Promoters are crucial expression elements in synthetic biology,and Bacillus licheniformis serves as an excellent chassis cell for industrial production.However,the diversity and quantity of available promoter elements...Promoters are crucial expression elements in synthetic biology,and Bacillus licheniformis serves as an excellent chassis cell for industrial production.However,the diversity and quantity of available promoter elements remain particularly limited.Existing promoters are categorized into constitutive and carbon source-inducible types,both exhibiting deficiencies in transcriptional strength and diversity of environmental signal responsiveness.As essential nutrients for microbial growth comparable to carbon sources,nitrogen sources hold significance.The development of nitrogen source-responsive promoters is vital for enriching synthetic biology toolkits and enhancing chassis cell performance.This study initially predicted nitrogen-responsive promoter elements through genomic analysis.Using enhanced green fluorescent protein(egfp)as a reporter gene,transcriptional initiation characteristics were evaluated.Results demonstrated that PglnR and Pgcv promoters could initiate transcription in response to sodium glutamate,with transcriptional intensities 200%and 100%higher than the control group at 36 h.Subsequently,these screened promoters(PglnR and Pgcv)were employed to mediate the expression of transglutaminase gene from Streptomyces mobaraensis.Under optimized conditions(37°C,10 g/L soluble starch,and 10 g/L glutamine),recombinant strains exhibited enhanced secretory expression.The maximum extracellular enzyme activity reached 2.61 U/mL.In fed-batch fermentation using a 5-L glass fermentor,the BL-TG3 recombinant strain achieved peak enzyme activity of 14.7 U/mL.The discovery,characterization,and application of novel nitrogen-responsive promoters establish a foundation for optimizing B.licheniformis expression systems.展开更多
Tannase has vital importance in several industries.However,tannase production employing tannic acid as a substrate is costly.This study optimized medium components,concentration of medium components and physical param...Tannase has vital importance in several industries.However,tannase production employing tannic acid as a substrate is costly.This study optimized medium components,concentration of medium components and physical parameters to yield maximum tannase production from Bacillus licheniformis AS1 in submerged fermentation utilizing low-cost agri-waste Citrus limetta(Mosambi)peels as a substrate.Tannase activity and stability parameters were also optimized.The produced crude tannase and partially purified tannase were used to reduce the bitterness(tannin content)from pomegranate juice and to remove the dye.B.licheniformis AS1 produced 0.361 U/mL under un-optimized conditions.During screening of medium components,Mosambi peels,yeast extract,potassium nitrate and sodium chloride was selected.The concentration of medium components(0.8%Mosambi peels,0.2%yeast extract,0.12%potassium nitrate,and 0.06%sodium chloride)was optimized using central composite design which yielded tannase up to 27.809 U/mL.Then,physical conditions were optimized(agita-tion,100μl inoculum size,40℃ temperature,pH 3,and 72 h of incubation)and yielded tannase up to 43.83±0.82 U/mL.The optimal conditions for tannase activity appeared at pH 8,at 40℃,and 10 min incubation period with 0.3%substrate concentration.The tannase showed highest stability at 40ºC and at pH 7.The maximum partial purified tannase activity was recorded at pH 8 and at 40℃,while enzyme stability was at pH 7 and a temperature of 40℃.The reduction in tannin content of pomegranate juice was noted after 2 h incubation at 37ºC.This enzyme was also effective for the partial removal of crystal violet dye.The tannase produced in this study was cost-effective due to utilization of low cost agri-waste and showed potential in various industrial applications.展开更多
[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, sp...[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.展开更多
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...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展开更多
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 lic...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.展开更多
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 co...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.展开更多
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 i...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.展开更多
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 activit...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.展开更多
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 ...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.展开更多
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 effi...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).展开更多
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 p...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.展开更多
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 fea...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.展开更多
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 model...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.展开更多
The present study deals with the kinetics of improved poly(3-hydroxybutyrate)(PHB)production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis.Production of biodegradable polymers is to eliminate use of ...The present study deals with the kinetics of improved poly(3-hydroxybutyrate)(PHB)production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis.Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact.For the current study,mutant strain(NA-21)&wild-type(IIB-isl19)were used for PHB production.Submerged culture with two-stage fermentation technique was used for PHB production.Results indicated that PHB production was improved with 300 mM of-HNO2.The superior mutant strain(NA-21)resulted in 2-fold more PHB as compared to the wild-type(IIB-isl9).It was selected,and resistance against L-cysteine HCl was developed.At 4 ppm concentration of L-cysteine HCl,PHB production by mutant strain(NA-cys4)was higher than its wild counterpart by 5.7-fold.Kinetic study of parameters including specific growth rate(μh−1),growth(Yx/s,Ys/x),product yield coefficients(Yp/s,Yp/x),volumetric rate constants(Qp,Qs,Qx)and specific rate constants(qp,qs,qx),were also accomplished.Moreover,Yp/x,Qp and qp=μ×Yp/x were found to be very significant as 1.254±0.06(g/g biomass),0.134±0.01(g/l/h)and 0.168±0.01(g/g/h),respectively.The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold.The highest PHB production during the study was 16.35±3.12 g/l which highlighted its significance(p≤0.05)and impact on the overall process.The variation in PBH yield between wild-type and mutant B.licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers.From the results,it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future.展开更多
