一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究

从堆肥中分离到一株对植物病原菌尖孢镰刀菌(Fusarium oxysporum)具有强烈抗菌活性并具有较广抗菌谱的细菌Q-12菌株。通过形态观察、生理生化实验1、6S rDNA同源性序列分析以及部分特异性基因序列分析,鉴定该菌为解淀粉芽孢杆菌。该菌的最适培养基组成为:葡萄糖5g/L,NH4Cl 1g/L,牛肉膏0.8g/L,氯化镁5g/L。最适培养温度为33℃,最适培养pH为6.0,最适培养时间为40h。     

Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03

The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides. The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography. Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic. The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2).     

Re-identification of the halotolerant, biosurfactant-producing Bacillus licheniformis strain JF-2 as Bacillus mojavensis strain JF-2

Bacillus strain JF-2 (ATCC 39307) is a halotolerant, biosurfactant-producing bacterium that was initially described as a member of the species Bacillus licheniformis based on a limited set of phenotypic characteristics. Here, genetic and phenotypic analyses were employed to determine the relationship of Bacillus strain JF-2 to other Bacillus strains. The restriction patterns with AluI and analysis of gyrA and 16S rRNA gene sequences grouped Bacillus strain JF-2 with B. mojavensis^T and not with B. licheniformis^T. DNA–DNA similarity showed JF-2 was 75% similar to B. mojavensis^T and only 11% similar to B. licheniformis^T. Both strain JF-2 and B. mojavensis^T required DNA for anaerobic growth, but B. licheniformis^T did not. B. mojavensis^T and strain JF-2 did not grow anaerobically in thioglycollate medium or aerobically with propionate while B. licheniformis^T grew under these conditions. DNA–DNA similarity, gene sequence data and phenotypic characteristics all support the assignment of JF-2 as a member of the species B. mojavensis.     

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.Lei Wang, Yun Tang and Shuo Wang contributed equally to this study.     

Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60 degrees C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade p-chlorobiphenyl, suggesting the existince of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra- and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA.     

Isolation and characterization of a Bacillus strain capable of degrading the extracellular glucan from Cellulomonas flavigena strain KU

P.A. BERTRAM, C.S. BULLER, G.C. STEWART AND J.M. AKAGI. 1993. Bacteria capable of utilizing the water-insoluble purified extracellular (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU by extracellular enzymes, were insolated and characterized. Enrichment cultures from a Winogradsky column were incubated anaerobically at 55C with curdlan as the sole source of carbon. Colonies surrounded by zones of clearing were selected from subcultures on solid curdlan media. One of the isolates was chosen for further study and identified by conventional methods, API-tests with calculation of similarity coefficients and ID-scores, estimation of mol% (G + C) and DNA-DNA liquid hybridization. The isolate is a facultatively anaerobic, facultatively thermophilic Bacillus sp. Identification at the species-level was not achieved. The isolate was characterized by some rare traits among bacilli, but it remains unresolved whether it defines a new taxon.     

一株可积累鸟苷菌株的纯培养条件研究

菌株Bacillus.subtilis.S3 68是以鸟苷生产菌株B .subtilis.A0 66为出发菌经诱变所得。对该菌株进行培养条件研究的过程中 ,发现该菌株可以在摇瓶纯培养条件下积累鸟苷。试验结果表明 :发酵过程中 ,腺嘌呤的用量 0 .3 5mg/ml时 ,发酵液中鸟苷积累量最大 ,培养基中腺嘌呤的用量高于或低于 0 .3 5mg/ml均不利于鸟苷产物的积累 ;培养基中味精、硫酸铵、硫酸镁、磷酸二氢钾及Mn2 +用量显著影响发酵液中鸟苷积累水平 ;培养基中生物素、蛋氨酸、精氨酸、组氨酸、氯化钙及Fe2 +、Zn2 +用量与鸟苷积累的相关性不显著     

