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.

生物炭载Bacillus subtilis菌粒对连作芹菜生长的影响

为解决土壤连作障碍对蔬菜生长发育的不利影响,本研究以连作3年的芹菜土壤为研究对象,采用随机区组试验,设置5种处理:单独施加生物炭(BC)、单独施加Bacillus subtilis菌剂(BS)、生物炭与菌剂混合施加(BB)、施加生物炭载B.subtilis菌粒(BP)、不施加生物炭与菌剂(CK),盆栽种植芹菜,测定其生长与品质指标,探究生物炭和B.subtilis对连作芹菜生长的影响。结果表明,BB、BP处理组芹菜的生长和品质指标都高于其他土壤处理组,BP处理组的芹菜鲜质量、可溶性蛋白含量以及纤维素含量均显著高于BB处理组,分别为60.25 g/棵、27.25 mg/g、2.48%,其他指标两处理间无显著差异。不同土壤处理组芹菜的生长和品质指标都高于CK处理,其中BP处理组各指标值最高,尤其是芹菜鲜质量,增幅高达226.56%。因此,施加生物炭载B.subtilis菌粒提高了连作芹菜生长指标与品质,为农业生产上缓解蔬菜连作障碍奠定了理论基础。

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。

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.

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.

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 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.

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。

Screening of Bacillus subtilis HAAS01 and Its Biocontrol Effect on Fusarium wilt in Sweet Potato

Fusarium wilt,a disease caused by Fusarium oxysporum f.sp batatas(Fob)is an important disease in sweet potato production.Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways.A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture.According to the biological characteristics,16S rDNA sequence analysis,and physiological and biochemical analysis,the Bacillus subtilis HAAS01 was named.A pot experiment was conducted for the biological control experiment of strain HAAS01,and the endogenous hormone content,antioxidant enzyme activity,soluble protein content,and related gene expressions of sweet potato plants were detected.The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions.In summary,Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.

Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88,isolated from Taptapani hotspring of odisha,India

Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present study baclerial strain isolated for extracellular L-asparaginase production from hotspring,identified by morphological,biochemical and physiological tests followed by t6S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.Result:The bacterial strain was identified as Bacillus sublilis strain hswx88(GenBank Accession Number JQ237656.1).The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88(23.8 IU/mL)was found to be 1.7 and 14.5 limes higher than the reference organism Pectobacterium carotovorum MTCC 1428(14.2 IU/mL)and Bacillus sp.BCCS 034(1.64 IU/mL).Conclusion:The isolate is eco-friendly and useful to produce bulk quantity of extracellular,thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.

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.

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.

On the Bacteriostatic Activity of Bacillus subtilis and Pyraclostrobin as Well as Their Mixtures to Grape Anthracnose and the Field Disease Control Efficiency

[Objective] To screen out the biological compound bactericides for grape anthracnose, reduce and replace the use of chemical pesticide. [Methods] The de- termination on the indoor bacteriostatic activity of different proportions of Bacillus subtilis and pyraclostrobin to grape anthracnose was carried out, and mycelial growth rate method was adopted to determine the toxicity of Bacillus subtilis and pyraclostrobin as well as their 5 mixtures to grape anthracnose. [Results] The EC50 of Bacillus subtilis and pyraclostrobin as well as their mixture combinations of 1：1, 1：2, 1：3, 1：4 and 1：5 to grape anthracnose were respectively 1.969 8, 1.527 4, 1.373 2, 1.294 8 and 1.247 3 μg/ml; the synergistic coefficients （SR） of the 5 mix- ture combinations to grape anthracnose were 1.70, 1.25, 1.13, 1.12 and 1.12, re- spectively, in which the synergistic effect of 1：1 was the largest. The indoor biologi- cal activity of pyraclostrobin（EC50 was 1.054 0μg/ml） was higher than that of Bacil- lus subtilis（EC50 was 15.017 5 μg/ml）. 50 d after the agentia（before the harvesting）, the investigation results showed that 1 000-fold dilution, 1 500-fold dilution and 2 000- fold dilution as well as each single dosage of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder all had better control efficiency to grape anthracnose after fruit setting and before bagging, in which the treatments of high concentration and middle concentration were higher than the treatments of low concentration and two single dosages： the highest control efficiency of high concentration was 90.03%, which was higher than all other treatments; the control efficiency of middle concen- tration was 87.01%, which was higher than that of low concentration and each sin- gle dosage; the control efficiency of low concentration was 84.11%, which was high- er than 1 000-fold dilution of 1 000×10^8 cfu/g Bacillus subti/i wettable powder （the control efficiency was 64.60%） and 2 000-fold dilution of 250 g/L Bacillus subti/i wettable powder （the control efficiency was 81.07%）. In addition, each treatment al- so had better control efficiency to other cluster diseases, such as white rot, etc., and the control efficiency was almost the same as that of anthracnose. [Conclusion] It was suggested that the prevention concentration of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold - 2 000-fold dilution.

Curing of the Bacillus subtilis Plasmid Using Sodium Dodecyl Sulfate

Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.

