Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

AIMS: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. METHODS AND RESULTS: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70 degrees C and resisted up to pH 3.0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. CONCLUSIONS: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160.05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.     

Optimization of Bacteriocin Production by Batch Fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2³_5-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3²_3-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10^7.3 to 10^7.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10⁴ times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Generation of Mini-Tn10 transposon insertion mutant library of Bacillus thuringiensis for the investigation of genes required for its bacteriocin production

Bacillus thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which inhibits the growth of several Gram-positive bacteria and particularly Bacillaceae . This study aimed to use the insertional transposon mutagenesis approach for disrupting and thus identifying genes associated with BF4 synthesis. Here, the mini-Tn 10 transposon was used to generate a library of B. thuringiensis mutants. Twenty thousand clones were screened for the search of mutants with affected bacteriocin synthesis. By molecular hybridization, it was demonstrated that the mini-Tn 10 transposition occurred in different sites. Clone MB1, containing a mini-Tn 10 single-copy insertion, lost the BF4 synthesis, but maintained its immunity to BF4. The flanking sequences surrounding the mini-Tn 10 insertion were cloned and sequenced. Homology searches of the surrounding ORFs revealed a strong similarity to a phage tail component, which allowed us to postulate that BUPM4 bacteriocin could be a phage tail-like one.     

Studies on bacteriocin (thermophilin T) production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ACA-DC 0040 in batch and fed-batch fermentation modes

Growth conditions that support bacteriocin (thermophilin T) production by Streptococcus thermophilus ACA-DC 0040 were identified. Synthesis of thermophilin T occurred during primary metabolic growth, while its specific rate of synthesis seemed to be optimal at T = 30°C. Thermophilin T activity rapidly decreased in the stationary phase, especially at high growth temperature (i.e. T = 42°C). In media with high content of complex nitrogen sources, high amounts of bacteriocin were detected in the growth environment, while about an 8-fold increase of thermophilin T titer and a 2-fold increase of specific synthesis rate was achieved when a fed-batch fermentation mode was applied.     

Optimization of bacteriocin production by batch fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2(3)(5-2) fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3(2)(3-1) fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27 degrees C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10(7.3) to 10(7.4) CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 x 10(4) times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Application of a mathematical model and Differential Evolution algorithm approach to optimization of bacteriocin production by Lactococcus lactis C7

The effect of pH and temperature on cell growth and bacteriocin production in Lactococcus lactis C7 was investigated in order to optimize the production of bacteriocin. The study showed that the bacteriocin production was growth-associated, but declined after reaching the maximum titer. The decrease of bacteriocin was caused by a cell-bound protease. Maximum bacteriocin titer was obtained at pH 5.5 and at 22°C. In order to obtain a global optimized solution for production of bacteriocin, the optimal temperature for bacteriocin production was further studied. Mathematical models were developed for cell growth, substrate consumption, lactic acid production and bacteriocin production. A Differential Evolution algorithm was used both to estimate the model parameters from the experimental data and to compute a temperature profile for maximizing the final bacteriocin titer and bacteriocin productivity. This simulation showed that maximum bacteriocin production was obtained at the optimal temperature profile, starting at 30°C and terminating at 22°C, which was validated by experiment. This temperature profile yielded 20% higher maximum bacteriocin productivity than that obtained at a constant temperature of 22°C, although the total amount of bacteriocin obtained was slightly decreased.     

Factors affecting the yield and properties of bacterial cellulose

Acetobacter xylinum E₂₅ has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel−), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young's modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303 Received 01 March 2002/ Accepted in revised form 18 July 2002     

Proteomic analysis of the bacteriocin thuricin 17 produced by Bacillus thuringiensis NEB17

Thuricin 17 is a recently discovered bacteriocin produced by Bacillus thuringiensis NEB17. The objective of this work was to conduct a proteomic analysis of this bacteriocin. The partial N- and C-terminal amino-acid sequences of thuricin 17 have now been determined using the Edman degradation and matrix-assisted laser desorption ionization-quadrapole time of flight mass spectrometry (MS)/MS. A hydrophobic cluster analysis indicates that thuricin 17 contains a hydrophobic region, potentially corresponding to a membrane associated domain. Based on time of production, this bacteriocin may be produced as a secondary metabolite. Interestingly, thuricin 17 shares the same N-terminal sequence, DWTXWSXL, with a previously reported bacteriocin, Bacthuricin F4, produced by B. thuringiensis ssp. kurstaki strain BUPM4. This is the first time two bacteriocins from different Bacillus species have been shown to share similar N-terminal sequences.     

Production of (R)-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana ZJB16007 in solid state fermentation using rice bran

Abstract

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key chiral intermediate for phenoxypropionic acid herbicide synthesis. In this study, to improve the production of R-HPPA with B. bassiana ZJB16007, the cultivation conditions in solid-state fermentation (SSF) were investigated. The effects of various substrates on R-HPPA production were evaluated and the process parameters were also optimized. The results showed that rice bran was the optimal substrate for R-HPPA production. The optimal medium components and cultivation conditions were: rice bran: silkworm chrysalis powder = 5.25: 2.25 (g: g), nutrient salts solution 12?mL which contained 50?g/L R-PPA, pH 5.0, and cultivated at 28?°C for 11 days. Under the optimized conditions, the transformation of R-HPPA was significantly improved and the yield of R-HPPA reached 77.78%, which was 15.14% higher than that of the control (67.55%). Therefore, SSF may serve as an alternative for R-HPPA production by B. bassiana ZJB16007.     

Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.     

