Analysis on 16S-ITS Marker from Bacillus licheniformis and Its Specificity

[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.

地衣芽孢杆菌(Bacillus licheniformis)对Cr^(6+)的吸附动力学研究

从污染土壤中分离出地衣芽孢杆菌(Bacilluslicheniformis),利用其死菌体对Cr6+溶液进行吸附动力学研究.在Ci=300mg/L、pH=2.5和θ=50℃条件下,吸附120min获得最大吸附量60.5mg/g.应用Langmuir和Freundlich吸附等温线研究,结果表明,Langmuir吸附等温线更为适合.动力学研究显示,地衣芽孢杆菌对Cr6+的吸附动力学可以用拟二级速度方程进行描叙.

利用Bacillus licheniformis NK-03合成聚谷氨酸及其合成酶基因pgsBCA的克隆

分离到一株能合成γ-聚谷氨酸(γ-PGA)的细菌,通过16SrRNA 序列同源性分析、计算机微生物分类鉴定系统 BIOLOG-System 4.20等方法,确立该菌株为地衣芽孢杆菌,命名为 Bacillus licheniformis NK-03.该菌合成的γ-PGA 所含的 L-谷氨酸单体可高达98%,且重均分子量(M_w)为1360000.另外,利用 pXMJ19质粒将 B.licheniformis NK-03的7-PGA 合成酶基因 pgsBCA 克隆到了 Escherichia coli JM109中,使其成功表达合成了重均分子量为42 433的γ-PGA.同时,通过比较 B.licheniformis NK-03与已报道菌株 pgsBCA 基因编码氨基酸序列的同源性,发现基因簇中 pgsC 基因编码的氨基酸序列最为保守.

Bacillus licheniformis 6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌 6 816中扩增了碱性蛋白酶基因 (apr) ,扩增的 1 14kb的DNA片段插入到大肠杆菌载体pET - 2 0b中 ,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM10 9(DE3)中得到表达。SDS -PAGE分析显示融合表达产物的分子量为 30kD ,同核酸序列测定所推导的值相符。表达产物占细胞总蛋白的 7 5 % ,重组菌的酶活比出发菌株提高了 3 3倍。研究发现 。

地衣芽孢杆菌(Bacillus licheniformis)对镉胁迫下泥蚶(Tegillarca granosa)的抗氧化酶活性和免疫相关基因表达的影响

本文研究一株地衣芽孢杆菌(Bacillus licheniformis)对提高泥蚶耐受镉(Cd2+)胁迫的作用,实验分析了不同浓度地衣芽孢杆菌对Cd2+胁迫下泥蚶(Tegillarca granosa)的存活率及组织中Cd残留量的影响;同时比较了地衣芽孢杆菌作用前后,泥蚶体内两类抗氧化酶,超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性,进一步通过Real-time PCR分析了三类免疫相关基因金属硫蛋白(MT)、小热休克蛋白分子(sHSP)和铁蛋白(ferritin)的表达情况。研究结果显示,地衣芽孢杆菌能降低泥蚶组织中Cd残留量,提高其存活率。并能有效促进泥蚶体内SOD和GSH-Px酶活性的增加,同时降低MT、sHSP和ferritin在mRNA水平上的表达。数据表明该地衣芽胞杆菌可通过调节泥蚶的免疫系统,起到降低Cd2+对泥蚶毒害的作用。

地衣芽孢杆菌(Bacillus licheniformis)HDYM-03亚麻脱胶粗酶液性质研究

以果胶为单一碳源,对分离自温水沤麻液中的地衣芽孢杆菌(Bacillus licheniformis)HDYM-03进行产酶发酵试验,探究脱胶酶粗酶液的酶学性质。研究表明:该粗酶液中的果胶酶和β-甘露聚糖酶的最适pH值分别为6.0和4.0,最适温度均为40℃,二者在pH值为4.0~6.0和30~40℃条件下保存0~60 h时,果胶酶能保持较高活力。

Bacillus licheniformis PF9 improves barrier function and alleviates inflammatory responses against enterotoxigenic Escherichia coli F4 infection in the porcine intestinal epithelial cells

Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus licheniformis(B.licheniformis)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of B.licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC F4.Methods:In the present study,the effects of B.licheniformis PF9 on the release of pro-inflammation cytokines,cell integrity and nuclear factor-κB(NF-κB)activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results:B.licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α),interleukin(IL)-8,and IL-6 during ETEC F4 infection.Furthermore,the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited.These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B.licheniformis PF9 treatment.The immunofluorescence and western blotting analysis revealed that B.licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1)and occludin(OCLN)in ETEC F4-infected IPEC-J2 cells.Meanwhile,the B.licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER)and cell permeability assay.Interestingly,B.licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including luxS,estA,estB,and elt)in ETEC F4.Conclusions:Collectively,our results suggest that B.licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways.Besides,B.licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410

The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows： elastolysis temperature 50 ℃, elastase concentration 1 × 10^4 U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasingelastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410:optimization of cultivation conditions using response surface methodology

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cul- tures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.

Mechanism and Application Effect of Bacillus licheniformis on Poultry

Bacillus licheniformis has the biological characteristics of strong resistance to stress, high temperature, high pressure, pH and bile salt, which also has unique advantage in application safety, antibacterial activity and stability. The recent research results on mechanism of B. licheniformis and its application effect in poultry production are elaborated in the paper.

溶剂稳定性蛋白酶产生菌Bacillus licheniformis YP1的发酵优化

溶剂稳定性蛋白酶产生菌Bacillus licheniformisYP1分离自油田土样。考察了碳源、氮源、金属离子等营养因素对YP1菌株发酵产溶剂稳定性蛋白酶的影响。YP1菌株发酵产胞外蛋白酶的最佳碳源为淀粉,果糖、甘露糖和乳糖显著抑制产酶;最佳氮源为酵母膏,干酪素、酵母粉和牛肉膏促进产酶,玉米浆和尿素显著抑制产酶。Mn2+可以显著促进酶活,Mg2+可以促进产酶,在初步优化的培养条件下,YP1菌株的胞外蛋白酶产量达980 U。

Production of Alkaline Protease by Bacillus Licheniformis

In this research the results of studies on optimization of alkaline protease production by Bacillus licheniformis are reported. The parameters, which were taken into consideration, are pH, temperature, time course of enzyme production, stirring rate and kinetics parameters. The effect of various carbon and nitrogen sources in culture medium compound on enzyme production was also considered The result of optimization revealed that maximum protease production was obtained at 37 ℃, pH equivalent tol 0.0 and with 150 rpm will occur after 72 hours. By comparing the effect of 5 carbon sources （maltose, glucose, starch, casein and lactose） in enzyme production, it has been known that using lactose will increase about 1.5 times enzyme production, compared to condition in which maltose is used. The result of studies on the effect of five nitrogen sources （i.e., peptone, tryptone, ammonium sulfate, urea and corn steep liquor） shows that corn steep liqour increases enzyme production more than others, while peptone can also be considered as a good nitrogen source; but, ammonium sulfate and urea reduce enzyme production considerably. It was concluded that protease production occurs in the stationary phase of growth. Studying the kinetics parameters resulted that the best model for the enzyme above is Lineweaver-Burk model according to which Km is 0.64 mmol and Vmax is 88 lamol/min.

A Two-step Biotechnological Process for Improving Nutrition Value of Feather Meal by Bacillus licheniformis S6

A two-step biotechnological process was developed using Bacillus licheniformis S6 to provide a simple and economical procedure which significantly improved feather meal nutrition value. Compared with IFM （initial feather meal） and CFM （commercial feather meal）, SFEFM （feather meal gained by solid fermentation and enzymolysis with continuous agitation） had a significant improvement （P〈0.05） in vitro digestibility, contents of oligopeptides and soluble protein released in digestive juice by pepsin- pancreatin digestion procedure, furthermore, some deficient essential amino acids in feather protein （histidine, methionine, lysine） were enhanced. Comapared with CFM, the oligopeptides released into digestive juice of ISFM （feather meal obtained by the biotechnological process described in the paper with intermittent shaking） was significantly enhanced （P〈O.05）, and its in vitro digestibility was statistically （P〉0.05） equivalent to CFM. The summary of the finding to IFM treatment and possible means of further improvements were also listed.

