Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Bacillus subtilis 168中亚硝酸盐还原酶基因的敲除及表型鉴定

该研究首先通过同源重组方式将Bacillus subtilis 168菌株nir基因位点置换为两端具有同向排列的loxP序列选择性标记基因,再运用Cre重组酶将两个loxP间的全部序列切除,成功敲除了Bacillus subtilis 168菌株亚硝酸盐还原酶(nitrite reductase,NiR)的3个亚基基因nasB、nasD和nasE。其中,敲除nasD基因的B.subtilis 168菌株在不同生长时期均未分解培养基中亚硝酸盐,表明该nasD基因缺陷型菌株NiR活性已被完全消除,不再具备降解亚硝酸盐的能力。

果胶酶产生菌 Bacillus subtilis Z-5的筛选及酶学性质研究

以果胶为唯一碳源,通过透明圈法和DNS法从上海海洋大学橘园的土壤中筛选出1株高产、有良好热稳定性和酸碱耐受性的产果胶酶野生型菌株Z-5。通过16S rDNA分子技术并综合生理生化特征确定为枯草芽孢杆菌(Bacillus subtilis)。对该果胶酶进行酶学性质研究,结果表明最佳酶活性的反应温度为60℃,最佳酶活性的反应pH为6.0,在此作用温度和作用pH下,酶活力为(879±2.733)U/mL。此外,该酶在40~50℃、pH 6.0~9.0稳定性良好。Ba^(2+)、Fe^(2+)、Ca^(2+)、Cu^(2+)对该酶表现出促进作用,Zn^(2+)、Mn^(2+)对该酶表现出抑制作用。其中Cu^(2+)促进作用最强,Zn^(2+)抑制作用最强,而Mg^(2+)、Hg^(2+)对该酶无明显影响。Bacillus subtilis Z-5所产的果胶酶具有应用于食品商用果胶酶的潜力。

Bacillus subtilis Z-5产果胶酶发酵条件的优化及其应用

为进一步提高果胶酶的产量,采用响应面法优化了Bacillus subtilis Z-5产果胶酶的发酵培养基和发酵条件,并将优化后得到的果胶酶应用于梨汁澄清实验。通过单因素实验探究9个因子对B.subtilis Z-5产果胶酶活力的影响,然后采用Plackett-Burman试验设计筛选出3个显著影响产酶的因素:酵母粉含量、pH、培养时间;再结合响应面设计法确定了最佳的发酵条件。结果表明最佳的发酵培养基成分是果胶10 g/L,酵母粉7.99 g/L,MgSO_(4)·7H2O 0.5 g/L;最佳的发酵条件为初始pH5.66,发酵温度37℃,培养时间72 h,接种量为6%(v/v),装液量50/250 mL,底物浓度为10 g/L;在此基础上,B.subtilis Z-5产果胶酶酶活力由879.00 U/mL提高到1549.62 U/mL,是优化前的1.76倍。在梨汁澄清实验中表明果胶酶的最适添加量为4 mL,最适酶解时间为2.5 h,最适酶解温度为65℃,最适酶解pH为6.0,此时透光率最大为79.77%,与商品酶相比,自制果胶酶是一种具有多种酶系复合酶,可作用于梨汁中含有的蛋白、淀粉等其他成分,使得梨汁更加澄清。本研究结果为果胶酶的工业化生产和应用提供了理论支撑。

Biotransformation of Shrimp Wastes by Bacillus subtilis OKF04 and Evaluation of Growth Promoting Effect in Crop Planting