Biosurfactant production by a thermophilic Bacillus subtilis strain

A strain of Bacillus subtilis was able to grow and produce a biosurfactant on 2% sucrose at 45°C. As a result of biosurfactant synthesis the surface tension of the medium was reduced from 68 dynes cm⁻¹ to 28 dynes cm⁻¹. The strain had the capacity to produce the biosurfactant at high NaCl concentrations (4%) and a wide range of pH (4.5–10.5). The biosurfactant retained its surface-active properties after heating at 100°C for 2 h and at different pH values (4.5–10.5). A maximum amount of biosurfactant was produced when urea or nitrate ions were supplied as nitrogen source. The use of the biosurfactant at high temperatures, acidic, alkaline and saline environments is discussed. As a result of its action, 62% of oil in a sand pack column could be recovered, indicating its potential application in microbiologically enhanced oil recovery. Received 28 March 1996/ Accepted in revised form 16 September 1996     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

枯草芽孢杆菌内切木聚糖酶的分离纯化与鉴定

目的：建立一种简便、快速的木聚糖酶分离和提取方法。方法：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,分离纯化枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中的木聚糖酶,进一步用薄层色谱和高压液相色谱对木聚糖酶进行鉴定。结果：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,从枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中分离得到了两种内切木聚糖酶,酶解桦木木聚糖的产要产物以木二糖和木三糖为主。结论：活性聚丙烯酰胺凝胶电泳和均质提取法相结合是一种新的分离纯化木聚糖酶的简便、有效方法。     

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization

The gene encoding copper-dependent laccase from Bacillus subtilis strain R5 was cloned and expressed in Escherichia coli. Initially the recombinant protein was produced in insoluble form as inclusion bodies. Successful attempts were made to produce the recombinant protein in soluble and active form. The laccase activity of the recombinant protein was highly dependent on the presence of copper ions in the growth medium and microaerobic conditions during protein production. The purified enzyme exhibited highest activity at 55 °C and pH 7.0. The recombinant protein was highly thermostable, albeit from a mesophilic source, with a half-life of 150 min at 80 °C. Similar to temperature, the recombinant protein was stable in the presence of organic solvents and protein denaturants such as urea. Furthermore, the recombinant protein was successfully utilized for the degradation of various synthetic dyes reflecting its potential use in treatment of wastewater in textile industry.

Abbreviations: ABTS,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CBB, Coomassie brilliant blue; SGZ, syringaldazine; DMP, 2,2-dimethoxy phenol.     

新型抑菌蛋白APn5抑制胡萝卜软腐欧文氏菌

[目的]魔芋软腐病是一种由胡萝卜软腐欧文氏菌引起的病害,严重影响了魔芋产业的发展.枯草芽胞杆菌BSn5所产生的蛋白APn5对胡萝卜欧文氏菌具有良好的抑菌效果,本文将对该蛋白进行鉴定和性质分析.[方法]用饱和度为30%的硫酸铵沉淀和超滤离心的方法从枯草芽胞杆菌BSn5的发酵上清液中分离纯化APn5蛋白.以滤纸片法检测APn5蛋白的抑菌谱.用不同的温度、pH条件和蛋白酶处理APn5蛋白,并检测其抑菌活性变化.测定菌株BSn5的生长周期,同步检测其发酵上清液的抑菌活性.利用基质协助激光解吸附离子化-飞行时间质谱(MALDI-TOF-MS)和电喷雾四极杆飞行时间串联质谱(Q-TOF)分析APn5蛋白.[结果]APn5蛋白对于魔芋软腐病菌具有较好的抑菌活性,抑菌谱较窄,主要对细菌性植物病原菌具有较强的抑制作用;对高温敏感,对蛋白酶E敏感,对蛋白酶K和胰蛋白酶部分敏感;在酸性环境下其抑菌活性稳定,碱性环境下活性不稳定.在菌株BSn5的生长周期中,该蛋白的抑菌活性不能稳定存在,其发酵上清液在对数生长期抑菌活性逐渐升高,进入稳定期后抑菌活性开始下降,直至完全消失.质谱分析未发现其与已知蛋白具有高度相关性.[结论]APn5蛋白具有不同于已报道的枯草芽胞杆菌所产生的抑菌物质的性质,推测为一种新型的抑菌蛋白.     

