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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Biotechnological and food synthetic biology potential of platform strain:Bacillus licheniformis

Bacillus licheniformis is one of the most characteristic Gram-positive bacteria.Its unique genetic background and safety characteristics make it have important biologic applications in the food industry,including,the biosyn-thesis of high value-added bioproducts,probiotic functions,biological treatment of wastes derived from food production,etc.In this review,these recent advances are summarized and presented systematically for the first time.In addition,we highlight synthetic biology strategies as a potential driver of developing this strain for wider and more efficient application in the food industry.Finally,we present the current challenges faced and provide our unique perspective on relevant future research directions.In summary,this review will provide an illumi-nating and comprehensive perspective that will allow an in-depth understanding of B.licheniformis and promote its more effective development in the food industry.

Isolation and characterization of cyclic lipopeptides from the marine-derived Bacillus licheniformis

Chromatographic separation of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new cyclic lipopeptides named ai-Cl6 surfactin （1） and ai-Cl4 surfactin （2）, together with iso-Cm5 surfactin and iso-Cl6 surfactin. The structures of the new cyclic lipopeptides were determined through extensive spectroscopic analysis. The sequences of the amino acids in cyclic nucleus were established by the ESI-MS/MS fragmentation, which provided an efficient method to detect lipopeptides from bacterium extracts without senaration

The Biocontrol Effects of the Bacillus licheniformis W10 Strain and Its Antifungal Protein Against Brown Rot in Peach

The biocontrol effects of Bacillus licheniformis W10 bacterial suspension and its antifungal protein on peach brown rot caused by Monilinia fructicola in storage peach fruits and the effects on fruit quality were investigated. The results showed that the fruit disease suppression of B. licheniformis W10 bacterial suspension and antifungal protein were significantly higher than that of the control. Inoculation of bacterial suspension and antifungal protein prior to M. fructicola gave a better biocontrol effect, and the higher concentrations of bacterial(1 × 1010 cfu · m L-1) and antifungal protein(3.0 mg · m L-1) performed better control effects. The environmental conditions, such as temperature and humidity, affected biocontrol effects of W10 bacterial suspension and antifungal protein. The influence of environment conditions on the activity of antifungal protein was less than that on bacterial suspension. Moreover, lower temperature(4 ℃) and relative humidity(RH 70%–75%) were favorable to prevent peach brown rot by W10 bacterial suspension and its antifungal protein. The W10 bacterial suspension and antifungal protein amended with calcium [0.1% Ca(NO3)2] could enhance the biocontrol effects, and obviously put off the occurrence of peach brown rot. In addition, the bacterial suspension and antifungal protein significantly reduced the natural decay rates of peach fruits during storage, and the effects were equal to carbendazim. Moreover, both W10 bacterial suspension and antifungal protein treatments did not have effects on external and internal fruit appearance, such as chromatic aberration parameter L* of flesh, flesh firmness, soluble solids content and weight loss. Therefore, the B. licheniformis W10 is a potential biocontrol factor for peach brown rot.

Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Bacitracin,a new type of cyclic peptide antibiotic,is widely used as the feed additive in feed industry.Branched chain amino acids(BCAAs)are the key precursors for bacitracin synthesis.In this research,soybean meal was served as the raw material to supply precursor amino acids for bacitracin synthesis,and enhanced production of bacitracin was attempted by engineering BCAA transporter BrnQ and its regulator Lrp in the bacitracin industrial production strain Bacillus licheniformis DW2.Firstly,our results confirmed that Lrp negatively affected bacitracin synthesis in DW2,and deletion of lrp improved intracellular BCAA accumulations,as well as the expression level of BCAA transporter BrnQ,which further led to a 14.71%increase of bacitracin yield,compared with that of DW2.On the contrary,overexpression of Lrp decreased bacitracin yield by 12.28%.Secondly,it was suggested that BrnQ acted as a BCAA importer in DW2,and overexpression of BrnQ enhanced the intracellular BCAA accumulations and 10.43%of bacitracin yield.While,the bacitracin yield decreased by 18.27%in the brnQ deletion strain DW2△brnQ.Finally,BrnQ was further overexpressed in lrp deletion strain DW2△lrp,and bacitracin yield produced by the final strain DW2△lrp::BrnQ was 965.34 U/mL,increased by 22.42%compared with that of DW2(788.48 U/mL).Collectively,this research confirmed that Lrp affected bacitracin synthesis via regulating the expression of BCAA transporter BrnQ and BCAA distributions,and provided a promising strain for industrial production of bacitracin.

Enhanced production of poly-γ-glutamic acid via optimizing the expression cassette of Vitreoscilla hemoglobin in Bacillus licheniformis

Poly-γ-glutamic acid(γ-PGA)is a natural polymer with various applications,and its high-viscosity hinders ox-ygen transmission and improvement of synthesis level.Vitreoscilla hemoglobin(VHB)has been introduced into various hosts as oxygen carrier,however,its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis.Here,we want to optimize the expression cassette of VHB for γ-PGA production.Firstly,our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation(Tat)signal peptides(SP_(YwbN),SP_(PhoD) and SP_(TorA))into VHB expression cassette,and the best per-formance was attained by SP YwbN from Bacillus subtilis,theγ-PGA yield of which was 18.53% higher than that of control strain,and intracellular ATP content and oxygen transfer coefficient(K_(L)a)were increased by 29.71% and 73.12%,respectively,indicating that VHB mediated by SP YwbN benefited oxygen transfer and ATP generation forγ-PGA synthesis.Furthermore,four promoters were screened,and P vgb was proven as the more suitable promoter for VHB expression andγ-PGA synthesis,andγ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%.Finally,WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation,and 46.39 g/Lγ-PGA was attained by glucose feeding,increased by 49.26%compared with the initial yield(31.01 g/L).Taken together,this study has attained an efficient VHB expression cassette for oxygen transfer andγ-PGA synthesis,which could also be applied in the production of other metabolites.

