Bacillus sp.B110胞内麦芽糖淀粉酶基因克隆与酶学特性

目的:对菌株Bacillus sp.B110的胞内麦芽糖淀粉酶BMAL进行基因克隆、异源表达、纯化及酶学性质研究,为后期开发新的淀粉加工用酶打下基础。方法:使用PCR技术对Bacillus sp.B110的胞内麦芽糖淀粉酶bmal基因序列进行全长克隆,异源表达,使用Ni2+-NTA进行纯化,再对其酶学特性进行测定,使用序列分析工具BioEdit、MEGA等对其氨基酸序列进行分析,使用AlphaFold2对其三级结构进行预测分析。结果:BMAL基因全长1770 bp,编码一个589氨基酸残基的蛋白。重组酶rBMAL经Ni2+-NTA亲和层析纯化后,SDS-PAGE电泳结果显示其分子量大小为63 kDa。氨基酸序列分析和三维建模表明BMAL与来源于B.subtilis 168和B.subtilis SUH4-2的麦芽糖淀粉酶有较高的一致性,且BMAL具有一个麦芽糖淀粉酶所独有的N端结构域以及由Asp328-Glu357-Asp424三个氨基酸残基所构成的催化中心。重组酶rBMAL最适反应温度为45℃,最适反应pH为6.0。重组酶rBMAL在30℃条件下保藏7 h残留酶活为60%,但在60℃条件下保藏2 h残留酶活力下降98%,说明BMAL对热敏感。重组酶rBMAL在4℃,pH7.0~9.5保藏12 h活性稳定。当存在1 mmol/L的金属离子Mg2+时,重组酶rBMAL活力提高36%,而Ni2+、Fe3+、Co2+、Cu2+、Zn2+、Al3+、Ca2+对重组酶rBMAL有抑制作用,酶活力减少85%~48%。有机溶剂和化学试剂甲醇、乙醇、丙酮、异腈、EDTA和SDS对重组酶rBMAL有较强的抑制作用,酶活力减少至32.3%~64.8%。底物特异性实验结果证实BMAL最适底物为环精糊。结论:Bacillus sp.B110的胞内麦芽糖淀粉酶BMAL具有良好的催化特性和pH稳定性,在面包烘焙工业上具有潜在的应用价值。

U(Ⅵ)在芽孢杆菌Bacillus sp.dwc-2上的矿化动力学研究

微生物诱导的U(Ⅵ)生物矿化会影响铀在环境中的化学形态和迁移行为,但目前对于生长和繁殖阶段的微生物诱导U(Ⅵ)矿化的动力学行为研究不多,相关的机制也不明确。本文从某拟用作极低放射性废物处置场场所的土壤中分离出1株芽孢杆菌Bacillus sp.dwc-2,考察了pH值、甘油磷酸钠(SGP)、U(Ⅵ)初始浓度等因素对Bacillus sp.dwc-2在生长繁殖过程中诱导U(Ⅵ)矿化动力学行为的影响,并对其矿化机制进行了探讨。结果表明,Bacillus sp.dwc-2可诱导U(Ⅵ)形成钠铀云母(NaUO_(2)PO_(4)·3H_(2)O)的晶体矿物,该过程作为一种酶促反应,受pH值、SGP浓度、U(Ⅵ)初始浓度等因素的显著影响;当U(Ⅵ)初始浓度从50 mg/L增加至150 mg/L时,刺激了处于生长迟滞期至对数期(培养0~12 h)菌体磷酸酶活性的表达和磷酸盐的代谢,使其诱导U(Ⅵ)的矿化行为发生在生长对数期(培养12 h);SGP浓度的增加促进了磷酸酶的催化反应速率,进而促进了U(Ⅵ)的矿化,当SGP的浓度从25 mmol/L增加到125 mmol/L时,Bacillus sp.dwc-2诱导U(Ⅵ)的矿化时间从其生长稳定期(培养24 h)提前至生长对数期(培养12 h);中性和碱性条件下,磷酸酶活性在生长迟滞期至对数期(培养0~12 h)快速增加,从而促进了磷酸盐的消耗和U(Ⅵ)的矿化,特别是在碱性条件下,沉积物在生长对数期(培养8 h)时即转化为明显的晶体矿物。

