Biosynthesis of Poly-(3-hydroxybutyrate) under the Control of an Anaerobically Induced Promoter by Recombinant Escherichia coli from Sucrose

Poly-(3-hydroxybutyrate) (PHB) is a polyester with biodegradable and biocompatible characteristics and has many potential applications. To reduce the raw material costs and microbial energy consumption during PHB production, cheaper carbon sources such as sucrose were evaluated for the synthesis of PHB under anaerobic conditions. In this study, metabolic network analysis was conducted to construct an optimized pathway for PHB production using sucrose as the sole carbon source and to guide the gene knockout to reduce the generation of mixed acid byproducts. The plasmid pMCS-sacC was constructed to utilize sucrose as a sole carbon source, and the cascaded promoter P_3nirB was used to enhance PHB synthesis under anaerobic conditions. The mixed acid fermentation pathway was knocked out in Escherichia coli S17-1 to reduce the synthesis of byproducts. As a result, PHB yield was improved to 80% in 6.21 g/L cell dry weight by the resulted recombinant Escherichia coli in a 5 L bed fermentation, using sucrose as the sole carbon source under anaerobic conditions. As a result, the production costs of PHB will be significantly reduced.     

甲烷氧化细菌生物催化合成聚-β-羟基丁酸酯的分子量调控

在甲烷氧化细菌Methylosinus trichosporium IMV3011细胞内生物催化合成聚-β-羟基丁酸酯(PHB)的过程中,对影响聚合物分子量的各种因素进行了研究.发现碳源、培养基组分NH4+,NO3-,HPO24-,Mg2+,某些导向PHB合成的关键中间产物以及PHB的提取方法均会对PHB的分子量产生影响.同时,通过对胞内PHB合成酶系中关键作用酶的活性变化进行研究,发现β-酮硫解酶催化着控制进入PHB循环入口的关键反应,而PHB分子量的变化则主要取决于PHB合成酶和PHB降解酶的协同作用.     

Understanding the Influence of Phosphatidylcholine on the Molecular Weight of Hyaluronic Acid Synthesized by Streptococcus zooepidemicus

The effect of phosphatidylcholine on the molecular weight properties of hyaluronic acid (HA) was studied in batch culture of Streptococcus zooepidemicus by adding phosphatidylcholine at the early stage of exponential phase. With the addition of 80 mg/L of phosphatidylcholine, maximum HA yield (2.47 g/L) and weight-average molecular weight (902.60 KDa) were achieved, increased by 17.4% and 67.1%, respectively, as compared to the control. Metabolic flux analysis was employed to study the mechanism of phosphatidylcholine on the molecular weight of HA. The normalized flux distribution maps based on fermentation data at phosphatidylcholine addition indicated that phosphatidylcholine resulted in higher flux flowing to the HA pathway and lower flux flowing to the glycolysis and biomass synthesis pathway, coupling with higher level of UDPNAG generation and extra regeneration of ATP. The GC-MS analysis of fatty acids in the plasma membrane showed that the addition of phosphatidylcholine could promote the mobility and permeability of the cell membrane, making the HA chain pass through the membrane more easily, thus decreasing the energy consumption. All these results led to higher molecular weight of hyaluronic acid.     

Enzymatic biodegradation of chemosynthetic atactic P(3HB) enhanced by an amorphous non‐biodegradable polymer: blend of atactic P(3HB) with PMMA

Communication: The phase structure and biodegradability were investigated for a blend of chemosynthetic atactic poly((R,S)‐3‐hydroxybutyrate), a‐P(3HB), and poly(methyl methacrylate), PMMA. The thermal analysis indicated that amorphous a‐P(3HB)/PMMA blends with 20 wt.‐% and 40 wt.‐% PMMA shows sophisticated phase behavior and is partially miscible. The depolymerase of natural poly((R)‐3‐hydroxybutyrate) purified from Alcaligenes faecalis T1 did not degrade chemosynthesized a‐P(3HB) at all in the pure state, but it degraded a‐P(3HB) in some a‐P(3HB)/PMMA blends. The results suggested that enzymatic degradation of a‐P(3HB) can be enhanced by an amorphous non‐biodegradable polymer.     