Inhibitory Effects of Bacillus subtilis on Staphylococcus aureus

[Objective] To produce drug resistance, seek non-toxic environmental so as to change the current biological drugs that did not excessive use of antibiotics. [Methods] A strain of Bacillus was purified and isolated from fresh and healthy in- testines of grass carps. Biochemical identification was carried out by conventional bacterial biochemical test method. Two pairs of primers were designed, 16S rRNA detection and sequencing analysis were carried out. Drug sensitive test was carried out by agar diffusion method. In vitro inhibition test on Staphylococcus aureus was carried out by Oxford cup method. [Results] The isolated bacterium had basically the same biochemical characters as Bacillus subtilis; and the homology reached 100%. Thus, the isolated bacterium was identified to be Bacillus subtilis. It was insensitive to amoxicillin, ampicillin, penicillin G and so on, but sensitive to amikacin, cefalexin, ciprofloxacin and cefradine. The inhibitory effects of Bacillus subtilis on Staphylococ- cus aureus were significant. The minimum inhibitory concentration （MIC） was 2.8×10^8×2^-5/ml and minimum bactericidal concentration （MBC） was 2.8×10^8×2^-2/ml. [Conclusions] The isolated Bacillus subtilis could be used to prevent and control diseases caused by Staphylococcus aureus, and reduce the abuse of antibiotics.

高产淀粉酶菌株Bacillus subtilis XC2的产酶条件及酶学性质

试验研究从高产淀粉酶菌株Bacillus subtilis XC2的产酶条件及酶学性质。通过单因素试验和正交试验,初步探究淀粉酶酶学性质并优化菌株发酵产酶条件。结果表明,菌株XC2产淀粉酶的反应最适温度为50℃,最适反应pH值为6.0,在30~50℃和pH值4.0~7.0时酶活稳定性较好,Fe^(2+)、Cu^(2+)和Zn^(2+)对酶活有促进作用,Na^(+)对酶活有抑制作用。菌株XC2产淀粉酶最佳条件为蔗糖添加量1.5%、牛肉膏添加量2.0%、K^(+)添加量0.6%、培养温度37℃、转速200 r/min、初始pH值7.0、发酵时间21 h。研究表明,对产酶条件优化后,Bacillus subtilis XC2产淀粉酶活力达984.5 U/mL。

Isolation and Identification of Triazophos-degrading Bacillus subtilis str. C-Y106 and Its Degradation Characteristics

[Objective] The aim was to isolate the triazophos-degrading strain and study its degradation characteristics. [Method] A triazophos-degrading bacterium strain C-Y106 was isolated from sludge in an aeration tank of triazophos manufacture. Then the strain C-Y106 was identified according to the morphology,physiological and biochemical characteristics,and 16S rRNA sequence analysis. The effect of medium with different nutrients on triazophos-degrading rate by C-Y106 was studied. [Result] The strain C-Y106 was identified as Bacillus subtilis. The strain C-Y106 could grow in the mineral salt medium with 40 mg/L of triazophos as the sole sources of carbon,Nitrogen and Phosphorus. The triazophos-degrading rate was the highest as 76.8% in the mineral salt medium with 40 mg/L of triazophos as the sole source of Phosphorus,after being incubated at 31 ℃,pH 8.0 and 150 r/min for 60 h. [Conclusion] The research had provided theoretical basis for the identification and purification of enzymes for triazophos degradation.

Antibacterial Activity of Bacillus subtilis and Properties of Protein Crude Extract

In order to get biological drugs with no resistance or toxic side effects and to reduce the use of antibiotics, a strain of Baci//us subtilis was isolated from animal intestine, and the isolate was identified by molecular biological method; in vitro an- tibacterial test of the isolate was performed using agar diffusion method; the optimal fermentation condition of the isoJate was screened by conventional culture method; the antibacterial crude protein of the isolate was extracted by saturated ammonium sulfate method; the physicochemical properties of antibacterial crude protein was de- tected by comparison method; The results showed that the isolate was B. subti/is, which had antibacterial effects on Staphy/ococcus aureus, streptococcus and swine erysipelas. The fermentation effect of the isolate was the best under the condition of temperature 30 ~C, pH 7, liquid volume 75 ml/250 ml, inoculation volume 20% and culture time 48 h. The antibacterial effect of the isolate was the best when extract- ed by 80% saturated ammonium sulfate. The antibacterial crude protein had strong resistance to heat and acid. Organic solvent and UV irradiation had some influences on antibacterial crude protein. Proteases had hydrolytic effects on antibacterial crude protein. The isolated B. subti/is can be used to prevent and control the diseases caused by S. aureus, streptococcus and swine erysipelas, and can regulate intesti- nal microecology by adding into expanded feeds.

Construction of Bacillus subtilis GMP Reductase Gene(guaC)Mutant

An integrated expression vector pGT9GH was constructed by using guaC gene of receptor strain as guide sequence for homologous recombination.And then,by using the method of double crossover recombination between plasmid and chromosome,the linearized plasmid pGT9GH were integrated into B.subtilis GJ06 to get B.subtilis GJ07 with guaC gene mutant,in which,one copy of riboflavin operon was inserted between 5' and 3' end of guaC gene.The homologous recombination events were confirmed by PCR methods,and then,the riboflavin yield of mutant strain GJ07 was measured.The results of shake flask culture showed that the production of riboflavin of GJ07 was 24.5% higher than that of GJ06 in 60 h.