Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis

Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l^–1 in a glutamic acid/citric acid medium. Poly(glutamic acid) reached 23 g l^–1 after 30 h.     

Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers

An empirical kinetic model is proposed for the batch production of poly(glutamic acid) from Bacillus subtilis IFO 3335. In addition, the proposed model was used to fit the kinetic data of poly(glutamic acid) production from other bacterial strains using different media, as well as kinetic data from different strains for the production of the exocellular biopolymers dextran, hyaluronic acid, xanthan, alginate, and the endocellular biopolymer polyhydroxybutyrate. The empirical model treats the biopolymer as a component of the biomass and fits the experimental biomass data using a sigmoidal relationship that includes the maximum specific growth rate, mu(max), and the substrate saturation parameter, K(S). An empirical parameter, the relative coefficient (r), quantifies, in relative terms, the degree of nongrowth-associated biopolymer formation.     

Poly(glutamic Acid) for Biomedical Applications

ABSTRACT:?

Paclitaxel is a widely used anti-cancer agent. Conjugates of paclitaxel with poly(glutamic acid) have shown great promise in preclinical trials, and clinical trials are now underway. Preclinical data suggest that more paclitaxel is preferentially delivered to tumor sites vs. nonconjugated paclitaxel. When poly(glutamic acid) is conjugated to other families of cancer drugs, similar improvements in effectiveness and reduced toxicity are observed. Optimization of poly(glutamic acid) for use in drug delivery applications is a key step in making this technology viable.     

High yield and cost‐effective production of poly(γ‐glutamic acid) with Bacillus subtilis

Poly(γ‐glutamic acid) (γ‐PGA) is a promising biopolymer with many potential industrial and pharmaceutical applications. To reduce the production costs, the effects of yeast extract and L ‐glutamate in the substrate for γ‐PGA production were investigated systematically at shake flask scale. The results showed that lower concentrations of yeast extract (40 g/L) and L ‐glutamate (30 g/L) were beneficial for the cost‐effective production of γ‐PGA in the formulated medium. By maintaining the glucose concentration in the range of 3–10 g/L via a fed‐batch strategy in a 10‐L fermentor, the production of γ‐PGA was greatly improved with the highest γ‐PGA concentration of 101.1 g/L, a productivity of 2.19 g/L·h and a yield of 0.57 g/g total substrate, which is about 1.4‐ to 3.2‐fold higher than those in the batch fermentation. Finally, this high‐density fermentation process was successfully scaled up in a 100‐L fermentor. The present work provides a powerful approach to produce this biopolymer as a bulk chemical in large scale.     

聚谷氨酸批式生物合成的主要影响因素研究

采用Bacillus subtilisNX-2菌株,在5 L发酵罐批式操作过程中生物合成γ-聚谷氨酸,实验考察了搅拌转速(300~1 000 r/min)及底物质量浓度对生物合成γ-聚谷氨酸产率的影响。结果表明:转速为400 r/min时,γ-聚谷氨酸产率最高,可高达21.78 g/L。在转速400 r/min的条件下,考察了葡萄糖和谷氨酸浓度对聚谷氨酸生物合成的影响,根据葡萄糖初始质量浓度为40 g/L的实验数据建立了动力学方程。并在葡萄糖初始质量浓度为30,40,50,60g/L的范围内,验证所建动力学方程的稳定性和实用性。结果表明,在以上葡萄糖初始质量浓度范围内,所建模型对B.subtilisNX-2批式生物合成γ-聚谷氨酸产率的预测值与实验值能较好的吻合。     

Kinetics of molecular weight reduction of poly(glutamic acid) by in situ depolymerization in cell-free broth of Bacillus subtilis

Poly(glutamic acid) (PGA) is a water soluble, biodegradable biopolymer that is produced by microbial fermentation. Recent research has shown that poly(glutamic acid) can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment. The molecular weight of microbial poly(glutamic acid) is generally larger than what is required for drug delivery. As such, molecular weight reduction is a necessary step in producing poly(glutamic acid) for this application. Poly(glutamic acid) produced by Bacillus subtilis IFO 3335 was subjected to in situ depolymerization in the cell-free fermentation broth. Molecular weight reduction was measured, and an empirical kinetic model was used to correlate the experimental data. The kinetic rate constant, k, was found to be 6.92 × 10⁻⁶ h⁻¹ at pH 7.0 and 37 °C, which were the optimum depolymerization conditions.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

产氨短杆菌与枯草杆菌发酵产肌苷比较试验

采用诱变得来的枯草杆菌GMI-741和产氨短杆菌GMA-2892在1.2L自控发酵罐上进行肌苷发酵试验,产氨短杆菌GMA-2802在种子培养基中培养15h后,菌浓度达1.0×1011个/ml,而同样条件下,枯草杆菌GMI-741菌浓度只有9.5×109个/ml.在1.2L自控发酵罐上发酵时,GMA-2802发酵周期54h,产肌苷达20.40g/L,发酵液主要原材料成本为441.1元/吨;GMI-741发酵周期60h,产肌苷达19.52g/L,发酵液主要原材料成本为559.1元/吨.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