二苯醚类除草剂降解菌Bacillus sp.Za微生物制剂的研发与初步应用

以二苯醚类除草剂高效降解菌株Bacillus sp.Za为材料制备微生物制剂,优化液体制剂保护剂的物质配比,筛选固体制剂的最适材料,对固体制剂进行初步应用并评价其降解效果。研究结果表明:(1)液体制剂保护剂(0.20%柠檬酸钠、0.20%羧甲基纤维素、0.30%KCl)可使活菌数提高35.71%,保存30 d的液体制剂对50 mg/L乳氟禾草灵的降解率为83.50%;(2)筛选得到猪粪有机肥作为固体制剂的最适材料,保存60d时固体制剂活菌数为8.26×10^(8)cfu/g,对土壤中10mg/kg乳氟禾草灵的降解率为85.52%;(3)添加固体制剂可有效缓解乳氟禾草灵残留对玉米所产生的药害。

食品污水中异养硝化-好氧反硝化菌Bacillus sp.JG441的筛选鉴定及其脱氮特性

高氨氮、高盐、含有机物的食品工业污水处理难度大、周期长,添加高效硝化菌株可以大幅提高污水处理效率。本研究通过高盐培养基,从腌渍食品厂排放污水中筛选出一株对复杂环境有较强耐受性的新型高效异养硝化-好氧反硝化菌株JG441,经鉴定为Bacillus sp.,对其脱氮条件、异养硝化、好氧反硝化、极端环境耐受和脱氮通路进行研究。结果表明,菌株JG441可以利用(NH_(4))_(2)SO_(4)和KNO_(3)为氮源进行异养硝化和好氧反硝化;在NaCl浓度为30g·L^(-1),苯酚浓度为400mg·L^(-1)时,24h NH_(4)^(+)-N去除率可达99%,在NH_(4)^(+)-N浓度为500mg·L^(-1)时,24h NH_(4)^(+)-N去除率为57.4%。菌株JG441脱氮能力强,在高盐含氮和成分复杂的污水处理方面具有较好应用潜力。

Bacillus sp.L1胞外蛋白酶EL1基因克隆及酶学性质分析

目的:对菌株Bacillus sp. L1分泌的胞外蛋白酶EL1进行分离纯化、基因克隆及酶学性质研究。方法:利用硫酸铵沉淀、阴离子交换层析从菌株Bacillus sp. L1的发酵液中分离纯化出胞外酶EL1,对其进行了质谱分析、基因克隆及酶学性质研究。结果:质谱结果提示EL1属于丝氨酸蛋白酶,该酶基因开放阅读框为1326 bp,蛋白序列共441个氨基酸,氨基酸序列与菌株Bacillus stratosphericus分泌的丝氨酸蛋白酶(WP007499449)同源性最高(98%)。该酶最适温度60℃,具有较好的热稳定性;最适pH8.0,在pH8.0时稳定性较好;Mn2+对其有一定的激活作用,Cu2+对其有一定的抑制作用。结论:Bacillus sp. L1分泌的胞外蛋白酶EL1具有较好的热稳定性,在碱溶液中可以保持较高的活力,具有潜在的工业应用价值。

Biodegradation of benzo[a]pyrene in soil by Mucor sp.SF06 and Bacillus sp.SB02 co-immobilized on vermiculite

Two indigenous microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, capable of degrading polycyclic aromatic hydrocarbons （PAHs） were co-immobilized on vermiculite by physical adsorption and used to degrade benzo[a] pyrene （BaP）. The characteristics of BaP degradation by both free and co-immobilized microorganism were then investigated and compared. The removal rate using the immobilized bacterial-fungal mixed consortium was higher than that of the freely mobile mixed consortium. 95.3% of BaP was degraded using the co-immobilized system within 42 d, which was remarkably higher than the removal rate of that by the free strains. The optimal amount of inoculated co-immobilized system for BaP degradation was 2%. The immobilized bacterial-fungal mixed consortium also showed better water stability than the free strains. Kinetics of BaP biodegradation by co-immobilized SF06 and SB02 were also studied. The results demonstrated that BaP degradation could be well described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10--200 mg/kg. The scanning electronic microscope （SEM） analysis showed that the co-immobilized microstructure was suitable for the growth of SF06 and SB02. The mass transmission process of co-immobilized system in soil is discussed. The results demonstrate the potential for employing the bacterial-fungal mixed consortium, co-immobilized on vermiculite, for in situ bioremediation of BaP.