大肠杆菌HB101感受态的热化学研究

利用LKB-2277生物活性检测系统,测定了在生理盐水中,采用氯化钙法建立大肠杆菌感受态过程中的代谢热输出。生理盐水中大肠杆菌及其感受态进行内源代谢的热化学方程满足:P=K~c+A,dc/dP~0=K'c^0,氯化钙处理后,K与P~0均增大,说明内源代谢活性增强,这与大肠杆菌感受态建立过程中一系列生理生化活性的调整是一致的,表明大肠杆菌感受态的建立很可能有其内在的调节机制,而不完全是人工诱导的。     

Biodegradation of microbial polyesters P(3HB) and P(3HB-co-3HV) under the tropical climate environment

The biodegradation of poly(3-hydroxybutyrate), P(3HB), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV) produced by a locally isolated bacteria identified as Erwinia sp. USMI-20 were carried out by using soil burial test and immersion test method at various places under the tropical environment in West Sumatra, Indonesia. The isolation of P(3HA)-degrading microorganisms was done by the in vitro rapid plate test method and was further characterized by using biochemical reactions. Our results showed that P(3HB) biodegraded at a rate of 3.6% per week in activated sludge, 1.9% per week in soil, 1.5% per week in lake water and 0.8% per week in Indian Ocean sea water. The degradation rates for P(3HB-co-3HV) were 17.8% per week in activated sludge, 6.7% per week in soil, 3.2% per week in lake water and 2.7% per week in Indian Ocean sea water. The biodegradation of both polymers were highest after burial into activated sludge with a half-life (T_1/2) of 14 weeks and the time for 100% degradation (T_100%) of 28 weeks for P(3HB), and a T_1/2 of 3 weeks and T_100% at 6 weeks for P(3HB-co-3HV). In this study, 10 bacteria which were responsible for the biodegradation of P(3HB) and P(3HB-co-3HV) film were isolated and identified from the various places studied under the tropical environment. They were Bacillus sp. FAAC-2202, Enterobacter sp. FAAC-2207, Bacillus sp. FAAC-2209 and Proteus sp. FAAC-2203 obtained from activated sludge, Bacillus sp. FAAC-2201 and Alcaligenes sp. FAAC-2210 from soil, Alcaligenes sp. FAAC-2205, Micrococcus sp. FAAC-2206 and Pseudomonas sp. FAAC-2208 from lake water and Proteus sp. FAAC-2204 from Indian Ocean sea water.     

Effect of Molecular Weight of Poly-(1,2-Dimethyl-5-Vinylpyridinium Methyl Sulfate) on Flocculation of Aqueous Kaolin Suspension

Effect of molecular weight of poly-(1,2-dimethyl-5-vinylpyridinium methyl sulfate) on flocculation of aqueous kaolin suspension is studied.     

The Influence of Mineral Ions on the Microbial Production and Molecular Weight of Hyaluronic Acid

This study aimed to evaluate the influence of the culture medium supplementation with mineral ions, focusing on the growth of Streptococcus zooepidemicus as well as on the production and average molecular weight (MW) of hyaluronic acid (HA). The ions were investigated in terms of individual absence from the totally supplemented medium (C+) or individual presence in the non-supplemented medium (C−), where C+ and C− were used as controls. Differences between the effects were analyzed using the Tukey's test at p < 0.05. The adopted criteria considered required the ions, whose individual absence attained at 80% or less of the C+ and their individual presence was 20% or more than the C−. The supplementation was either inhibitory or acted in synergy with other ions, when the individual absence or presence was 20% higher than C+ or 20% lower than C−, respectively. Results showed that the effects of C+ or C− were equal for both the production of HA and its yield from glucose. However, C+ showed to be beneficial to cell growth while the individual absence of Na⁺ was beneficial to the production of HA. The highest MW of HA (7.4 × 10⁷ Da) was observed in the individual presence of Na⁺ in spite of the lowest HA concentration (0.65 g.L⁻¹). These results suggest that the quality of HA can be modulated through the mineral ion supplementation.     