In this study,we proposed a reliable and sustainable technique for the clean utilization of shrimp wastes,which can yield a solid inoculant of Bacillus subtilis OKF04 containing micronutrients at low cost without the risk of contamination.Study of the culture conditions revealed that the head of shrimp Litopenaus vannamei and the wheat bran acted as suitable substrates for the growth of B.subtilis OKF04.With 60%initial moisture content,30℃culture temperature,and 5%inoculation amount,followed by 48 hours of fermentation and 0.5%soluble starch added during the drying process(50℃for 6h),a solid B.subtilis OKF04 inoculant with a spore amount of 2.4×10^(10)CFU g^(-1)and a high amino acid content was obtained.The solid B.subtilis OKF04 inoculant was applied to cultivate pakchoi under pot experiment.As the result,of adding to,the size of stems and leaves,nutritional composition,and physiological activity of pakchoi were significantly(P<0.05)enhanced by solid B.subtilis OKF04 inoculant.B.subtilis OKF04 also significantly(P<0.05)increased the soil’s nutrient content and improved its microbial composition.Furthermore,pakchoi cultivated with a low dose of solid B.subtilis OKF04 inoculant(0.05 g kg^(-1)soil)resulted in the best results.This study provides a new method for the preparation of microbial inoculants with solid waste shrimp heads.

Bacillus subtilis fmbR抗菌物质的分离和鉴定

【目的】对Bacillus subtilis fmbR产生的抗菌物质进行分离和鉴定研究,以确定抗菌物质的组成和结构。【方法】采用HPLC和TLC层析对Bacillus subtilis fmbR抗菌物质进行分离纯化,通过ESI-MS和MALDI-MS分析对抗菌物质的组成和结构进行初步鉴定。【结果】HPLC层析表明了Bacillus subtilis fmbR抗菌物质含有保留时间与surfactin相似的成分。TLC层析和原位酸解证明了Bacillus subtilis fmbR抗菌物质含有闭合肽键类的物质,其中之一为相对迁移率Rf与标样surfactin相近的组分。采用ESI-MS分析检测到Bacillus subtilis fmbR抗菌物质含有分子量与surfactinA相同的m/z1009.1、m/z1023.2和m/z1037.0等3种同系物;通过MALDI-MS分析获得[M+H]+为m/z 3403.95抗菌物质,该物质分子量与Bacillus subtilis 168产生的细菌素subtilosin的m/z3403.3相同。【结论】Bacillus subtilis fmbR抗菌物质由C13～C15的3种surfactinA同系物和一种羊毛硫抗生素subtilosin组成。

Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定

对BacillussubtilisfmbJ脂肽类抗菌物质的分离和鉴定进行了系统研究。通过HPLC层析确定BacillussubtilisfmbJ抗菌物质由多种组分构成,其中含有保留时间与surfactin相似的成分。通过TLC层析和原位酸解确定BacillussubtilisfmbJ抗菌物质含有两个具有闭合肽键类的物质,其中之一为迁移率Rf与标样surfactin非常相近的组分。通过ESI-MS分析检测到BacillussubtilisfmbJ抗菌物质含有分子量与fengicin相同的m/z1449.9、m/z1463.8、m/z1477.8、m/z1491.9和m/z1505.9五种同系物,和分子量与surfactin相同的m/z1008.8、m/z1022.8和m/z1036.8三种同系物。

Screening for the Strain Highly Producing Antagonistic Substance from Bacillus subtilis B47 by UV Mutagenesis

[Objective] The aim of this study was to breed new strains which have higher inhibitory effects on the pathogens of watermelon fusarium wilt.[Method] The endophytic Bacillus subtilis B47 strain was obtained from tomato stems by UV mutagenesis for two consecutive times,then genetic stability as well as physiological and biochemical properties of mutant strains were studied.[Result] The antibacterial activity of all the three mutant strains F303,F304 and F305 was higher than that of B74 strain.After subculture of 10 successive generations,the antibacterial activity of all the three mutant strains for the pathogens of watermelon fusarium wilt decreased,but the antibacterial activity of F305 strain decreased the least,indicating its best genetic stability among the tested strains.The antibacterial circle diameter of F305 strain was 5 mm larger than that of wild strain B47 under the same condition.The mutant strain F305 was in logarithmic growth phase within 36 h and in stationary phase within 36-96 h,while its optimum growth temperature was 35 ℃.F305 strain could grow in sodium salt with the concentration of 1%-10%,but it grew best at the concentration of 1%.Physiological and biochemical responses of F305 strain were in accordance with those of wild strain B47.[Conclusion] This study lays the foundation for the factorial production of antagonistic substance by B47 strain and new methods of preventing from the pathogens watermelon fusarium wilt.