枯草芽孢杆菌B47菌株高产抗菌物质的培养基及发酵条件优化

枯草芽孢杆菌Bacillus subtilis B47菌株为番茄内生细菌, 也是玉米小斑病拮抗菌, 能产生对玉米小斑病菌有强烈抑制作用的抗菌物质。以B47菌株发酵液的无菌滤液对玉米小斑病菌的抗菌活性为检测指标, 测定B47菌株产抗菌物质培养所需的最佳碳、氮源和无机盐, 并通过正交试验法对该菌株产抗菌物质的培养基配方和摇瓶发酵条件进行优化。研究结果表明, B47菌株产抗菌物质最佳碳、氮源和无机盐分别为蔗糖、酵母浸膏和MgSO4·7H2O, 最优培养基是YSB (Yeast extract-sucrose-beef extract)培养基, 其配方为: 蔗糖2%, 酵母浸膏2%, 牛肉浸膏1.5%, MgSO4?7H2O 0.06%, FeSO4·7H2O 0.000 9%, 最优发酵条件组合为: 30 °C, pH 7.0, 170 r/min摇床培养6 d, 接种量为1%, 装液量为40 mL/200 mL。     

枯草芽孢杆菌纤溶酶基因在转基因烟草组织中的分泌表达

克隆枯草芽孢杆菌纤溶酶(Bacillussubtilisfibrinolyticenzyme,BSFE)基因及其前导肽序列。通过农杆菌EHA105介导转化,获得转基因烟草植株。其BSFE的表达水平为叶片42.97±28.59U·g-1FW、茎15.14±10.57U·g-1FW和根25.55±14.71U·g-1FW。其内源BSFE信号肽可在转基因烟草中行使蛋白转运功能,使BSFE具有分泌表达特性。这一系统可用于建立利用植物组织分泌表达外源蛋白的系统模型。     

一株分离自绿化废弃物堆肥枯草芽孢杆菌的常压室温等离子诱变及其发酵条件

【背景】为了提高堆肥降解有机废弃物的效率,高效堆肥菌剂成为了研究热点,其中以真菌应用的研究为多,但真菌也有对氧气和底物敏感等缺点,细菌对堆肥的作用开始被研究。本实验室以羧甲基纤维素钠(CMC-Na)为底物,从绿化废弃物堆肥中筛选得到枯草芽孢杆菌(Bacillussubtilis,B.subtilis) BL03,它具有较好的纤维素分解能力,能提高绿化废弃物堆肥中纤维素降解和腐殖质合成的速度。【目的】进一步提高B.subtilisBL03的纤维素酶生产能力。【方法】利用常压室温等离子(Atmospheric and room temperature plasma,ARTP)诱变BL03菌,通过CMC-刚果红固体培养基观察水解透明圈,以及液体发酵后检测酶活力的方法进行3轮筛选;通过连续多代培养观察突变株的遗传稳定性;通过梯度温度、p H培养研究突变株发酵的最适生长温度、培养基初始pH;利用正交设计方法研究适合突变株发酵培养的工业级原料配方。【结果】筛选到2株正突变株,酶活力分别提高了69%和72%;连续10代培养稳定,验证了突变株的遗传稳定性;其中酶活力最高的突变株BLA3890最适培养温度为37°C、培养基初始pH为5.0-6.5,研究得到较经济的发酵培养基配方。【结论】ARTP诱变B. subtilis BL03后得到的突变株BLA1973和BLA3890在绿化废弃物堆肥或其他纤维素降解行业具有进一步研究和应用的价值。     

Enhanced poly(L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation

Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4?g/L (untreated molasses) to 36.9?g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20?g/L. This technique generated approximately 95.4?g/L PMA with a productivity of 0.57?g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

Degradation of proteins during the fermentation of African locust bean (Parkia biglobosa) by strains of Bacillus subtilis and Bacillus pumilus for production of Soumbala

AIMS: To examine isolates of Bacillus subtilis and B. pumilus predominant in Soumbala for their ability to degrade African locust bean proteins (ALBP). METHODS AND RESULTS: Agar diffusion test in casein and ALBP agar was used for screening of isolates. The profiles of water-soluble proteins and free amino acids (FAA) during the fermentation of ALBP by the Bacillus isolates were studied by SDS-PAGE and cation exchange chromatography. The profile of soluble proteins changed with the fermentation time and varied depending on the isolate. The quantity of total FAA and essential FAA such as lysine was increased sharply between 24 and 48 h of fermentation and differed among the isolates. Simultaneously, a pH increase was observed. Cysteine, methionine, leucine, isoleucine, tyrosine and phenylalaline appeared during fermentation. CONCLUSION: The Bacillus isolates studied degraded ALBP leading to a profile of soluble proteins and FAA specific for each isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of Soumbala.     

Commercial riboflavin production by recombinant Bacillus subtilis: down-stream processing and comparison of the composition of riboflavin produced by fermentation or chemical synthesis

A novel process for riboflavin production using a recombinant Bacillus subtilis strain has been developed. Here we describe a down-stream processing procedure to obtain riboflavin qualities having a minimal content of 96% (‘feed-grade’) and 98% (‘food/pharma-grade’) riboflavin, respectively. Compared to riboflavin produced by chemical synthesis, products with improved chemical purity were obtained. All compounds representing more than 0.1% of the final products were identified. Feed-grade riboflavin material ex fermentation contained small amounts of amino acids and amino sugars and the biosynthetic riboflavin precursor dimethyl-ribityl-lumazine. All other side products found were derived from riboflavin, resulted from the purification procedure and were also found in riboflavin obtained by chemical synthesis. The Bacillus-produced riboflavin does not contain DNA. The data presented here were used to obtain product approval for the commercial application in the USA, Japan and the UK. Received 22 July 1998/ Accepted in revised form 8 November 1998