Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus

A phytoplankton-lytic (PL) bacterium, Bacillus cereus, capable of lysing the bloom-forming cyanobacterium Aphanizomenon flos-aquae was isolated from Lake Dianchi of Yunnan province, China. This bacterium showed lytic activities against a wide range of cyanobacteria/algae, including A. flos-aquae, Microcystis viridis, Microcystis wesenbergi, Microcystis aeruginosa, Chlorella ellipsoidea, Oscillatoria tenuis, Nostoc punctiforme, Anabaena flos-aquae, Spirulina maxima, and Selenastrum capricornutum. Chlorophyll a contents, phycocyanin contents, and photosynthetic activities of the A. flos-aquae decreased evidently in an infected culture for a period. Bacterium B. cereus attacked rapidly A. flos-aquae cells by cell-to-cell contact mechanism. It was shown that the lysis of A. flos-aquae began with the breach of the cyanobacterial cell wall, and the cyanobacterial cell appeared abnormal in the presence of the PL bacterium. Moreover, transmission electron microscope examinations revealed that a close contact between the bacterium and the cyanobacterium was necessary for lysis. Some slime extrusions produced from B. cereus assisted the bacterial cells to be in close association with and lyse the cyanobacterial cells. These findings suggested that this bacterium could play an important role in controlling the Aphanizomenon blooms in freshwaters.     

一株地衣芽胞杆菌产碱性蛋白酶条件优化

【背景】碱性蛋白酶是众多芽胞杆菌的发酵产物,是工业上极其重要的一类酶。【目的】利用酪素培养基从环境样品中筛选出一株产碱性蛋白酶的菌株,对传代次数、发酵的碳源、氮源、金属离子、磷酸盐、初始pH、接种量和温度进行优化,提高其产碱性蛋白酶的能力并降低发酵成本。【方法】采用革兰氏染色法、扫描电镜、生理生化试验、16S rRNA基因序列对分离的菌株进行鉴定;采用单因素、Plackett-Burman、最陡爬坡和响应面试验优化碱性蛋白酶的发酵条件,使用Minitab对试验数据进行分析。【结果】经鉴定分离菌株为地衣芽胞杆菌,命名为Bacillus licheniformis NWMCC0046。优化后的发酵培养基组成为（g/L）:豆粕50.00,葡萄糖10.00,酵母浸膏13.46,CaCl₂ 0.50,Na₂HPO₄·12H₂O 4.00,KH₂PO₄ 0.30;优化后的培养条件为:pH 7.5,34.81℃,接种量4.13%。在此条件下,摇瓶发酵48 h时碱性蛋白酶...     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53

Rice bran and yeast extract were found to be the best combination of carbon and nitrogen sources for the production of carboxymethycellulase (CMCase) by Bacillus subtilis subsp. subtlis A-53. Optimal concentrations of rice bran and yeast extract for the production of CMCase were 5.0% (w/v) and 0.10% (w/v), respectively. Optimal temperature and initial pH of medium for cell growth of B. subtilus subsp. subtilis A-53 were 35 °C and 7.3, whereas those for the production of CMCase by B. subtilus subsp. subtilis A-53 were 30 °C and 6.8. Optimal agitation speed and aeration rate in a 7 L bioreactor were 300 rpm and 1.0 vvm, respectively. The optimal agitation speed and aeration rate for the production of CMCase by B. subtilus subsp. subtilis A-53 were lower than those for cell growth. The highest productions of CMCase by B. subtilus subsp. subtilis A-53 in 7 and 100 L bioreactors were 150.3 and 196.8 U mL⁻¹, respectively.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

