脂肪酸Bacillus pumilus羟基化调控基因fadD-like的克隆与序列分析

利用短小芽孢杆菌(Bacillus pumilus)的ω-1-羟基脂肪酸高产突变菌株M-F641、M-F81、M-F8272及B.pumilus20075的总DNA为模板,通过Primer Premier5.0软件设计引物,对决定长链脂肪酸无效降解途径中的关键酶基因fadD-like基因进行克隆,得到4条长约1 720 bp的fadD-like基因全序列;利用MEGA3.1、DNAStar等软件进行序列分析结合脂酰辅酶A合成酶系统进化分析,结果表明其与fadD基因具有较高的相似性,克隆得到的核苷酸为编码脂酰辅酶A合成酶的fadD基因序列;研究结果为长链脂肪酸高效转化生产ω-1-羟基脂肪酸提供基础。

羟基脂肪酸生产菌Bacillus pumilus对油酸的代谢特性和产物分析

一株具有转化油酸羟基化的微生物Bacillus pum ilus BD-12(记作BP BD-12),在摇瓶条件下分批深层培养,研究了菌株在以胰蛋白胨为主要氮源、葡萄糖为基本碳源的条件下对油酸(OA)的代谢特性,并对转化OA生成羟基脂肪酸的规律及产物进行了分析测定。结果表明:在只有葡萄糖作碳源条件下,细胞生长良好,且无羟基脂肪酸生成,OA对菌株BP BD-12细胞生长有抑制作用;用40 h细胞菌龄添加OA条件下其转化产物为ω-1、ω-2和ω-3-羟基脂肪酸,其转化率和羟基脂肪酸得率最大值分别达到63%和12.3%;通过气质联用方法对产物组分分析,其ω-1-羟基脂肪酸占总羟基脂肪酸的70%,细胞培养过程中未有其它羟基脂肪酸检出。因此,菌株BP BD-12是一株较理想的ω-1-羟基脂肪酸工业化生产菌株。

降解苯并[a]芘的Bacillus pumilus strain Bap 9菌株的分离、鉴定与降解特性

从长期受烧烤影响的土壤中,分离筛选出一株能利用苯并[a]芘作为唯一碳源和能源的高效降解菌Bap9,通过16S rRNA基因序列分析和部分生理生化特征分析,鉴定为Bacillus pumilus.摇瓶试验结果表明,菌株Bap9能在20d内将40mg/L的苯并[a]芘降解27.3%.通过对OD600和苯并[a]芘残留浓度的检测分析,考察了pH值、接种量和装液量对菌株生长和降解性能的影响.结果表明,菌株在pH值为6.0—9.0的条件下均能生长并能降解苯并[a]芘,降解的最适pH值为8.0,最佳接种量为5%,最佳装液量为10ml/50ml.

Bacillus pumilus菌剂与化肥配施对油麦菜生长和品质的影响

Bacillus pumilus菌株隶属芽孢杆菌属,其有利于促进植物的生长和营养吸收。为探讨该菌株对植物生长及营养品质的应用价值,将该菌株与化肥进行不同配施处理,进行油麦菜盆栽试验。结果表明,B.pumilus菌剂与化肥配施对油麦菜有促生作用且提高,其品质,氮磷肥与菌剂配施使油麦菜株高、鲜重和干物质量比对照分别高26.54%、353.95%和303.83%,根长、根鲜重、根干物质量分别高210.18%、163.89%和328.30%;化肥与菌剂配施对油麦菜的促生效果较单施菌剂亦有显著增加,菌剂与化肥配施处理后维生素C和总糖含量比对照分别高100.00%和283.50%,同时对油麦菜中的钾、镁、钠含量有一定的影响。另外,B.pumilus菌株具有解有机磷及无机磷的能力,这可能是其促进植株生长的作用机制。可见,B.pumilus菌剂与化肥配施后的促生效果较好,可促进油麦菜的生长及其品质的提高。