Biosorption of Cu(Ⅱ) on extracellular polymers from Bacillus sp.F19

Biosorption can be an effective process for the removal of heavy metals from aqueous solutions.The adsorption of Cu(Ⅱ) from aqueous solution on the extracellular polymers (EPS) from Bacillus sp.(named MBFF19) with respect to pH,incubation time,concentration of initial Cu(Ⅱ),and biosorbent dose was studied.Biosorption of Cu(Ⅱ) is highly pH dependent.The maximum uptake of Cu(Ⅱ) (89.62 mg/g) was obtained at pH 4.8.Biosorption equilibrium was established in approximately 10 min.The correlation coeffcient of mor...

Bacillus sp.JY-23发酵制备胶原蛋白酶工艺优化

【目的】胶原蛋白具有特殊的三螺旋结构,对常见的蛋白酶具有抗性,只能被特定蛋白酶(胶原蛋白酶)水解。微生物胶原蛋白酶由于其较低的要求和较高的生产率,在胶原废物的高值化利用中发挥着重要作用。本研究拟通过单因素优化及响应面法优化,提高胶原蛋白酶产生菌的产酶量。【方法】通过单因素试验探究培养时间、温度、pH、接种量、装液量、碳源、氮源及金属离子等对胶原蛋白酶产生菌Bacillus sp.JY-23的产酶影响;在此基础上利用PB试验筛选出显著影响因素,最后通过CCD试验确定最佳发酵产酶工艺条件。【结果】通过PB试验筛选出葡萄糖、酵母浸粉、接种量和Fe^(2+)等4个显著影响因素,通过响应面法确定了最佳产酶发酵工艺:培养时间36 h、培养温度37℃、培养基pH值8.0、接种量2.97%,葡萄糖19.60 g/L、酵母浸粉25.80 g/L、Fe^(2+)0.75 mmol/L、NaCl 10 g/L。在该优化条件下,Bacillus sp.JY-23所产胶原蛋白酶活力达到28.13 U/mL,较优化前的酶活(5.94 U/mL)提高4.73倍。【结论】经过单因素、PB设计和响应面逐级优化,显著提高了Bacillus sp.JY-23发酵产胶原蛋白酶的酶活,为该菌株工业化应用于胶原蛋白活性肽开发提供了理论基础。

MICROCALORIMETRY ON BACILLUS SP.NTT-61

The fundamental growth thermograms of Bacillus sp.NTT-61 have been de- termined by microcalorimetric method.From these growth curves we got some thermo- kinetics data of its growth and its thermodynamic properties.

Selection and Molecular Biological Identification of a Strain of Bacillus sp. Inhibiting the Growth of Saprolegnia ferax

Based on the theory of biological control of Saprolegnia ferax,antagonism test of nine strains of Bacillus sp. to S. ferax JL was carried out. Bacillus sp.BA1 was screened to have significantly inhibitory effects on the growth of S. ferax JL( P 【 0. 05). Then,the effects of Bacillus sp. BA1 on different sources of S. ferax were carried out. Results showed that BA1 also had significantly inhibitory effects on S. ferax 6#,10# and S2( P 【 0. 05). Sequence of 16 S r DNA of BA1 was analyzed; and homologous alignment analysis showed that BA1 had more than 99% similarity with Bacillus cereus. Therefore,it could be concluded that strain BA1 was B. cereus,which significantly inhibited the growth of S. ferax and could be used as the biological control agent for S. ferax diseases in aquaculture.

Mechanism and Application of Plant Rhizospheric Bacillus sp. for Growth Promotion and Disease Prevention

In order to promote the development and application of environmental-friendly,efficient and safe beneficial Bacillus sp.preparations,the paper summarizes and systematically elaborates the colonization of Bacillus sp.in host plants and the mechanism of synergistic effect on disease prevention of host plants,further reviews the application of rhizospheric Bacillus sp.in promoting the growth of agricultural and forestry crops and controlling plant diseases,and prospects the scientific issues and application of plant rhizospheric Bacillus sp.in the future.

In vitro and in silico studies on fibrinolytic activity of nattokinase： A clot buster from Bacillus sp.