In Maotai-flavor Baijiu production,reducing lactic acid(LA)can alleviate microbial imbalance and flavor disharmony caused by LA accumulation.Current methods for reducing LA mainly focus on physical removal and ferment...In Maotai-flavor Baijiu production,reducing lactic acid(LA)can alleviate microbial imbalance and flavor disharmony caused by LA accumulation.Current methods for reducing LA mainly focus on physical removal and fermentation parameter control,but they address only the symptoms,not the underlying cause.This study selected Lactobacillus panis antagonistic bacteria to control LA production at its source by inhibiting L.panis growth and analyzed its antimicrobial substances and mechanisms.Firstly,a high-throughput screening method for L.panis antagonists was developed based on lactate dehydro-genase,which correlates LA concentration with reduced nicotinamide adenine dinucleotide(NADH).Subsequently,a total of 34 antagonists were screened,with Bacillus licheniformis BL-4 exhibiting the highest inhibition rate(62.25%)against L.panis.Moreover,the primary antimicrobial substance,antimicrobial peptide antiL24,was purified from B.licheniformis BL-4 and evaluated for its activity and sequence.Finally,the mechanism of antiL24 against L.panis was analyzed by using microstructural analyses and transcriptomic profiling,revealing that antiL24 disrupts the cell wall and membrane of L.panis and affects genes involved in energy metabolism and protein synthesis.This study proposes a novel strategy for regulating LA concentration in Maotai-flavor Baijiu production,with the potential to enhance its quality.展开更多
基金funded by the National Key Research&Development Program of China(2020YFA0907700)the Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)+2 种基金Wuxi Industrial Innovation Research Institute Pilot Technology Pre-research Project(XD24024)the National Natural Foundation of China(32172174)the Jiangsu Funding Program for Excellent Postdoctoral Talent(2024ZB371).
文摘Promoters are crucial expression elements in synthetic biology,and Bacillus licheniformis serves as an excellent chassis cell for industrial production.However,the diversity and quantity of available promoter elements remain particularly limited.Existing promoters are categorized into constitutive and carbon source-inducible types,both exhibiting deficiencies in transcriptional strength and diversity of environmental signal responsiveness.As essential nutrients for microbial growth comparable to carbon sources,nitrogen sources hold significance.The development of nitrogen source-responsive promoters is vital for enriching synthetic biology toolkits and enhancing chassis cell performance.This study initially predicted nitrogen-responsive promoter elements through genomic analysis.Using enhanced green fluorescent protein(egfp)as a reporter gene,transcriptional initiation characteristics were evaluated.Results demonstrated that PglnR and Pgcv promoters could initiate transcription in response to sodium glutamate,with transcriptional intensities 200%and 100%higher than the control group at 36 h.Subsequently,these screened promoters(PglnR and Pgcv)were employed to mediate the expression of transglutaminase gene from Streptomyces mobaraensis.Under optimized conditions(37°C,10 g/L soluble starch,and 10 g/L glutamine),recombinant strains exhibited enhanced secretory expression.The maximum extracellular enzyme activity reached 2.61 U/mL.In fed-batch fermentation using a 5-L glass fermentor,the BL-TG3 recombinant strain achieved peak enzyme activity of 14.7 U/mL.The discovery,characterization,and application of novel nitrogen-responsive promoters establish a foundation for optimizing B.licheniformis expression systems.
文摘Tannase has vital importance in several industries.However,tannase production employing tannic acid as a substrate is costly.This study optimized medium components,concentration of medium components and physical parameters to yield maximum tannase production from Bacillus licheniformis AS1 in submerged fermentation utilizing low-cost agri-waste Citrus limetta(Mosambi)peels as a substrate.Tannase activity and stability parameters were also optimized.The produced crude tannase and partially purified tannase were used to reduce the bitterness(tannin content)from pomegranate juice and to remove the dye.B.licheniformis AS1 produced 0.361 U/mL under un-optimized conditions.During screening of medium components,Mosambi peels,yeast extract,potassium nitrate and sodium chloride was selected.The concentration of medium components(0.8%Mosambi peels,0.2%yeast extract,0.12%potassium nitrate,and 0.06%sodium chloride)was optimized using central composite design which yielded tannase up to 27.809 U/mL.Then,physical conditions were optimized(agita-tion,100μl inoculum size,40℃ temperature,pH 3,and 72 h of incubation)and yielded tannase up to 43.83±0.82 U/mL.The optimal conditions for tannase activity appeared at pH 8,at 40℃,and 10 min incubation period with 0.3%substrate concentration.The tannase showed highest stability at 40ºC and at pH 7.The maximum partial purified tannase activity was recorded at pH 8 and at 40℃,while enzyme stability was at pH 7 and a temperature of 40℃.The reduction in tannin content of pomegranate juice was noted after 2 h incubation at 37ºC.This enzyme was also effective for the partial removal of crystal violet dye.The tannase produced in this study was cost-effective due to utilization of low cost agri-waste and showed potential in various industrial applications.