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

To optimize culture conditions for xylanase production by solid state fermentation （SSF） using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K2HPO4, 2.5 g/L; KH2PO4, 1.0 g/L; NaCl, 0.1 g/L; （NH4）2SO4, 2.0 g/L, CaCl2-2H2O, 0.005 g/L; MgCl2.6H2O, 0.005 g/L; and FeCI3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity （142.0 ±0.47 U/g DM] was obtained on the 6th day at 30℃ The optimized paddy husk to liquid fermentation medium ratio was 2：9, and the optimized culture temperature was 40℃. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized （224.2 U/g DM） in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production （290.7 U/g DM） was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum （326.5±0.34 U/g DM）. Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Thermostable alkaline protease production from Bacillus pumilus D-6 by using agro-residues as substrates

Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i>against Phytopathogenic Rna-Viruses

Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.

<i>Bacillus pumilus</i>: Possible Model for the Bioweapon <i>Bacillus anthracis</i>

The misuse of Bacillus anthracis as a bioweapon continues to be a serious concern. Medical personnel and researchers are served well if appropriate non-pathogenic anthrax simulants can be used as countermeasures in preparative planning. While there are several accepted simulants of B. anthracis, the addition of another model organism would be beneficial. This investigation was undertaken to evaluate the suitability of B. pumilus as a simulant for B. anthracis. All organisms were grown on AK Agar #2 to foster sporulation. Optimum conditions for spore formation were determined for B. pumilus as well as for currently used anthrax surrogates B. atrophaeus and B. thuringiensis. Spore dimensions were determined by scanning electron microscopy. Comparative antibody binding studies using commercially available anti-Bacillus antisera were completed with the simulants as well as with a negative control organism, Clostridium sporogenes. We report that B. pumilus sporulated readily (2.9 × 1010 viable spores per plate), had appropriate spore size (1.24 μm × 0.59 μm) and reactivity with anti-Bacillus antibodies. The characteristics of B. pumilus determined in this study suggest this organism represents a novel, suitable model for B. anthracis.

Growth Performance,Physicochemical Properties of Meat of Broiler Chicken’s Supplemented with Bacillus pumilus

This study examined that the efficiency of Bacillus pumilus(BP)on growth performance and meat quality of broiler chickens.A total of day old 240 male broiler chicks were purchased from local hatchery Hyderabad.The chicks were allocated into four groups with six replicates of 10 birds in each replicate.Four diets were prepared for experiment,which included control as basal diet,BP-0.1(CONT+0.1%BP),BP-0.2(CONT+0.2%BP)and AGP(CONT+zinc bacitracin 20 ppm)as antibiotic growth promotor.The results showed that live body weight(LBW),average daily gain was recorded higher(P<0.01)in birds fed diet with BP-0.1 than control and BP-0.2 groups,as well as ADFI also increased in BP-0.1 than control group.BP-0.1 supplementation significantly reduced feed conversion ratio(FCR)than other groups(P<0.01).Both moisture and crude protein(CP)content influenced by supplementation of BP-0.1 than other groups.However,birds fed diet with BP-0.1 had least fat content in breast meat compared with the other groups(P<0.01).In addition,BP-0.1probiotic-fed birds had the lowest(P<0.01)pH,cooking loss(CL)and drip loss(DL)as compared to control,BP-0.2 and AGP groups.The water holding capacity(WHC)were found higher in BP-0.1 as compared to other groups(P<0.01).In conclusion,Bacillus pumilus probiotic supplementation is a promising approach for substitution of antibiotics in broiler diet for enhancement of growth performance and physiochemical properties of meat.