BACKGROUND： Nattokinase （NK） is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation. METHODS： In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed. RESULTS： Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues offibrinogen （THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321） and 8 residues ofNK （GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222） are involved in intact binding. CONCLUSIONS： A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

Evaluation of Bacillus sp. MZS10 for decolorizing Azure B dye and its decolorization mechanism

To evaluate decolorization and detoxification of Azure B dye by a newly isolated Bacillus sp. MZS 10 strain, the cultivation medium and decolorization mechanism of the isolate were investigated. The decolorization was discovered to be dependent on cell density of the isolate and reached 93.55% （0.04 g/L） after 14 hr of cultivation in a 5 L stirred-tank fermenter at 2.0 g/L yeast extract and 6.0 g/L soluble starch and a small amount of mineral salts. The decolorization metabolites were identified with ultra performance liquid chromatography-tandem mass spectroscopy （UPLC-MS）. A mechanism for decolorization of Azure B was proposed as follows： the C=N in Azure B was initially reduced to -NH by nicotinamide adenine dinucleotide phosphate （NADPH）-dependent quinone dehydrogenase, and then the -NH further combined with -OH derived from glucose to form a stable and colorless compound through a dehydration reaction. The phytotoxicity was evaluated for both Azure B and its related derivatives produced by Bacillus sp. MZS 10 decolorization, indicating that the decolorization metabolites were less toxic than original dye. The decolorization efficiency and mechanism shown by Bacillus sp. MZS10 provided insight on its potential application for the bioremediation of the dye Azure B.

Enhanced microbial degradation of pyrene using biosurfactant isolated from Bacillus sp.SS105

The present research work highlights the biosurfactant application toward enhanced pyrene degradation by Pseudomonas sp.Lipopeptide-type biosurfactant was produced from Free Air CO_(2) Enriched(FACE)soil bacterium Bacillus sp.SS105.The different concentrations of biosurfactant at different time intervals were optimized for maximum pyrene degradation and quantified through analytical methods such as the scanning process of UV-Vis spectrophotometer and GC-MS analysis.In the absence of biosurfactant,Pseudomonas sp.could degrade only 8%of pyrene at the concentration of 5 mg per 100 ml,while the degradation rate enhanced by 95%in presence of biosurfactant(5 mg/100 ml of culture).This result demonstrated that biosurfactant produced from Bacillus sp.SS105 has potential to enhance the bioavailability and biodegradation of pyrene by Pseudomonas sp.

Degradation characteristics of two Bacillus strains on the Microcystis aeruginosa

The degradation kinetics of strains P05 and P07 and the degradation effects of mixed strain on Microcystis aeruginosa were studied. The results showed that: (1) The degradation processes of strains P05 and P07 on Microcystis aeruginosa accorded with the first-order reaction model when the range of Chl-a concentration was from 0 to 1500 μg/L. (2) The initial bacterium densities had a strong influence on the degradation velocity. The greater the initial bacterium density was, the faster the degradation was. The degradation velocity constants of P05 were 0.1913, 0.2175 and 0.3092 respectively, when bacterium densities were 4.8×10 5, 4.8×10 6, 2.4×10 7 cells/ml. For strain P07, they were 0.1509, 0.1647 and 0.2708. The degradation velocity constant of strain P05 was higher than that of P07 when the bacterium density was under 4.8×10 5 cells/ml, but the constant increasing of P07 was quicker than that of P05. (3) The degradation effects of P05 and P07 strains did not antagonize. When the concentration of Chl-a was high, the degradation effects of mixed strain excelled that of any single strains. But with the decrease of the Chl-a concentration, this advantage was not clear. When the concentration was less than 180 μg/L, the degradation effects of mixed were consistent with that of strain P07.

Combined Use of NMR, LC-ESI-MS and Antifungal Tests for Rapid Detection of Bioactive Lipopeptides Produced by Bacillus

The science and technology interact with the art in several ways. Biotechnological coupled with analytical approaches can play an important role in protecting and preserving cultural heritage for future generations. Many microorganisms influenced by environmental conditions are the main responsible for biological contamination in built heritage. Biocides based on chemical compounds have been used to mitigate this problem. Thus, it is vitally important to develop proper remediation actions based on environmentally innocuous alternative. Bacillus specie is emerging as an optimistic alternative for built heritage treatment due to their capacity to produce secondary metabolites with antagonistic activities against many fungal pathogens. Therefore, the intent of this work was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using spectroscopic (NMR) and chromatographic techniques (LC-ESI-MS). The high antifungal activity obtained for Bacillus sp. active compounds produced in this study confirms the great potential to suppress biodeteriogenic fungi growth on historical artworks. Additionally, the proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and could be used as a viable alternative to be employed for screening and production of new green biocides.

