Properties of the biosurfactant produced byBacillus licheniformis strain JF-2

Summary The activity of the biosurfactant produced byBacillus licheniformis strain JF-2 was quantified using a unit defined as the amount of the acid-precipitated biosurfactant that lowered the surface tension by 10 mN/m. One unit was equivalent to 37 g/ml of the acid-precipitated biosurfactant. Acid precipitation was very effective in the removal of the biosurfactant from the spent medium. Among the solvents tested methanol was the most efficient in extracting the surfactant activity from acid-precipitated material. Thin-layer chromatography of the acid-precipitated biosurfactant revealed four components, two of which contained a lipid moiety and one of which contained an amino group. The methanol-soluble fraction also contained these four components. Studies suggested that all four components were needed for full activity. The lowest interfacial tensions against octane were observed when NaCl concentrations were 50 g/l or greater. Calcium concentrations greater than 25 g/l significantly increased the interfacial tension     

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.     

Re-identification of the halotolerant, biosurfactant-producing Bacillus licheniformis strain JF-2 as Bacillus mojavensis strain JF-2

Bacillus strain JF-2 (ATCC 39307) is a halotolerant, biosurfactant-producing bacterium that was initially described as a member of the species Bacillus licheniformis based on a limited set of phenotypic characteristics. Here, genetic and phenotypic analyses were employed to determine the relationship of Bacillus strain JF-2 to other Bacillus strains. The restriction patterns with AluI and analysis of gyrA and 16S rRNA gene sequences grouped Bacillus strain JF-2 with B. mojavensis^T and not with B. licheniformis^T. DNA–DNA similarity showed JF-2 was 75% similar to B. mojavensis^T and only 11% similar to B. licheniformis^T. Both strain JF-2 and B. mojavensis^T required DNA for anaerobic growth, but B. licheniformis^T did not. B. mojavensis^T and strain JF-2 did not grow anaerobically in thioglycollate medium or aerobically with propionate while B. licheniformis^T grew under these conditions. DNA–DNA similarity, gene sequence data and phenotypic characteristics all support the assignment of JF-2 as a member of the species B. mojavensis.     

Anaerobic Production of a Biosurfactant by Bacillus licheniformis JF-2

Bacillus licheniformis JF-2 anaerobically produced a biosurfactant when grown in a glucose-mineral salts medium containing yeast extract and NaNO₃. Surface tension of the medium was reduced from 70 to 74 mN/m to as low as 28 mN/m due to the production of an anionic biosurfactant.     

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.     

产生物表面活性剂的地衣芽孢杆菌种子培养条件研究

本文对产生物表面活性剂的油藏地衣芽孢杆菌种子培养条件进行优化.通过测定种子培养液中菌体浓度和发酵液的菌浓及表面张力,研究温度、通气量、接种龄、接种量对种子生长和发酵产生物表面活性剂的影响.确定了种子适宜培养条件为装液量100 mL/250 mL,12层纱布封口,于50 ℃、180 r/min摇床培养14 h.以10％接种量接种发酵,发酵24 h的发酵液表面张力降至最低,为22.6 mN/m.在该条件下培养种子,可缩短种子培养时间,实现提前接种发酵并高产生物表面活性剂.     

Recovery and purification of the lipopeptide biosurfactant of Bacillus subtilis by ultrafiltration

Surfactin, a lipopeptide biosurfactant produced as micelles by Bacillus subtilis, was recovered from the fermentation broth by ultrafiltration with a 30 kDa MWCO membrane. The retained surfactin micelles were then ruptured and collected in the permeate by adding methanol to 50% (v/v). The final yield of surfactin was 95%.     

Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis.

Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues L-Gln, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13-15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.     

Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans

Aims: To isolate the biologically active fraction of the lipopeptide biosurfactant produced by a marine Bacillus circulans and study its antimicrobial potentials. Methods and Results: The marine isolate B. circulans was cultivated in glucose mineral salts medium and the crude biosurfactant was isolated by chemical isolation method. The crude biosurfactants were solvent extracted with methanol and the methanol extract was subjected to reverse phase high‐performance liquid chromatography (HPLC). The crude biosurfactants resolved into six major fractions in HPLC. The sixth HPLC fraction eluting at a retention time of 27·3 min showed the maximum surface tension‐reducing property and reduced the surface tension of water from 72 mNm^?1 to 28 mNm^?1. Only this fraction was found to posses bioactivity and showed a pronounced antimicrobial action against a panel of Gram‐positive and Gram‐negative pathogenic and semi‐pathogenic micro‐organisms including a few multidrug‐resistant (MDR) pathogenic clinical isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of this antimicrobial fraction of the biosurfactant were determined for these test organisms. The biosurfactant was found to be active against Gram‐negative bacteria such as Proteus vulgaris and Alcaligens faecalis at a concentration as low as 10 μg ml^?1. The biosurfactant was also active against methicillin‐resistant Staphylococcus aureus (MRSA) and other MDR pathogenic strains. The chemical identity of this bioactive biosurfactant fraction was determined by post chromatographic detection using thin layer chromatography (TLC) and also by Fourier transform infrared (FTIR) spectroscopy. The antimicrobial HPLC fraction resolved as a single spot on TLC and showed positive reaction with ninhydrin, iodine and rhodamine‐B reagents, indicating its lipopeptide nature. IR absorption by this fraction also showed similar and overlapping patterns with that of other lipopeptide biosurfactants such as surfactin and lichenysin, proving this biosurfactant fraction to be a lipopeptide. The biosurfactant did not show any haemolytic activity when tested on blood agar plates, unlike the lipopeptide biosurfactant surfactin produced by Bacillus subtilis. Conclusions: The biosurfactant produced by marine B. circulans had a potent antimicrobial activity against Gram‐positive and Gram‐negative pathogenic and semi‐pathogenic microbial strains including MDR strains. Only one of the HPLC fractions of the crude biosurfactants was responsible for its antimicrobial action. The antimicrobial lipopeptide biosurfactant fraction was also found to be nonhaemolytic in nature. Significance and impact of the study: This work presents a nonhaemolytic lipopeptide biosurfactant produced by a marine micro‐organism possessing a pronounced antimicrobial action against a wide range of bacteria. There is a high demand for new antimicrobial agents because of the increased resistance shown by pathogenic micro‐organisms against the existing antimicrobial drugs. This study provides an insight into the search of new bioactive molecules from marine micro‐organisms.     

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

生物表面活性剂生产菌的鉴定及其发酵优化和产物性质研究

由中国科学院南海海洋研究所提供的一株生物表面活性剂生产菌,经菌落、菌体形态和16S rDNA序列分析,鉴定为芽孢杆菌属,命名为Bacillus SCUT09.初步优化了该菌株的培养条件,最佳碳、氮源分别为木薯淀粉、牛肉膏,最利于Bacillus SCUT09生长和生物表面活性剂积累的条件为:NaCl 1%,pH 6.5...     

L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

枯草芽孢杆菌BI1产抗真菌脂肽培养条件的优化

采用Plackett—Burman设计对枯草芽孢杆茵BI1产抗菌脂肽培养条件中相关影响因素(即种龄、接种量和pH值)的效应进行评价并筛选出重要的影响因素,然后用Box-Behnken设计及响应面分析确定了重要影响因素的最佳条件,优化后的溶血透明圈直径达到17.5 mm,比优化前的13.75 mm提高了27.27%。     

Levan-type FOS production using a Bacillus licheniformis endolevanase

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 °C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.     

Increased flux through the TCA cycle enhances bacitracin production by Bacillus licheniformis DW2

Applied Microbiology and Biotechnology - The dodecapeptide antibiotic bacitracin, produced by several strains of Bacillus licheniformis and Bacillus subtilis, is widely used as an antibacterial...     

能量代谢工程促地衣芽胞杆菌DW2高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性环肽类抗生素,目前广泛应用于兽药领域。能量代谢在微生物高效合成目的代谢产物中具有重要作用。文中以杆菌肽工业生产菌株地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,首先构建了呼吸链分支途径细胞色素bd泛醇氧化酶基因cydB缺失菌株,发现cydB缺失后杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了10.97%和22.96%。接着,证实了强化表达另外一条呼吸链分支途径——细胞色素aa3氧化酶基因qoxA能够提高杆菌肽合成水平,其杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了18.27%和34.00%。强化ADP合成供给也是促进胞内ATP积累的有效策略,结果表明强化表达腺苷激酶DcK和腺苷酸激酶AdK均可以提高杆菌肽效价和胞内ATP浓度,其中强化表达DcK效果较好,其杆菌肽效价相比对照提高16.78%。最后,通过组合代谢工程育种,在基因cydB缺失菌DW2ΔcydB基础上整合表达了qoxA和dck,得到工程菌株DW2-CQD(DW2ΔcydB::qoxA::dck),发酵结果表明,DW2-CQD杆菌肽效价达到954.25 U/mL,相比于对照菌株提高了21.66%,单位菌体杆菌肽效价为2.11 U/CFU,相比对照提高了11.05%。此外,DW2-CQD胞内ATP浓度为39.54 nmol/L,相比于对照提高了49.32%。结果证实能量代谢工程是提高杆菌肽发酵水平的有效策略,提供了一株具有工业化应用前景的杆菌肽生产菌株。     

Development of an asporogenic Bacillus licheniformis for the production of keratinase

AIMS: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. METHODS AND RESULTS: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. CONCLUSIONS: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.