzyme appears to serve an anabolic function and, therefore, must be considered as a possible route for the incorporation of inorganic nitrogen into an organic form.     

Antitumoral and Antimicrobial Activity of Surfactin Extracted from Bacillus subtilis KLP2015

International Journal of Peptide Research and Therapeutics - Surfactin lipopeptide (SLP) from Bacillus subtilis KLP2015 (Accession number KT459335) was extracted and purified by ammonium sulphate...     

Physical Properties and Kinetic Behavior of a Cephalosporin Acetylesterase Produced by Bacillus subtilis

An esterase that deacetylates cephalosporins was recovered from the supernatant of a Bacillus subtilis culture. It was partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme had a temperature optimum between 40 and 50 C and a pH optimum of 7.0. The molecular weight was estimated by gel filtration to be 190,000. The enzyme was very stable and retained greater than 80% of its activity after storage in solution at 25 C for 1 month. The esterase exhibited Michaelis-Menton kinetics with the substrates 7-aminocephalosporanic acid (7-ACA) and 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin); the K(m) values were 2.8 X 10(-3) and 8.3 X 10(-3) M, respectively. The products of 7-ACA deacetylation were weak competitive inhibitors, and a K(i) value of 5.0 X 10(-2) M was determined for acetate and of 3.6 X 10-2 M for deacetyl-7-ACA. Weak product inhibition did not prevent the deacetylation reaction from going to completion. A 5-mg/ml solution of partially purified esterase completely hydrolyzed (greater than 99.5%) a 24-mg/ml solution of 7-ACA in 3 h. Because of the kinetic properties and excellent stability, this enzyme may be useful in an immobilized form to prepare large quantities of deacetylated cephalosporin derivatives.