Biosynthesis of raw starch degradingβ-cyclodextrin glycosyltransferase by immobilized cells of Bacillus licheniformis using potato wastewater

The study was sought to enhance the synthesis of thermal stableβ-cyclodextrin glycosyltransferase(β-CGTase)using potato wastewater as a low-cost medium and assess the degree to which it is efficient for industrial production ofβ-cyclodextrin(β-CD)from raw potato starch.Thermophilic bacteria producingβ-CGTase was isolated from Saudi Arabia and the promising strain was identified as Bacillus licheniformis using phylogenetic analysis of the 16S rRNA gene.Alginate-encapsulated cultures exhibited twice-fold ofβ-CGTase production more than free cells.Scanning electron microscopy(SEM)of polymeric capsules indicated the potential for a longer shelf-life,which promotes the restoration of activity in bacterial cells across semi-continuous fermentation ofβ-CGTase production for 252 h.The optimal conditions forβ-CGTase synthesis using potato wastewater medium were at 36 h,pH of 8.0,and 50°C with 0.4%potato starch and 0.6%yeast extract as carbon and nitrogen sources,respectively.The purified enzyme showed a specific activity of 63.90 U/mg with a molecular weight of∼84.6 kDa as determined by SDS-PAGE analysis.The high enzyme activity was observed up to 60°C,and complete stability was achieved at 75°C.High levels of activity and stability were shown at pH 8.0,and the pH range from 7.0–10.0,respectively.The enzyme has an appreciable affinity for raw potato starch with a Km of 5.7×10−6 M and a Vmax of 87.71μmoL/mL/min.β-CD production was effective against 25 U/g of raw potato starch.The outcomes demonstrated its feasibility to develop a fermentation process by integrating the cost-effective production ofβ-CGTase having distinctive properties forβ-CD production with ecofriendly utilization of potato wastewater.

Establishment of Kinetics Models for Batch Fermentation Process of β-mannase with Bacillus licheniformis HDYM-04

In order to improve the yield of β-mannase and to investigate the rules of fermentation production, a high-yield β-mannase producing strain, Bacillus licheniformis HDYM-04, was used to investigate the kinetics models based on the optimal fermentation conditions： HDYM-04 strain was fermented at 37℃ for 30 h with agitation speed at 300 r/min and aeration rate at 3 L/min in a 5 L fermenter, the initial addition amount of konjac flour was 2%（w/v）, the initial pH of medium was 8.0, and the inoculum concentration was 6.7%（v/v）. Three batch fermentation kinetic models were established （cell growth kinetic model, substrate consumption kinetic model, product formation kinetic model） bases on Logistic and Luedeking-Piret equations. To be specific, cell growth kinetic model was dX/dt =0.431X （1- X/ 15.522 ）, substrate consumption kinetic model was -ds/dt =1.11 dX/dt ＋0.000 2 dP/dt ＋0.000 8X, and product formation kinetic model was dP/dt=133.1 dX ＋222.87X. The correlation coefficients R^2 of the three equations were 0.990 21, 0.989 08 and 0.988 12, respectively, which indicated a good correlation between experimental values and models. Therefore, the three equations could be used to describe the processes of cell growth, enzyme synthesis and substrate consumption during batch fermentation using B. licheniformis strain HDYM-04. The establishment of batch fermentation kinetic models （cell growth kinetic model, substrate depletion kinetic model, product formation kinetic model） could lay the theoretical foundation and provide practical reference for the applica- tion of HDYM-04 in fermentation industry.

产香风味地衣芽孢杆菌(Bacillus licheniformis)的分离及发酵代谢产物分析

从生产用大曲和酒醅中分离纯化得到1株细菌,根据该菌株的形态特征、生理生化指标和16S rDNA序列同源性分析结果将其鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以本地高粱为底物进行固态发酵闻香和代谢发酵风味产物分析。结果表明,该菌株发酵产香风味物质丰富,GC-MS分析定性检测出40多种发酵代谢产物,包括吡嗪类、酸类、酯类、醇类、酮类及芳香族化合物等,其中,四甲基吡嗪、3-羟基-2-丁酮、2,3-丁二醇、2,3-丁二酮、1,3-丁二醇、愈创木酚、乙酸、丁酸、3-甲基丁酸等成分含量较高。