水体中添加两种菌剂对凡纳滨对虾存活、生长及消化酶活力的影响

向水体中添加不同浓度的短小芽孢杆菌CGMCC 1004(Bacillus pumilus)和胶红酵母菌CGMCC1013(Rhodotorula mucilaginosa)以研究凡纳滨对虾(Litoenaeus vannamei)体长、体质量、存活率、胃蛋白酶、肝胰腺淀粉酶和肝胰腺脂肪酶的影响,以及需钠弧菌(Vibrio natriegens)的感染效果。结果表明:向水体中添加104CFU/mL短小芽孢杆菌能提高对虾体长、存活率,但结果不显著,但对体质量增长率具有显著提高作用;水体中添加104CFU/mL短小芽孢杆菌对提高需钠弧菌感染后对虾成活率有一定的提高作用;水体中添加104CFU/mL短小芽孢杆菌能显著提高胃蛋白酶活性,但是对肝胰腺淀粉酶活性有一定抑制作用;添加复合菌剂比添加单一菌种的作用要好;亦证明了向水体中添加细菌的方式对凡纳滨对虾的体长、体质量、存活率以及水质指标影响比较小,但是对消化酶具有一定的提高作用。

不同碳源对苯并[a]芘降解菌生长和降解性能影响的研究

采用从受户外烧烤影响的土壤中分离出的苯并[a]芘降解菌Bacillus pumilus strain Bap9,利用摇床实验研究了不同外加碳源对其生长和苯并[a]芘降解性能的影响。结果表明,苯并[a]芘初始浓度为40 mg/L时,蔗糖、葡萄糖、麦芽糖的存在会抑制菌株对苯并[a]芘的降解,可溶性淀粉的影响不明显。以醋酸钠为外加碳源可明显促进菌株的降解作用,添加40 mg/L的醋酸钠,20天后可将菌株Bap9的降解率提高了8.8%,但过量的碳源会抑制菌株对苯并[a]芘的降解。添加适量的低分子量菲(PAH)作为共代谢底物,菲的促进作用大于同浓度的醋酸钠,添加40 mg/L的菲,20天后菌株对苯并[a]芘的降解率提高了26.4%。

短小芽孢杆菌E601传代和中子辐照后的菌落形态变化

为研究短小芽孢杆菌E601不同形态菌落分布情况及中子辐照对其影响,对短小芽孢杆菌不同形态菌落进行了反复传代,并采用CFBR-Ⅱ快中子脉冲反应堆分别对不透明菌落进行了低、中、高剂量的一次中子辐照和二次中子辐照,发现:(1)在正常传代条件下,E601短小芽孢杆菌有半透明型和不透明型菌落存在;(2)经反复传代,半透明菌落可产生各占一半的半透明型与不透明型两种菌落形态,而不透明菌落基本不产生半透明菌落;(3)中子辐照对菌落分布影响较大,随着一次中子辐照剂量的升高,半透明菌落所占的比例逐渐增加;当中子二次辐照剂量最高时,半透明菌落所占比例最大。因此认为E601短小芽孢杆菌之所以能产生两种不同形态菌落,主要是由半透明菌落的不稳定性决定的。在中子辐照中,半透明菌落型菌株耐辐照能力高于不透明型菌落菌株。

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。

直接/间接作用模式下一株短小芽孢杆菌对蒙脱石层间域特性的影响研究

为探讨直接/间接作用模式下土壤常驻菌对蒙脱石层间距、层电荷及层间介质的影响,从绵阳地区紫色土壤中分离选取1株短小芽孢杆菌,利用透析袋,建立土壤菌与蒙脱石"接触"与"非接触"体系进行相互作用实验。作用5 d后的结果表明,蒙脱石样品层间距变小,样品M-1和M-2的d001值从1.486 nm分别减小到1.246 nm和1.257 nm,单位半晶胞层电荷数减少,且ξMMT>ξM-2>ξM-1,FTIR分析显示作用后蒙脱石样品出现来自蛋白质酰胺Ⅱ带及蛋白质分子甲基的新特征峰,层间水分子3434 cm-1、1637 cm-1处的峰发生偏移,此外阳离子交换容量也发生明显改变。土壤菌的生物作用能改变蒙脱石的层间距、层电荷及层间介质,使其结构和性质发生改变。对比2种作用方式与作用后蒙脱石层间域特性的改变分析可知,接触作用对蒙脱石层间域特性的改变效果强于非接触作用。

Screening and Identification of Antagonistic Strain against Phytophthora sojae

[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.