Genomic and metabolomic analysis of Bacillus licheniformis with enhanced poly-γ-glutamic acid production through atmospheric and room temperature plasma mutagenesis

Poly-γ-glutamic acid is an extracellular polymeric substance with various applications owing to its valuable properties of biodegradability,flocculating activity,water solubility,and nontoxicity.However,the ability of natural strains to produce poly-γ-glutamic acid is low.Atmospheric and room temperature plasma was applied in this study to conduct mutation breeding of Bacillus licheniformis CGMCC 2876,and a mutant strain M32 with an 11%increase in poly-γ-glutamic acid was obtained.Genome resequencing analysis identified 7 nonsynonymous mutations of ppsC encoding lipopeptide synthetase associated with poly-γ-glutamic acid metabolic pathways.From molecular docking,more binding sites and higher binding energy were speculated between the mutated plipastatin synthase subunit C and glutamate,which might contribute to the higher poly-γ-glutamic acid production.Moreover,the metabolic mechanism analysis revealed that the upregulated amino acids of M32 provided substrates for glutamate and promoted the conversion between L-and D-glutamate acids.In addition,the glycolytic pathway is enhanced,leading to a better capacity for using glucose.The maximum poly-γ-glutamic acid yield of 14.08 g·L^(–1)was finally reached with 30 g·L^(–1)glutamate.

CcpA mutants infuence selective carbon source utilization by changing interactions with target genes in Bacillus licheniformis

The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources.This process is mainly governed by the global regulator catabolite control protein A(CcpA),which can recognize and bind to multiple target genes that are widely distributed in metabolic pathways.Although the DNA-binding domain of CcpA has been predicted,the infuence of key amino acids on target gene recognition and binding has yet to be uncovered.In this study,the impact of Lys31,Ile42 and Leu56 on in vitro protein–DNA interactions and in vivo carbon source selective utilization was investigated.The results showed that alanine substitution of Lys31 and Ile42,located within the 3rd helices of the DNAbinding domain,signifcantly weakened the binding strength between CcpA and target genes.These mutations also lead to alleviated repression of xylose utilization in the presence of glucose.On the other hand,the Leu56Arg mutant in the 4th helices exhibited enhanced binding afnity compared with that of the wild-type one.When this mutant was used to replace the native one in B.licheniformis cells,the selective utilization of glucose over xylose increased.This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins.This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.

饲料中添加地衣芽孢杆菌对鲤鱼生长性能的影响

该试验旨在研究鲤(Cyprinus carpio)的饲料中添加不同水平的地衣芽孢杆菌(Bacillus licheniformis)对其生长性能的影响,确定鲤养殖中最适地衣芽孢杆菌添加水平。试验选择480尾体重相近的鲤随机分成4个处理组,每组4个重复,每个重复30尾。各组鲤依次投喂含有0%、0.1%、0.2%和0.4%地衣芽孢杆菌的饲料。驯养2周,进行60 d的正式试验。试验结束测定各组鲤的生长性能。结果显示,与对照组比较,试验组投喂含0.2%和0.4%地衣芽孢杆菌的饲料,可以显著提高其试验末重、增重率,显著降低了饲料系数(P<0.05);投喂含有0.1%、0.2%和0.4%地衣芽孢杆菌的饲料,可以显著提高特定生长率(P<0.05);投喂含有0.2%地衣芽孢杆菌的饲料可以显著提高蛋白质效率(P<0.05)。研究结果表明,在鲤的养殖中添加适量的地衣芽孢杆菌可以明显改善其生长性能,且地衣芽孢杆菌的最适添加量为0.2%。

16S与gyrB基因联合建树快速鉴定海水中的一株地衣芽胞杆菌

在保守序列高度相似的细菌鉴定中,单独使用16S rDNA/RNA序列进行比对和构建进化树通常无法准确鉴定到种,需要增加测序基因数并对多基因进行分析。为实现快速鉴定,课题组对16S与gyrB基因联合建树的方法进行了研究,将海洋来源的一株杆菌,分别用通用引物扩增16S和gyrB基因并测序,在GeneBank进行序列比对后,选择各菌种保藏中心16S和gyrB基因均相似的菌株,取16S和gyrB基因序列,采用Paup*4.0构建进化树。使用16S与gyrB拼接序列构建的进化树中属于同一种的菌株均很好的聚合在一枝,种间分枝自展值均高于98,分类结构准确,筛选得到的杆菌与地衣芽胞杆菌(Bacilluslicheniformis)聚合在一枝,自展值为100,鉴定为地衣芽胞杆菌。经生理生化试验验证,该菌株与地衣芽胞杆菌特征完全一致,使用16S和gyrB基因联合建树得到的鉴定结果准确且快速简便。