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

一株枯草芽孢杆菌的分离鉴定、生物学特性及其对水质净化的作用

[背景]随着水产养殖业的发展和养殖集约化程度的提高,养殖水环境日趋恶化,养殖动物病害频发,而水产益生菌因其环境友好、安全而被广泛应用于水产养殖中.[目的]从南美白对虾养殖池底泥分离枯草芽孢杆菌,探究其体外生物学特性及对水质的净化作用,以期扩充微生态制剂的种质资源.[方法]采用稀释涂布平板法分离菌株,通过形态学观察、生理...     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

枯草芽孢杆菌BS-26菌株纤溶酶的性质分析及活性组分的分离纯化

[目的]溶栓疗法是血栓性疾病安全且有效的治疗手段,从微生物中寻找溶栓药物是一种理想有效的途径,枯草芽孢杆菌(Bacillus subtilis)BS-26菌株发酵液具有很强的体外纤溶活性,本文分析了发酵液中纤溶酶的性质并对活性组分进行了分离纯化.[方法]利用纤维蛋白平板法检测纤溶酶活性,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow阴离子交换层析和聚丙烯酰胺制备电泳等方法,进行分离纯化.[结果]此菌株产生的纤溶酶在50℃以下和pH5.0～11.0范围内具有较好的稳定性,最适作用温度为42℃;最适pH值为9.0;Mg2 、Ca2 对此酶有明显的激活作用,而Cu2 能完全抑制酶的活性;174.2μg/mL的苯甲基磺酰氟、1000μg/mL的鸡卵类粘蛋白和1000μg/mL大豆胰蛋白酶抑制剂能完全抑制酶活性,初步说明此酶属于丝氨酸蛋白酶类;体外溶纤作用表明,该酶溶解纤维蛋白的方式是直接溶解,而不是通过激活纤溶酶原.从该菌株的发酵液中获得了一种纤溶酶组分,比活力达8750 U/mg,回收率为3.2%,所获得样品纯度相对于发酵液提高了41倍,该酶在SDS-PAGE中是单肽链蛋白,分子量为32 kDa.[结论]获得了一种纤溶酶的单一组分,为纤溶酶发酵产品的大规模纯化及进一步研制和开发新的溶栓药物提供重要理论依据.     

一株多功能芽孢杆菌的鉴定

【摘 要】 目的 从土壤筛选所得1株芽孢杆菌并对该菌进行鉴定.方法 通过表型特征、生理生化特性和16S rDNA序列同源性分析（Gen Bank登录号为: JN609382）。结果 形态学观察：菌落呈圆形,白色微黄,中央凸起,半透明,长时间培养容易形成褶皱;革兰染色为阳性;芽孢中生,周生鞭毛,细长且多。生理生化特征：参照《常见细菌手册》和《伯杰氏细菌鉴定手册》,其中接触酶反应、V-P测定、卵磷脂以及革兰染色呈阳性;能够利用D-葡萄糖、D-木糖、D-甘露醇产酸,并能使淀粉水解;不能利用丙酸盐,不能形成吲哚,不能水解酪氨酸,生理生化试验结果显示该芽孢杆菌与枯草芽孢杆菌的特征一致。分子生物学鉴定：在Gen Bank上经过BLAT分析,其与枯草芽孢杆菌的相似性最高（99％）。结论 鉴定该菌为枯草芽孢杆菌。     

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

Gene cloning and characterization of a second alanine racemase from Bacillus subtilis encoded by yncD

Alanine racemase activity was investigated in Bacillus subtilis. A putative second alanine racemase gene (yncD) was cloned in parallel with the previously identified alanine racemase gene, dal. Each of the B. subtilis genes, dal and yncD complemented the Escherichia coli Alr- DadX- double mutant alanine auxotrophic strain MB2159 in vivo, restoring the prototrophic phenotype. Alanine racemase activity was also detected in vitro in cell-free extracts prepared from cultures of E. coli MB2159 harboring plasmids expressing either of the cloned B. subtilis genes and preliminary characterization of enzyme activity is presented.