一株自中国大鲵的副炭疽芽孢杆菌分离和鉴定

[目的]对陕西某大鲵养殖场患病的中国大鲵腹水中分离培养得到的一株蜡样芽孢杆菌群细菌疑似菌株进行鉴定,明确该菌生长特性和种类。[方法]无菌解剖患病大鲵,取肠道、腹水、皮肤等各部位的样品均质稀释并分离纯化,从腹水中获得疑似蜡样芽孢杆菌群细菌的纯菌株,命名为SHOU-BC01。对该菌株进行形态与染色特性、培养与生化特性、生物膜形成能力、芽孢形成、药敏检测、全基因组测序等试验鉴定,并根据测序结果进行平均核苷酸相似度(average nucleotide identity,ANI)、数字DNA-DNA杂交(digital DNA-DNA hybridization,dDDH)、多位点序列分型(multilocus sequence typing,MLST)、全基因组SNP聚类和毒力因子分析。[结果]菌株SHOU-BC01为革兰氏阳性杆菌,表面粗糙;具有蛋白酶、卵磷脂酶和溶血酶活性;能够发酵L-阿拉伯糖、D-核糖、D-木糖等多种糖类,能利用色氨酸、丙酮酸盐等;有较强生物膜形成能力;120 h的芽孢形成率达到70.60%;该菌株对青霉素G、头孢噻吩、万古霉素等15种抗生素耐药,对哌拉西林、头孢唑啉、庆大霉素等25种抗生素敏感;根据生物学特性结合ANI、dDDH及全基因组SNP聚类分析,鉴定菌株SHOU-BC01为副炭疽芽孢杆菌(Bacillus paranthracis),经MLST分型,该菌株属于ST205序列型;该菌株含有鞘磷脂酶、CytK和NheC毒素、多糖荚膜、PlcR-PapR群感效应系统及Ⅶ型分泌系统等毒力因子。[结论]成功从中国大鲵腹水中分离出副炭疽芽孢杆菌,丰富了大鲵副炭疽芽孢杆菌数据。     

Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group

Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12 095 bp), pFR12.5 (12 459 bp) and pFR55 (55 712 bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study of the molecular basis of the conjugative process in Gram positive bacteria, particularly due to the similarity with known conjugation systems. It is also a contribution to the expansion of the non-pathogenic B. cereus plasmid gene pool.     

反复冻融对枯草芽孢杆菌St-zn-34抑制链格孢菌活性的影响北大核心CSCD

【目的】以从冬枣分离的内生枯草芽孢杆菌(Bacillus subtilis St-zn-34)为供试菌株,明确反复冻融对菌体形态及其发酵滤液抑制枣缩果病初侵染病菌(Alternaria alternata)活性的变化。【方法】对供试菌株分批发酵后进行反复冻融,采用梯度稀释计数法、滤纸片法分别测定活菌数量和芽孢含量、发酵滤液抑菌活性,对供试菌株的形态变化进行电镜观察。【结果】发酵培养中pH值、活菌量、芽孢量及抑菌活性随发酵时间的增加均呈先上升后下降的趋势,其中发酵60 h发酵滤液抑菌活性最大,对此时的发酵液反复冻融,冻融3次枯草芽孢杆菌活菌量和发酵滤液抑菌活性依次减少,以后再冻融差异不显著(P>0.05),电镜观察发现随冻融次数增加,菌体变小,表面凹陷、扭曲,胶状物流出。抑菌谱检测发现发酵滤液对12种植物病原菌具有抑菌能力。不同温度和蛋白酶处理发酵滤液表明,低于60℃以下,抑菌活性与对照相比差异不显著;80℃以上抑菌活性随温度上升而下降,与对照相比差异显著;蛋白酶K可降低抑菌活性。【结论】反复冻融影响细菌形态并降低发酵滤液的抑菌活性。     

Hymenopteran specificity of Bacillus thuringiensis strain PS86Q3

The biological activity of Bacillus thuringiensis (Bt) strain PS86Q3 against five Hymenopteran species was determined by means of bioassays adapted to each species. Four species of sawfly that are important pests of conifers (Diprion pini, Gilpinia hercyniae and Pristiphora abietina) or ornamental plants (Arge rosae), as well as the non-target honeybee, Apis mellifera, were studied. Two out of the four sawfly species tested were found to be sensitive to PS86Q3 crystals or spore/crystal suspensions. A sporulated culture of this strain was moderately active on D. pini, and a complete bioassay with solubilized crystals was performed to estimate the LC₅₀ of 4.9 mg/ml. Pristiphora abietina was also found to be sensitive to PS86Q3, with an LC₅₀ of 1.6 mg/ml. By contrast, at the concentrations tested, PS86Q3 did not prove active on the remaining sawflies, G. hercyniae and A. rosae. The strain was administered orally to check its effects on honeybees which were fed sucrose solutions supplemented with a PS86Q3 sporulated suspension, in a field assay using commercial beehives. No significant differences in larval mortality (as deduced by comparing the number of larvae, pupae and empty cells) were found between the Bt and control treatments. On the basis of the results presented here, the suitability of PS86Q3 for the control of Hymenopteran pests, particularly sawflies, in terms of both potency and environmental safety, is discussed.     