Influence of pH on growth and bacteriocin production byLactococcus lactis subsp.lactis 14ONWC during batch fermentation

The influence of pH on growth, and lactic acid and bacteriocin production byLactococcus lactis subsp.lactis 140 NWC was studied during batch fermentation in a lactose-based complex medium. Growth and lactic acid production were modelled using a simple logistic equation while substrate consumption was found to be a function growth and lactic acid production rate. The optimal pH for growth and lactic acid production was between 6.0 and 6.5. Bacteriocin production showed primary metabolite kinetics. pH had a dramatic effect on the production of the bacteriocin, lactococcin 140. A maximum activity of 15.4 × 10⁶ AU (arbitrary units) 1^–1 was obtained after 7 h at pH 5.5. Maximum bacteriocin activity was achieved before the end of growth and was followed by a decrease in activity, which was due to adsorption to the cells of the producing organism, possibly followed by degradation by specific proteases. Both bacteriocin production and degradation rates were higher at pH 5.0 and 5.5, resulting in sharper activity peaks than at pH 6.0 or 6.5. On the basis of the experimental results a qualitative model for bacteriocin production is proposed.     

Statistical optimization for production of carboxymethylcellulase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> DL-3 by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/DL-3 from rice bran using response surface method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

海南近海水域产碱性蛋白酶菌株的分离筛选及发酵条件优化

【背景】微生物蛋白酶在工业生物技术上具有广阔的应用前景。在微生物蛋白酶中,碱性蛋白酶占全球酶总产量的50%以上,获取产碱性蛋白酶的新微生物资源意义重要。【目的】在海南近海贝类养殖基地海泥中筛选获得高产碱性蛋白酶的菌株,对其生长特性进行探究并优化菌株产酶条件,获得新的蛋白酶生产资源。【方法】以酪素培养基为筛选培养基,采用形态学结合系统发育分析鉴定菌株,通过响应面实验优化菌株的产酶条件。【结果】筛选获得一株高产碱性蛋白酶的菌株F3,鉴定为粘质沙雷氏菌（Serratia marcescens）。菌株在最优产酶条件下发酵酶活达到（339.36±4.30） U/mL。【结论】筛选获得的菌株粘质沙雷氏菌F3有较好的产碱性蛋白酶的能力。     

一株高产洛伐他汀红曲霉的筛选与液态发酵条件优化

【背景】洛伐他汀(lovastatin)是红曲霉的次生代谢产物,是重要的临床用降血脂药物。在液态发酵条件下,红曲霉的洛伐他汀产量较低,难以满足工业化生产的要求。【目的】筛选获得一株高产洛伐他汀的红曲霉株,并通过优化液态发酵条件提高洛伐他汀的产量。【方法】从红曲米中筛选获得一株高产洛伐他汀的红曲霉株,依据形态学特征、生理生化特性及18S rRNA基因序列分析对分离菌株进行鉴定;通过响应面法对其产洛伐他汀的液态发酵条件进行优化。【结果】获得一株产洛伐他汀的紫红曲霉(Monascus purpureus M4),该菌在甘油57.80g/L、酵母浸粉5.52 g/L、接种量为6.90%条件下,洛伐他汀产量(173.60 mg/L)较优化前提高了4.8倍。【结论】菌株M4产洛伐他汀最优液态发酵条件的建立,为洛伐他汀的大规模生产及该菌株的工业化应用提供了技术支撑。     

Enhanced production of lactococcin 972 in chemostat cultures

Lactococcus lactis subsp. lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products. This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation. In this work, we report on the conditions to optimize biomass and Lcn972 production. In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production. The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration. In chemostat cultures, steady states were achieved even at dilution rates higher than _max, due to the strong wall growth. Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing. Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.     

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making.     

Mathematical evaluation of plantaricin formation supports an auto-induced production mechanism

The production rate of a bacteriocin, produced by Lactobacillus plantarum TMW1.25 and previously named plantaricin1.25, was studied during pH-constant batch fermentations under various growth media conditions. The growth of L. plantarum and production of bacteriocin during the retardation phase were modelled, using 11 different empirical and mechanistic approaches. The optimal pH for bacteriocin production was 4.5. Among the different nitrogen sources tested, yeast extract was the most important, on the basis of the fact that the maximum growth rate decreased 16% without yeast extract, and only 7.2% or 8.1% without meat extract or peptone respectively. However, the change of nitrogen source did not have a significant effect on bacteriocin production. The progression of plantaricin1.25 production during the retardation phase and growth of L. plantarum TMW1.25 could be described by a structured model in which the bacteriocin concentration induces its own production. Among those models not implementing bacteriocin induction, only the one with an exponential increase of bacteriocin yield per unit biomass was suitable to describe bacteriocin production. Computer-aided evaluation of experimental data appears to be helpful in elucidating the relationship between the growth of lactic acid bacteria and bacteriocin production. Received: 22 May 1998 / Received last revision: 9 November 1998 / Accepted: 14 November 1998     

C55 bacteriocin produced by ETB‐plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB‐positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB‐positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB‐negative but not pETB‐positive strains, including TY4. Additionally, a TY4? strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46‐47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4? with orf46‐47 strain exhibited complete resistance to bacteriocin, whereas the TY4? with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co‐cultured with S. aureus strains was also investigated. When TY4 or TY4? was co‐cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co‐cultured with TY4 was significantly less than when 209P was co‐cultured with TY4?, whereas the proportion of TY4? with orf46‐48 co‐cultured with TY4 was greater than with TY4?. These results suggest that the C55 bacteriocin produced by pETB‐positive strains affects the proportion of each strain when pETB‐positive and ‐negative strains co‐exist.