Bacillus subtilis B2产1-脱氧野尻霉素(DNJ)发酵条件优化

为探索制备含有1-脱氧野尻霉素(1-deoxynojimycin,DNJ)的降血糖功能性食品的新方法,以产DNJ的菌株Bacillus subtilisB2为初始菌株,以价廉的农产品加工副产物豆渣为原料,通过对发酵条件的优化,获得富含功能因子DNJ的发酵液。结果表明:接种量对α-葡萄糖苷酶抑制活性影响不明显,当接种量大于104个/mL时,发酵液的α-葡萄糖苷酶抑制活性均在20以上。而豆渣浓度、发酵温度、培养基初始pH值及发酵时间对发酵液α-葡萄糖苷酶抑制活性影响较大,在豆渣质量浓度30mg/mL,初始pH值6～8,发酵温度40℃的条件下发酵48h,发酵液表现出较高的α-葡萄糖苷酶抑制活性。以Bacillus subtilisB2发酵豆渣的发酵液为原料开发含DNJ的降糖功能食品,可以大大提高豆渣的利用价值,为降糖功能食品的开发利用开辟新途径。

枯草芽孢杆菌(Bacillus subtilis)B-912对采后柑桔果实青、绿霉病的抑制效果

用从土壤中分离到的枯草芽孢杆菌 B- 912作为生防拮抗菌 ,主要研究在不同贮藏温度下不同接种时间的 B- 912活菌液、热处理液和过滤液对柑桔青、绿霉病的抑制效果。B- 912活菌液的抑菌效果最佳 ,热处理液和过滤液次之 ,但均显著地好于对照。较高的贮藏温度有利于拮抗菌对病菌的抑制作用 ,活菌液处理的果实在 2 5℃下的发病率及病斑直径均小于 15℃下的果实 ,2 4 h后接种病菌孢子的果实其发病率及病斑直径一般都高于 4 8h后接种的果实。实验证明 ,枯草芽孢杆菌的抑菌机理主要是产生抗菌素。

辣椒内生枯草芽孢杆菌(Bacillus subtilis)BS-2和BS-1防治辣椒炭疽病研究

辣椒内生枯草芽孢杆菌(Bacillus subtilis)BS-莉和BS-1,对胶孢炭疽菌(Colletotrichum gloeosporioides)引起的辣椒苗和果炭疽病有良好的防治效果,菌株培养液对苗炭疽病的防效分别为81.5％～93.3％和66.1％～79.2％;对果炭疽病的防效分别为80.0％～100％和60.0％～100％,喷施菌株培养液后间隔24h以上接种病原菌的防病效果比两者同时接种的高。菌株外分泌物对病菌菌丝生长、分生孢子产生和萌发及附着胞形成均有明显的抑制作用。菌株菌体及其胞外分泌物均有防病作用,菌体可能起主导作用。另外,2菌株培养液浸种,对辣椒还有明显的促生效果。BS-2菌株的防病和促生效果均比BS-1高。

Screening of Two Biocontrol Strains of Bacillus subtilis against Ginseng Soil-borne Disease and Their Antifungal Activities

[ Objective] The paper was to obtain biocontrol strains with good control effects against ginseng soil-borne disease through screening. [ Method] Dilu- tion plate method and plate confrontation culture method were used to isolate and screen biocontrol bacteria from the rhizosphere soil of diseased ginseng. The strains were identified through morphology, physiological and biochemical characteristics and 16S rDNA. [ Result ] With Rhizoctonia solani, Fusarium oxysporum and Fu- sarium solani as the indicator strains, two biocontrol strains B59 and X1 with strong antagonistic effects were screened from the rhizosphere soil of diseased ginseng in Tieli farm of Heilongjiang Province, and they were identified to be Bacillus subtilis. The inhibition rates of two biocontrol strains against eight different fungi were all greater than 90%. The primary study indicated that B59 and X1 strains could secrete antifungal active substances. [ Conclusion] Two biocontrol Bacillus subti- lis strains 1359 and X1 all had strong antagonistic effect against ginseng soil-borne disease, which had certain potential for development and utilization.