贝莱斯芽孢杆菌Bv-303对水稻白叶枯病菌的拮抗活性及其应用

本研究明确了一株新型贝莱斯芽孢杆菌(Bacillus velezensis) Bv-303菌株对黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv. oryzae,Xoo)的拮抗活性及其对水稻白叶枯病(bacterial-blight,BB)的生物防治效果。采用牛津杯法测定了菌株Bv-303发酵上清液(cell-free supernatant, CFS)对白叶枯病菌体外拮抗的活性及其稳定性;通过对接种白叶枯病菌的水稻叶片进行喷雾处理,在水稻体内测试了该菌株发酵液(cell-culture broth,CCB)、发酵上清液及菌悬液(cell-suspension water,CSW)对白叶枯病菌的抑制效果;并统计了该菌株对水稻种子发芽率与幼苗生长的影响。结果表明,在体外,菌株Bv-303发酵上清液对白叶枯病菌的生长抑制率可达85.7%–88.0%,对热、酸、碱、紫外线等具有较好的稳定性;在水稻叶片上,喷施该菌株的发酵液、发酵上清液及菌悬液均能提高植株对白叶枯病的抗性,其中发酵液的效果最佳,抗病性提高率高达62.7%;且发酵液对水稻种子萌发和幼苗生长均没有副作用。因此,菌...     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定

【目的】为了筛选防治水稻条斑病(bacterial leaf streak,BLS)的生防细菌。【方法】以水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的模式菌株RS105为靶标菌,采用平板稀释和抑菌圈法,从空心菜根际土壤中筛选到一株对RS105具有拮抗作用的细菌菌株504。通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对菌株504进行了鉴定。利用牛津杯法测定504对植物病原黄单胞菌的拮抗活性及其无菌发酵液拮抗活性的稳定性。通过PCR扩增预测504编码合成脂肽类和聚酮类化合物的合成相关基因。采用苗期水稻注射接菌法来评价水稻组织中504对Xoc的拮抗活性。【结果】菌株鉴定结果表明504为贝莱斯芽孢杆菌,命名为Bacillusvelezensis504。抑菌实验显示,B.velezensis504对黄单胞菌属的细菌具有较好的抑菌活性,对水稻白叶枯病菌(X. oryzae pv. oryzae,Xoo)的拮抗效果最显著。基因预测结果显示,B. velezensis 504含有fenA、dhbA、sfrA、bmyA、beaS、dfnA及bacA等编码脂肽类和聚酮糖类抑菌化合物的基因簇。其无菌发酵液的活性物质耐高温和蛋白酶降解,但不耐强酸、强碱,在pH值为5.5–8.9时仍具有稳定的拮抗活性。在高感水稻品种原丰早上,B. velezensis 504对Xoc在水稻叶片中引起的水渍症状具有显著的抑制作用。【结论】B. velezensis 504能够特异性拮抗黄单胞菌,在黄单胞菌引起的细菌性病害的生物防治中将具有较大的应用潜力。     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