短小芽孢杆菌BPS48对马铃薯生长的影响

以天薯11号为指示品种,研究了短小芽孢杆菌(Bacillus pumilus)BPS48对马铃薯生长期间病害及产量的影响。结果表明,马铃薯用短小芽孢杆菌(Bacillus pumilus)BPS48可湿性粉剂300倍液灌根后,病毒病发病率较轻;折合产量最高,为14 200 kg/hm^2,较空白对照增产2 250 kg/hm^2,增产率18.8%。用芽孢杆菌可湿性粉剂45 g/L浓度下浸种30 min后,马铃薯折合产量为10 600 kg/hm^2,较空白对照减产1 350 kg/hm^2,减产率11.3%。

生防菌诱导水稻抗病性相关酶活性的变化

将生防菌bio—2和稻瘟病菌（Magnaporthe grisea）接种于正处于分蘖期的水稻植株上，测定其与植物抗病有重要相关的苯丙氨酸解氨酶（Phenlyalanine ammonia lyase PAL）、过氧化物酶（Peroxidase，POD）和多酚氧化酶（Polyphenoase，PPO）活性的变化．结果显示，接种处理后，POD活性明显高于对照，在接种后第5d。稻瘟病菌、稻瘟病菌＋生防菌bio-2处理组的POD活性达到最大值，但到接种后第8d开始略低于对照；稻瘟病菌＋生防菌bio-2、生防菌bio-2处理组，其叶片内的PAL活性仅在第1—2d内高于对照，其后均低于对照；在稻瘟病菌＋生防菌bio-2、生防菌bio-2处理组，其PPO活性除在第2d高于对照外，其余均低于对照．

短小芽孢杆菌阿魏酸酯酶的分离纯化及性质研究

通过硫酸铵沉淀、离子交换层析和疏水层析,对短小芽孢杆菌(Bacillus pumilus)发酵液中的阿魏酸酯酶进行分离纯化,并测定其部分酶学性质。结果表明,阿魏酸酯酶的纯化倍数为5.06,回收率达到14.8%。该酶的相对分子质量约为28.6 ku;最适反应温度为50℃,最适pH为5.8,在40~45℃和pH 5.8~8.2都能保持很高的稳定性。K^+、Co^(2+)、Ca^(2+)和DTT对酶活有明显的促进作用,而Cu^(2+)、乙醇和苯甲基磺酰氟严重抑制了其酶活。以阿魏酸甲酯为底物,测得Km为0.25 mmol/L,Vmax为6.27 U/(min·mg)。

产碱性蛋白酶菌株Zkud202-4发酵条件的研究

对土壤中分离的一株产耐盐碱性蛋白酶的短小芽孢杆菌(Bacillus pumilus)Zkud202-4株的发酵条件进行了研究,通过单因素试验和正交试验,确定了最适产酶培养基组成:pH9.0、基础培养基中加入质量分数1.5%的葡萄糖为碳源、1.5%的蛋白胨为氮源、0.01%的Ca2+为金属离子.最适发酵条件:接种量V(种子培养液)∶V(培养基)=2%、发酵温度36℃、发酵时间45 h.优化后菌株Zkud202-4碱性蛋白酶的酶活力达到383 U/mL.

用三种不同方法保藏短小芽孢杆菌的比较研究

据前人研究,不同微生物应采取不同的方法进行保藏。我们利用短小芽孢杆菌产生碱性蛋白酶的特点,以其酶活性变化为指标比较了冷冻干燥法、沙土法及定期移植法三种不同方法的优劣,考查不同保藏方法对微生物生理活性的影响,为微生物的菌种保藏工作提供依据。