Rheology,oxygen transfer,and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis

Poly(glutamic acid) (PGA) is a water-soluble, biodegradable biopolymer that is produced by microbial fermentation. Recent research has shown that PGA can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment. A fundamental understanding of the key fermentation parameters is necessary to optimize the production and molecular weight characteristics of poly(glutamic acid) by Bacillus subtilis for paclitaxel and other applications of pharmaceuticals for controlled release. Because of its high molecular weight, PGA fermentation broths exhibit non-Newtonian rheology. In this article we present experimental results on the batch fermentation kinetics of PGA production, mass transfer of oxygen, specific oxygen uptake rate, broth rheology, and molecular weight characterization of the PGA biopolymer.     

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.     

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。     

稀土元素对谷氨酸发酵的影响

通过对谷氨酸发酵培养基中添加稀土元素的研究,确定了几种能对发酵产酸有提高作用的稀土元素及其浓度,其中La^3 、Ce^4 、Nd^3 离子浓度分别为100mg/L、10mg/L、1mg／L时能提高产酸水平,而Sm^3 、Y^3 离子对发酵基本上没有影响。     

Fuzzy supervisory control of glutamic acid production

In glutamic acid fermentation, the molasses feeding policy and time of penicillin addition significantly affected glutamic acid production, and a fuzzy supervisory control system was developed for their quasi-optimal regulation.From the trend of the experimental data, production rules and membership functions of fuzzy inference were devised to determine the quasi-optimum molasses feeding policy and penicillin addition time. A computer with multitasking operating system was used for the construction of the control system with fuzzy inferencing, which decided the control policy every minute, and the feed rate was controlled automatically. The pattern of residual sugar concentration was almost the same as that of maximum glutamic acid production under manual operation. Using the computer control system, stable production was maintained at the highest level of 71 to 75 g/L. (c) 1994 John Wiley & Sons, Inc.     

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.     

基于自联想神经网络的谷氨酸发酵故障诊断

研究了用自联想神经网络对谷氨酸发酵进行故障诊断。自联想神经网络采用一种带有瓶颈层的特殊结构,且具有单位总增益。在经过大量样本的训练之后,各变量之间能够建立起内在联系。输入信息通过瓶颈层前的压缩及瓶颈层后的解压缩过程,信息中的精华将被提取。应用自联想神经网络对发酵过程变量进行预处理,可以准确及时的进行谷氨酸发酵故障诊断。     

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.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

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.     

Multi‐armed poly(L‐glutamic acid)‐graft‐oligoethylenimine copolymers as efficient nonviral gene delivery vectors

Background

The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low‐toxic, highly efficient gene delivery.

Methods

A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi‐armed poly(L ‐glutamic acid) backbone. The molecular structures of multi‐armed poly(L ‐glutamic acid)‐graft‐OEI (MP‐g‐OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP‐g‐OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP‐g‐OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney‐293 cells for their cytotoxicity and transfection efficiency.

Results

The particle sizes of MP‐g‐OEI/DNA complexes were in a range of 109.6–182.6 nm and the zeta potentials were in a range of 29.2–44.5 mV above the N/P ratio of 5. All the MP‐g‐OEI copolymers exhibited lower cytotoxicity and higher gene transfection efficiency than PEI25k in the absence and presence of serum with different cell lines. Importantly, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay revealed that the cytotoxicity of MP‐g‐OEI copolymers varied with their molecular weight and charge density, and two of MP‐g‐OEI copolymers (OEI600‐MP and OEI1800‐MP) could achieve optimal transfection efficiency at a similar low N/P ratio as that for PEI25k.

Conclusions

MP‐g‐OEI copolymers demonstrated considerable potential as nonviral vectors for gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

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.     

Poly(gamma-L-diaminobutanoic acid), a novel poly(amino acid), coproduced with poly(epsilon-L-lysine) by two strains of Streptomyces celluloflavus

Two poly(ɛ- l -lysine) (ɛ-PL) producer strains of Streptomyces celluloflavus secreted a novel polymeric substance into their culture broths along with ɛ-PL. Three types of HPLC analysis plus one- and two-dimensional ¹H and ¹³C nuclear magnetic resonance experiments revealed that the secreted substance was poly(γ- l -diaminobutanoic acid) (γ-PAB), an l -α,γ-diaminobutanoic acid ( l -DAB) homopolymer linking between γ-amino and α-carboxylic acid functional groups. The γ-PABs from the two strains had an identical chemical structure, and the same number-average molecular weight of 2100–2200. No copolymers composed of the two amino acids l -DAB and l- lysine were found in either of the broths from the producers. Both strains coproduced high levels of the two poly(amino acid)s in the presence of SO₄²⁻ at pH 4.0 and 4.5 L min⁻¹ aeration in a 5-L jar fermentor. γ-PAB exhibited strong inhibitory activities against various yeasts and weaker actions against bacteria than ɛ-PL. γ-PAB may have various biological functions similar to ɛ-PL, and the use of γ-PAB along with ɛ-PL would be advantageous for technical applications in various fields.     

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery

N_ω-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N_ω-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N_ω-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8–40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.Revisions requested 8 April 2005; Revisions received 6 May 2005     

琥珀酸放线杆菌发酵培养基的优化

对琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC1593发酵产生琥珀酸培养基的主要成分,及其含量进行优化。通过单因素试验,得出发酵培养基中葡萄糖、酵母膏和玉米浆的含量对产生琥珀酸有显著影响;采用响应面法(RSM),得出多元二次回归方程拟合的三种因素与琥珀酸含量间的函数关系,并根据优化结果与实验,CGMCC1593产琥珀酸达到41.69g/L。     

Influences of cultural medium component on the production of poly(γ-glutamic acid) byBacillus sp. RKY3

In this study, the cultural medium used for the efficient production of γ-PGA with a newly isolatedBacillus sp. RKY3 was optimized. It was necessary to supplement the culture medium withl-glutamic acid and an additional carbon source in order to induce the effective production of γ-PGA. The amount of γ-PGA increased with the addition ofl-glutamic acid to the medium. The addition of 90 g/Ll-glutamic acid to the medium resulted in the maximal yield of γ-PGA (83.2 g/L). The optimum nitrogen source was determined to be peptone, but corn steep liquor, a cheap nutrient, was also found to be effective for γ-PGA production. Both the γ-PGA production and cell growth increased rapidly with the addition of small amounts of K₂HPO₄ and MgSO₄·7H₂O.Bacillus sp. RKY3 appears to require Mg²⁺, rather than Mn²⁺, for γ-PGA production, which is distinct from the production protocols associated with other, previously reported bacteria.Bacillus sp. RKY3 may also have contributed some minor γ-PGA depolymerase activity, resulting in the reduction of the molecular weight of the produced γ-PGA at the end of fermentation.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.