重组大肠杆菌细胞不对称还原4-氯乙酰乙酸乙酯合成(R)-(+)-4-氯-3-羟基丁酸乙酯

 从赭色掷孢酵母(Sporobolomyces salmonicolor ZJU0105)中克隆出NADPH依赖型醛基还原酶基因,构建了重组大肠杆菌E.coli BL21(pET28-ALR0105), 该工程菌可以高效地表达醛基还原酶. 将重组细胞用于催化4-氯乙酰乙酸乙酯不对称还原,合成出具有光学活性的(R)-(+)-4-氯-3-羟基丁酸乙酯. 实验发现,在加入适量辅酶及辅酶再生酶的条件下,利用重组细胞催化还原反应可以获得比使用赭色掷孢酵母更高的转化率、产率和ee值,得到了几乎是光学纯的(R)-(+)-型产物,从而解决了酵母细胞催化此类反应ee值较低的问题. 考察了辅酶及共底物的添加、底物和产物的浓度、pH值、温度以及菌体密度等因素对还原反应的影响. 结果表明,不对称还原反应必须在辅酶NADPH和辅酶再生酶系及共底物葡萄糖的参与下进行; 底物和高浓度的产物对还原反应有一定的抑制作用; 当pH＞6.0时,反应的转化率及产率都显著降低; 高密度重组细胞可以减小底物的抑制作用.     

Poly(L-lactide) Nano- and Microfibers by Electrospinning: Influence of Poly(L-lactide) Molecular Weight

Summary: The electrospinning technique based on single and multi-jet systems was applied for poly(L -lactide) (PLA) nano- and microfibers as well as fibrous mats manufacture; the latter with dimensions suitable for the tensile tests. The PLA's employed were prepared by the controlled ring-opening polymerization of the L,L -lactide (LA) monomer. The resulting fibers thickness was correlated with molecular weights of PLA's and viscosities of spinning solutions. The scanning electron microscopic, thermal, and tensile characteristics of the polymeric materials and fibrous mats were also examined.     

ESI-MS测定牛血清白蛋白分子量过程中的pH影响

在电喷雾质谱测定牛血清白蛋白分子量的过程中,适当调整样品溶液的酸度,可提高分析测试的灵敏度.在选定最佳样品浓度的基础上,通过适当加入冰醋酸来调整测试样品的pH值,准确测定了标准蛋白质——牛血清白蛋白的分子量.     

Influence of Molecular Weight on Mild Steel Corrosion Inhibition Effect by Polyvinyl Alcohol in Hydrochloric Acid Solution

The corrosion inhibition of mild steel in hydrochloric acid solution in the presence of three different molecular weights of polyvinyl alcohol (PVA) designated as PVA-I, PVA-II, and PVA-III corresponding to 14,000, 72,000, and 125,000 g mol^?1, respectively, was investigated using electrochemical impedance spectroscopy, linear polarization resistance (LPR), and potentiodynamic polarization techniques at 25°C. It was found that PVA of different molecular weights inhibited the corrosion of mild steel in the acid environment. Inhibition efficiency (η%) increases with increase in concentration of the polymers. LPR measurements clearly show that inhibition efficiency increases with increasing molecular weight in the order PVA-III > PVA-II > PVA-I. Polarization curves indicate that PVA functions as a mixed inhibitor affecting both the anodic metal dissolution and cathodic hydrogen evolution partial reactions of the corrosion process. The experimental data obtained fitted well into Langmuir adsorption isotherm model. Physical adsorption mechanism is proposed from the thermodynamic (free energy of adsorption) parameters obtained.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

A Study of the Molecular Weight Distribution of Poly(Amino Acid)s Synthesized by Diphenyl Phosphoryl Azide

Abstract

Direct polycondensations of β-benzyl-l-aspartate (Asp.Bz) and β-benzyl-l-glutamate (Glu.Bz) were carried out in the presence of diphenyl phosphoryl azide (DPPA) as a condensation agent and triethyl amine (TEA). Poly(amino acid)s were obtained by this convenient approach whose structure was confirmed by IR and ¹H-NMR spectroscopy. The effects of the monomer concentration, the polymerization time and temperature, the ratios [DPPA]/[monomer] and [TEA]/[monomer], and the solvent used on the molecular weight distribution of the polymer were studied. When the monomer concentrations were higher than 0.2 g/mL, poly(Asp.Bz) with a bimodal molecular weight distribution was obtained (M_w of 37,000 and M_w/M_n of 1.68). The polycondensations carried out in THF or in bulk provided the highest molecular weight (M_w ? 40,000). Several other amino acids were also polymerized by DPPA.     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