Bacillus subtilis UN_(13)产α-淀粉酶酶学性质研究

对Bacillus subtilis UN13所产生的α-淀粉酶酶学性质进行初步研究。结果表明：该酶是一种中温酸性α-淀粉酶；其最适温度为50℃，在40-60℃范围内稳定性较好；最适pH为5．5，在pH值为5．0～7．5范围内较稳定；Ca^2＋和Mn^2＋对该酶有激活作用，Cu^2＋，Zn^2＋，K^＋和EDTA则抑制该酶的活性；动力学研究表明该酶的Km值为1．7039mg／mL，Vmax值为3．1615mg／min·mL。

枯草芽孢杆菌(Bacillus subtilis)B10对采后草莓果实病害的抑制效果

主要研究使用生物防治的方法来控制草莓果实采后病害以达到防腐保鲜的目的。以枯草芽孢杆菌(Bacillus subtilis)B10作为拮抗菌,以新鲜草莓果实为试验材料,测定了不同贮藏温度、不同接种时间的B10活菌液、菌悬液、热处理液和过滤液对草莓果实人工接种灰霉病的抑制效果;以及不同浓度菌悬液对果实自然病害的抑制效果。结果表明,B10活菌液、菌悬液对病害的抑制效果较好,热处理液和过滤液次之,但均显著好于对照;在25℃和15℃的不同温度条件下,较高的贮藏温度有利于拮抗菌对病原菌的抑制作用;在接种拮抗菌之后8h和16h分别接种病原菌的不同时间下,拮抗菌接种时间早,抑制效果好;B10菌悬液对草莓果实自然病害有抑制作用,且浓度越高该作用越显著。

Separation and Purification of Recombinant Hirudin Variant 3 from Bacillus subtilis

[ Objective] The research aimed to get the optimized separation and purification conditions of the hirudin produced from Bacillus subtilis DB403 （pUBH5）. [Method] Through the systemic pretreatment, preliminary chromatography and fine chromatography. [Result]The optimized separation and purification conditions were that： Supernatant was treated by trichloroacetic acid, then by ultrafiltration desalt and anion exchange chromatography. Strong anion Q F. F. was better than weak anion DEAE F.F. The proper balanced solution was Tris-HCI （ pH 8.0）. The proper conductivity was 6 ms/cm. The maximum applied sample was 240 ATU/ml to matrix of strong anion Q F. F. This optimized procedure was magnified in strong anion exchange HiPrep 16/10Q with the 90% recovery and 70.2% purity. The purification of gel filtration of Sephacryl S-100 to hirudin was not relative to flow rate within certain scope. The application size of sample was 10 ml. The purity checked by HPLC was 95.1%, and the recovery was 93%, and the band of SDS-PAGE was single. [ Conclusion] The research provided the reference of the further industrialization separation and purification of hiruin.

Screening of Biocontrol Strain Bacillus subtilis by N^+ Ion Beam Implantation

[Objective] This study was to investigate the effect of N＋ ion beam implantation on the survival rate and mutation rate of biocontrol strain Bacillus subtilis. [Method] The factors influencing B. subtilis ion beam implantation, including culture time, dilution concentration, solvent, drying time of mycoderm were optimized. B. subtilis cells were implanted by using ion beam at dose of 2.0×10^14～4.0×10^14 ions/cm2 and the energy of 30 kev. Then the methods of culturing colonies confronting each other on plate and Oxford cup diffusion were used to screening strains. [Result] The optimal parameters were found as follows： culture in liquid for 20-24 h, dilution with sterile water to 106 cells/ml and drying time of 60 min for sample preparation; the optimal N＋ ion beam implantation dose of 2.0×10^14～4.0×10^14 ions/cm2 at the energy of 30 kev, the survival rate of 8.43%-26.71% and the mutation rate of 3.50%-5.43%. [Conclusion] This study provided reference for ion beam implantation mutation of B. subtilis.