文摘[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.
基金Grants from COMRA(Grant No.DY125-15-T-01 and SOA(Grant No.2010319123366025-4)National High Technology Development Project(863 Project,Grant No.2011AA10A202-2)National Key Technologies R&D Program(Grant No.2011BAE06B04)
文摘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
基金supported by the Agriculture and Agri-Food Canada,AAFC’s IOP project,Manitoba Pork and Swine Innovation PorcCanada Foundation for Innovation(CFI)supported by the Chinese Scholarship Council(CSC).
文摘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.
基金Project (No. Y304203) supported by the Natural Science Foundationof Zhejiang Province, China
文摘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.
文摘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.
基金Supported by Three New Agriculture Project of Jiangsu Province(SXGC[2012]2012)
文摘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.
文摘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.
基金financially supported by the National Basic Research Program of China(973 Program,No.2014CB846003)the National Natural Science Foundation of China(No.41372346,21477129)
文摘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).
基金Deanship of Scientific Research at King Khalid University through research groups program,Grant No.R.G.P.1/241/41.
文摘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.
文摘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.
基金Supported by Educational Commission of Heilongjiang Province of China(11551z011)
文摘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.
基金the Researchers Supporting Project,King Saud University,Riyadh,Saudi Arabia for funding this work through the project number(RSP-2024R437).
文摘The present study deals with the kinetics of improved poly(3-hydroxybutyrate)(PHB)production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis.Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact.For the current study,mutant strain(NA-21)&wild-type(IIB-isl19)were used for PHB production.Submerged culture with two-stage fermentation technique was used for PHB production.Results indicated that PHB production was improved with 300 mM of-HNO2.The superior mutant strain(NA-21)resulted in 2-fold more PHB as compared to the wild-type(IIB-isl9).It was selected,and resistance against L-cysteine HCl was developed.At 4 ppm concentration of L-cysteine HCl,PHB production by mutant strain(NA-cys4)was higher than its wild counterpart by 5.7-fold.Kinetic study of parameters including specific growth rate(μh−1),growth(Yx/s,Ys/x),product yield coefficients(Yp/s,Yp/x),volumetric rate constants(Qp,Qs,Qx)and specific rate constants(qp,qs,qx),were also accomplished.Moreover,Yp/x,Qp and qp=μ×Yp/x were found to be very significant as 1.254±0.06(g/g biomass),0.134±0.01(g/l/h)and 0.168±0.01(g/g/h),respectively.The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold.The highest PHB production during the study was 16.35±3.12 g/l which highlighted its significance(p≤0.05)and impact on the overall process.The variation in PBH yield between wild-type and mutant B.licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers.From the results,it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future.
基金supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)+1 种基金the Natural Science Foundation of Jiangsu Province(BK20202002)the Fundamental Research Funds for the Central Universities(JUSRP124034).
文摘In Maotai-flavor Baijiu production,reducing lactic acid(LA)can alleviate microbial imbalance and flavor disharmony caused by LA accumulation.Current methods for reducing LA mainly focus on physical removal and fermentation parameter control,but they address only the symptoms,not the underlying cause.This study selected Lactobacillus panis antagonistic bacteria to control LA production at its source by inhibiting L.panis growth and analyzed its antimicrobial substances and mechanisms.Firstly,a high-throughput screening method for L.panis antagonists was developed based on lactate dehydro-genase,which correlates LA concentration with reduced nicotinamide adenine dinucleotide(NADH).Subsequently,a total of 34 antagonists were screened,with Bacillus licheniformis BL-4 exhibiting the highest inhibition rate(62.25%)against L.panis.Moreover,the primary antimicrobial substance,antimicrobial peptide antiL24,was purified from B.licheniformis BL-4 and evaluated for its activity and sequence.Finally,the mechanism of antiL24 against L.panis was analyzed by using microstructural analyses and transcriptomic profiling,revealing that antiL24 disrupts the cell wall and membrane of L.panis and affects genes involved in energy metabolism and protein synthesis.This study proposes a novel strategy for regulating LA concentration in Maotai-flavor Baijiu production,with the potential to enhance its quality.