Highly Regioregular Poly-3-alkylthiophenes: Influence of the Structure on Photoluminescence

Summary.   A class of regioregular poly-3-alkylthiophenes was synthesized via a new and simple synthetic procedure. These polymers exhibit a regioregularity as high as 95–100% and show different thermal behaviour depending on the length of the side chain. The high conjugation length reached is responsible for a red-orange emission with photoluminescence (PL) quantum efficiencies of about 45% in solution. The study of the PL of spin coated films at different temperatures allows to correlate the PL spectra with the structure of the polymers as detected by XRD. Received June 23, 2000. Accepted (revised) August 21, 2000     

高效凝胶渗透色谱法研究超氧化物歧化酶的分子量

本文采用凝胶渗透色谱仪，对修饰后的牛血ＳＯＤ进行测定。仪器：美国ＷａｔｅｒｓＡＬＣ／ＧＰＣ２４４型高效液相色谱仪，ＳｈｏｄｅｘＰ－８２葡聚精为高分子标样。采用紫外和示差折光双检测器进行测定。所得数据与经典的凝胶电泳法完全一致，为此类高分子材料的Ｍｎ、ＭＷ和Ｄ值的测定，建立了准确、快速的测定方法。     

Low frequency electric polarizability and zeta-potential of Escherichia coli HB101 (K-12) cells during inactivation with ethanol

The electric properties of bacteria determine their non-specific interactions with the environment, in particular their pathogenic activity. The electric polarizability of Escherichia coli HB101 (K-12 strain) was studied while inactivation with ethanol (20–40 vol.%). The current investigation might be regarded as a continuation of previous research on the polarizability of E. coli at lower ethanol concentration (≤ 20 vol.%) and higher frequencies (≥ 20 kHz). The bacteria polarizability at low frequencies (<10⁴ Hz) shows anomalies (unexpected increase in the polarizability at certain ethanol concentrations), while the parameter decreases with an increase in the ethanol concentration at higher frequencies. We investigated for the possible reasons causing the anomalies — in our case reduced to the medium dielectric permittivity, the average cell length and the surface electric charge density distribution, related to bacterial lipopolysaccharides. We suggest a hypothesis for the molecular mechanism of changing the surface charge of E. coli, carried by lipopolysaccharides, induced by the non-ionic ethanol.      

(1R,3R)-1-氨基-1,3-二羧基环丁烷衍生物桥联环糊精与一组低分子量多肽之间的包合行为

近年来,有关环糊精(CD)衍生物的合成、结构表征和应用方面的研究已引起越来越多化学家的关注[1～3].包含两个空腔的CD二聚体因其独特的理化性质而成为环糊精化学研究的一个热点[4,5].     

A preliminary study on the development and characterisation of enzymatically grafted P(3HB)-ethyl cellulose based novel composites

In the present study, a novel enzyme-based grafting of poly(3-hydroxybutyrate) [P(3HB)] onto the ethyl cellulose (EC) as a backbone polymer was developed under a mild and ecofriendly environment and laccase was used as a grafting tool. The resulting composites were characterised using various instrumental and imaging techniques. The high intensity of the 3,358 cm^?1 band in the FTIR spectra showed an increase of hydrogen–bonding interactions between P(3HB) and EC at that distinct wavelength region. The morphology was examined by scanning electron microscopy, which showed the well dispersed P(3HB) in the backbone polymer of EC. X-ray diffraction pattern for P(3HB) showed distinct peaks at 2-theta values of 28°, 32°, 34°, 39°, 46°, 57°, 64°, 78° and 84°. In comparison with those of neat P(3HB), the degree of crystallinity for P(3HB)-g-EC decreased. The tensile strength, elongations at break and Young’s modulus of P(3HB)-g-EC reached the highest levels in comparison to the film prepared with pure P(3HB) only, which was too brittle to measure any of the above said characteristics. Results obtained in the present study suggest P(3HB)-g-EC as a potential candidate for various biotechnological applications, such as tissue engineering and packaging.