Preliminary Study on Garlic Root Exudates Influences to the Growth of Pomegranate Wilt Pathogen(Ceratocystis fimbriata)and Bacillus subtilis

[Objectives] This paper aims to explore the possibility to intercrop garlic with pomegranate tree to control pomegranate wilt.[Methods] Root exudates of garlic is cultivated in 1/5 concentration of MS solution and distilled water is examined in lab to test their effect to growth of mycelia of pomegranate wilt pathogen（Ceratocystis fimbriata）and multiplication of Bacillus subtilis.[Results] The result shows that garlic root exudates whatever cultivated in MS solution or distilled water could not inhibit or promote mycelia growth of C.fimbriata.However,garlic root exudates cultivated in both methods effectively promote multiplication of B.subtilis.[Conclusions] It is suggested that intercropping garlic with pomegranate tree by combining application B.subtilis could be a promising way to prevent pomegranate wilt spread in practice.

枯草芽孢杆菌(Bacillus subtilis (Cohen))B-903菌株抗菌物质对植物病原真菌的抑制作用

作者从郑州苹果园中分离到枯草芽孢杆菌的一株强拮抗菌株。研究认为该菌株产生的抗菌物质对镰刀菌等12种病原真菌具有抑制作用,抑菌谱广。其抑菌机理主要是融解细胞壁,造成原生质泄漏,同时也使孢子和菌丝畸形。田间小区试验证明,以B-903培养滤液浸种可有效防治棉炭疽病等棉苗病害,并促进棉苗生长发育;果园喷雾防治苹果轮纹病和叶斑病的效果相当或优于化学药剂。

脂肪酶产生菌Bacillus subtilis FS321的分离鉴定及其产酶条件的初步优化

从北方堆肥试样中分离筛选得到1株中度耐热脂肪酶产生菌,编号为FS321。对该菌株进行产酶条件的初步优化,得到了摇瓶发酵条件:产酶培养基M4,发酵周期为48～60 h,摇瓶发酵温度为37℃,通气量为25 mL/250 mL锥形瓶。所产脂肪酶在50℃,pH 9.0时表现最高活性。为了鉴定该菌株,分析该菌的细胞脂肪酸谱,克隆测序了其16S rDNA基因序列,系统进化树及细胞脂肪酸组分分析均表明,FS321与Bacillus subtilis具有最紧密的亲缘关系。

枯草芽孢杆菌(Bacillus subtilis)在刺参养殖中的益生作用

运用生化方法分析了不同浓度枯草芽孢杆菌制剂对养殖水质、刺参的生长、消化酶活性、非特异性免疫酶活性的影响。结果显示,与对照组相比,添加枯草芽孢杆菌制剂,能够显著降低养殖水体中的硝酸盐和亚硝酸盐,去除率最高可达42.28%和22.33%,对水体中氨氮含量和化学需氧量影响不显著;实验组刺参特定生长率显著提高,枯草芽孢杆菌添加量为6×10–6时,SGR提高40.74%,添加量为6×10–6时,淀粉酶活性提高30.27%,添加量为5×10–6时,蛋白酶活性提高53.93%,达到显著水平差异,但脂肪酶活性无显著提高;非特异性免疫酶(过氧化物酶、碱性磷酸酶、溶菌酶、超氧化物歧化酶)活性均显著高于对照组,枯草芽孢杆菌添加6×10–6效果最好,分别提高135%、27%、396%、192%。本研究表明,添加适当的枯草芽孢杆菌能够有效改善刺参养殖水体水质,提高机体消化酶和非特异性免疫酶活性,从而促进刺参生长。