地衣芽孢杆菌(Bacillus licheniformis)10236发酵条件的优化

为了提高菌株生物量、降低生产成本,通过单因素试验筛选最佳碳源种类、氮源种类、无机盐种类、培养温度、接种量和初始pH值,通过正交试验筛选氮源、氮源和无机盐的最佳配比。结果表明:培养该菌的最佳培养基配方为葡萄糖1%、黄豆粉3.5%、玉米淀粉3%、MnSO4·H2O 0.03%、CaCO30.3%、NaCl 0.5%;最佳发酵条件为发酵温度37℃、接种量10%、初始pH值7.0。利用优化后的发酵条件,在10 t发酵罐上进行了发酵水平验证,该菌芽孢数最低达到了2.02×1010 CFU/mL,较原来发酵条件下的发酵水平至少提高了2.2倍。优化后的发酵条件,大大提高了地衣芽孢杆菌10236的发酵芽孢数,降低了生产成本。

Characterization and Biosorption Performance of Silver by Bacillus Licheniformis

The demand of silver is increasing rapidly in recent decades, because silver and its related products are widely used in modern industry and decoration. It is necessary to recover silver from waste water using an efficient and environmental friendly method due to its environmental and economic benefits. In this paper, we eliminated the interference of Cl-and light conditions, and then studied the characterization and biosorption performance of silver by Bacillus licheniformis. The max biosorption amount was 87.4 mg/g(dry weight) with the initial Ag+concentration of 100 mg/L at pH 6.0. XRD pattern showed that the product was an amorphous compound. SEM/EDS-mapping and FT-IR results implied that phosphate, amino and carboxyl groups located on the cell walls involved in the biosorption of Ag^+. The XPS spectra result showed that the value of EB of Ag 3d_(5/2_(367.51 eV) corresponded to the energy values for Ag(Ⅰ), and indicated Ag^+ adsorbed to the surface of cell still maintained mono-valence. The results confirm that B. licheniformis just adsorb Ag+ but cannot covert soluble Ag^+ to silver nano-particles(AgNP).

Characterization of the chemical fungicides-responsive and bacterial pathogen-preventing Bacillus licheniformis in rice spikelet

Seed-borne bacterial pathogens cause severe yield loss and biotoxin contamination in rice,leading to increasing concern on the global food supply and environmental safety.Plant native microbes play an important role in defending against diseases,but their actions are often influ-enced by the chemical fungicides applied in the field.Here,Bacillus licheniformis mmj was isolated from rice spikelet,which uniquely showed not only fungicide-responsiveness but also broad-spectrum antimicrobial activity against major rice bacterial pathogens including Xanthomonas oryzae pv.oryzae,Burkholderia plantari and Burkholderia glumae.To understand the hallmark underlying the environmental adaptation and anti-microbial activity of B.licheniformis mmj,the genome sequence was determined by SMRT and subjected to bioinformatics analysis.Genome sequence analysis enabled the identification of a set of antimicrobial-resistance and antibacterial activity genes together with an array of harsh environment-adaptive genes.Moreover,B.licheniformis mmj metabolites were analyzed with gas chromatography coupled to triple quadrupole mass spectrometry,and the volatile components that were linked with the antimicrobial activity were preliminarily profiled.Collectively,the present findings reveal the genomic and metabolic landscapes underlying fungicide-responsive B.licheniformis,which offers a new opportunity to design harsh environment-adaptive biopesticides to cope with prevalent bacterial phytopathogens.

Recent biotechnological advances and future prospective of Bacillus licheniformis as microbial cell factories

Bacillus licheniformis is a characteristic Gram-positive bacterium originally found in soil.This microorganism has long been utilised as a workhorse for production of industrial enzymes or high value-added chemicals.With ever-increasing understand-ing on this strain and the maturation of the genetic technique,important advances have recently been made in developing B.licheniformis as an excellent chassis cell for synthetic biology.Here,we provide an overview of updated understanding on genome information,anaerobic metabolism,industrial applications of this strain.The state-of-art B.licheniformis genetics,especially its synthetic biology advances in biosensor,expression system and artificial metabolic pathways are illustrated.Finally,perspectives are offered for the limitations and challenges to be addressed to improve B.licheniformis as microbial cell factories.