苏云金芽胞杆菌rocE基因的转录调控

【目的】rocE基因编码精氨酸降解途径中的精氨酸通透酶,通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt) rocE基因的转录活性,明确rocE基因的转录调控机制。【方法】通过RT-PCR确定rocE基因所在基因簇的转录单元;β-半乳糖苷酶活性测定分析rocE基因启动子(ProcE)的转录活性;采用同源重组技术敲除BtHD73菌株的rocE基因;通过融合His标签的方法在大肠杆菌中表达纯化RocR蛋白的HTH结构域;通过凝胶阻滞实验明确RocR与rocE基因启动子的结合作用。【结果】在M9培养基中,精氨酸可诱导ProcE的转录活性;在SSM培养基和精氨酸诱导培养基中,与出发菌株HD73相比,ProcE在sigL (编码Sigma54因子)突变体和rocR突变体中的转录活性显著下降。RocR-HTH蛋白与ProcE有结合作用。rocE基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响。rocE缺失突变体的芽胞形成率为65.5%,HD73出发菌株为85.7%,显著性分析结果表明差异显著(P0.05)。【结论】rocE基因的转录活性受Sigma54的控制,并受RocR正调控。rocE基因的缺失影响菌株的芽胞形成率。     

Characterization of a cryptic plasmid pPZZ84 from Bacillus pumilus

The complete nucleotide sequence of a cryptic plasmid pPZZ84 from Bacillus pumilus strain ZZ84 was determined. Plasmid pPZZ84 is 6817 bp long with GC content of 36.7%. Seven putative open reading frames were identified. ORF7 shows 91% and 90% amino acid identity with rep proteins of pSH1452 and pPL1, respectively, members of rolling-circle replication (RCR) pC194-family. A typical pC194-family double strand origin (dso), a single-stranded origin (sso) and rap (regulator aspartate phosphatase) proteins were also identified in the plasmid. These results imply that pPZZ84 belongs to the Bacillus subtilis species group of small rolling circle (BsSRC) replicating plasmids. The plasmid copy number of pPZZ84 in B. pumilus ZZ84 was estimated to be 46 per cell, more than that of other BsSRC plasmids in their hosts.     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

苏云金芽胞杆菌抗菌脂肽初步鉴定及抑菌活性测定

植物枯萎病是影响作物生长的重要因素，利用植物内生菌拮抗病原菌生长，从而降低其危害程度是目前研究的热点。本研究从健康的番茄植株中筛选分离得到一株对番茄枯萎病病原菌有较强拮抗作用的内生细菌B-R1,通过形态学、生理生化以及分子生物学检测分析，鉴定该菌株为苏云金芽胞杆菌(Bacillus thuringiensis)。为进一步探索该菌株的生防作用，首先将菌株发酵后离心，得到发酵上清液，采用盐酸沉淀法对其活性物质进行粗提，并检测其对大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)以及尖孢镰刀菌(Fusarium oxysporum)的抑菌活性。通过薄层色谱法(TLC)、傅里叶红外光谱(FI-TR)、高分辨液相色谱-质谱联用仪(LC-MS)分析并鉴定活性物质的结构。结果表明，苏云金芽胞杆菌对尖胞镰刀菌有良好的抑制作用，结构分析初步鉴定抗菌物质中含有丰原素(fengycin),属于脂肽类抗生素。     

一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性

【背景】感染产气荚膜梭菌会引起动物坏死性肠炎,通常使用抗生素进行预防和治疗。随着我国饲料禁抗、养殖减抗的实施,寻找绿色微生态制剂及其代谢产物成为当前研究的热点。【目的】旨在研究前期筛选的一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性。【方法】检测了菌株生长曲线、代谢物质的抑菌特性及细菌素基因簇mRNA表达。【结果】枯草芽孢杆菌BS-2代谢物质对革兰氏阴性菌无抑制作用,而对革兰氏阳性菌具有较强的抑菌性能,并且对产气荚膜梭菌的抑菌性能在2-12 h内迅速增长,在12-24 h内抑菌性能较稳定;该抑菌性能不受胃蛋白酶、胰蛋白酶、蛋白酶K的影响,具有良好的热稳定性;进一步分析抑菌物质基因簇mRNA表达,发现枯草芽孢杆菌BS-2抑制产气荚膜梭菌的活性可能与表面活性素(surfactin)和美杀菌素(mersacidin)表达有关。【结论】枯草芽孢杆菌BS-2对产气荚膜梭菌具有较强的抑制作用,可能通过抑菌物质surfactin和mersacidin表达发挥作用。