Identification of Biocontrol Strain HJX1 and Determination of Its Antifungal Spectrum

A biocontrol strain HJX1,which had good inhibitory effect against Fusarium oxysporum f. sp. cubense,was identified as Bacillus subtilis based on traditional morphology,16 S rRNA gene sequence and fatty acid profiles. The sequence of 16 S rRNA gene had 99% homology to B. subtilis according to the DNA fragment of 1 521 bp amplified with a pair of universal primers,P0 and P6,of bacteria. Confront culture with pathogenic fungi showed that this strain had good inhibitory effect against 10 pathogenic fungi.

芽孢杆菌糖基水解酶GH489的表达及生物活性研究

为了探究芽孢杆菌(Bacillus sp.)糖基水解酶GH489的生物活性,通过设计特异性引物扩增得到目的基因gh489,采用大肠杆菌原核表达系统对重组GH489蛋白进行表达,利用组氨酸标签对目的蛋白进行分离纯化,并检测芽孢杆菌糖基水解酶GH489对二斑叶螨的杀螨活性。结果表明,由大肠杆菌BL21(DE3)表达的重组GH489蛋白为可溶性蛋白质,其蛋白质分子质量约为57 kDa。纯化后的GH489重组蛋白显示出良好的杀螨活性,处理24 h后,二斑叶螨的半致死浓度(LC_(50))为30.296μg/mL,处理48 h后,二斑叶螨的半致死浓度(LC_(50))为21.212μg/mL。

生物炭水凝胶固定微生物处理含酚废水

筛选和分离出一种高效的苯酚降解菌Bacillus sp.26,以西瓜生物炭水凝胶作为载体,用海藻酸钠作为交联剂包埋该菌株.通过实验研究对比灭活的固定化Bacillus sp.26细胞对苯酚的吸附;固定化Bacillus sp.26细胞对苯酚的吸附和降解;游离Bacillus sp.26细胞对苯酚的降解能力.结果表明:Bacillus sp.26游离菌株具有高效降酚能力,可在1.0 d内降解浓度高达800 mg/L的高盐含酚废水;生物炭水凝胶固定化Bacillus sp.26同样显示出高效降解苯酚废水的能力,且具有优良的循环使用性能,在循环使用3次后,对1200 mg/L苯酚废水的降解效率仍达到100%;固定化后的Bacillus sp.26在循环使用过程中性能不断提高,说明炭水凝胶为菌株提供良好的生长和增殖环境.生物炭水凝胶固定微生物的方法具有易于回收、分离和重复使用的特点,具有潜在的应用价值.所筛选出的菌株和发展的固定化方法,为固定化微生物技术处理苯酚废水提供新的材料和思路.

Isolation of Cr(Ⅵ) reducing bacteria from industrial effuents and their potential use in bioremediation of chromium containing wastewater

The present study was aimed to assess the ability of Bacillus sp.JDM-2-1 and Staphylococcus capitis to reduce hexavalent chromium into its trivalent form.Bacillus sp.JDM-2-1 could tolerate Cr（Ⅵ）（4800 μg/mL） and S.capitis could tolerate Cr（Ⅵ）（2800 μg/mL）.Both organisms were able to resist Cd^2＋（50 μg/mL）,Cu^2＋（200 μg/mL）,Pb^2＋（800 μg/mL）,Hg^2＋（50 μg/mL） and Ni2＋（4000 μg/mL）.S.capitis resisted Zn^2＋ at 700 μg/mL while Bacillus sp.JDM-2-1 only showed resistance up to 50 μg/mL.Bacillus sp.JDM-2-1 and S.capitis showed optimum growth at pH 6 and 7,respectively,while both bacteria showed optimum growth at 37°C.Bacillus sp.JDM-2-1 and S.capitis could reduce 85% and 81% of hexavalent chromium from the medium after 96 h and were also capable of reducing hexavalent chromium 86% and 89%,respectively,from the industrial effuents after 144 h.Cell free extracts of Bacillus sp.JDM-2-1 and S.capitis showed reduction of 83% and 70% at concentration of 10 μg Cr（Ⅵ）/mL,respectively.The presence of an induced protein having molecular weight around 25 kDa in the presence of chromium points out a possible role of this protein in chromium reduction.The bacterial isolates can be exploited for the bioremediation of hexavalent chromium containing wastes,since they seem to have a potential to reduce the toxic hexavalent form to its nontoxic trivalent form.