Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol

Poly-3-hydroxybutyrate （PHB） can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol （0.1%） was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages： a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients （ammonium, nitrate, phosphorus, copper, iron （Ⅲ）, magnesium or ethylenediamine tetraacetate （EDTA）） in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle （TCA）. It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% （w/w） from the initial yield of 12% （w/w） after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5 × 10^6Da.

SYNTHESIS OF NOVEL BLOCK COPOLYMERS OF POLY(3-HYDROXYBUTYRIC ACID)WITH POLY(ETHYLENE GLYCOL)THROUGH ANIONIC POLYMERISATION

A novel kind of copolymer with ABA-type block structure was synthesized by anionic ring-opening polymerization of beta-butyrolactone (beta-BL) in the presence of a PEG-based dicarboxylates as macroinitiators which were prepared by the esterification of aliphatic cyclic anhydride and poly(ethylene glycol) (PEG) oligomers (M-n = 2000, 4000 and 6000) and conversion of potassium dicarboxylates. The resultant copolymers as well as the intermediates were characterized by IR, H-1-NMR and GPC.

吡唑化合物pyr3干预对压力负荷诱导的大鼠左心室肥厚的影响

目的：探讨瞬时受体电位通道C亚族3亚型（TRPC3）的选择性阻滞剂吡唑化合物Ethyl-1-（4-f2，3，3一trichloroacrylamide）phenyl）-5-（trifluoromethyl）-1H—pyrazole-4-carboxylate（pyr3）的体内干预对压力负荷大鼠左心室肥厚（LvH）程度的影响。方法：采用腹主动脉缩窄手术建立左心压力负荷模型。30只200—240g雄性SD大鼠随机分为五组（n=6）：假手术对照组、手术对照组、假手术后全程给药组、手术后后期给药组，手术后全程给药组。6周后，计算各组大鼠左心室质量指数、左心室心肌细胞横径，逆转录多聚酶连反应（RT—PCR）、免疫组织化学和蛋白免疫印迹检测TRPC3和钙调神经磷酸酶A13信使核糖核酸（mRNA）和蛋白的表达水平。结果：与假手术对照组相比，手术对照组左心室肥厚相关指标（左心室质量指数和左心室心肌细胞横径）及TRPC3、钙调神经磷酸酶AB蛋白和mRNA的表达显著升高；假手术后全程给药组左心室肥厚相关指标无显著变化，而TRPC3和钙调神经磷酸酶AB蛋白及mRNA的表达量显著升高，差异均有统计学意义（P均〈0．05）。与手术对照组比较，手术后全程给药组左心室肥厚相关指标、TRPC3及钙调神经磷酸酶A13蛋白和mRNA表达均明显降低（P均〈0．05）；手术后后期给药组左心室肥厚相关指标及钙调神经磷酸酶AB蛋白和mRNA的表达下降，差异均有统计学意义（P均〈0．05），而TRPC3的蛋白和mRNA表达未见显著降低。结论：pyr3体内干预能够一定程度上阻止压力负荷导致的大鼠左心室肥厚，降低压力负荷大鼠左心室TRPC3和钙调神经磷酸酶AB表达的上调。

LC-MS^n测定2-[[(2′-氰基联苯基)-4-基]甲基]氨基-3-硝基苯甲酸乙酯中的不纯物

The impurities in ethyl-2-[[2′-cyanobiphenyl-4-yl] methyl] amino]-3-nitrobenzoate,an intermediate for synthesis of candesartan cilexiti,were detected by LC-MSn. The structural assignment of these impurities was carried out by LC-MSn using electrospray ionization source and an ion trap mass analyzer. The formation of the impurities was discussed. Also the fragmentation pathways of these compounds were studied.

Effect of poly(3-hydroxyalkanoates) as natural polymers on mesenchymal stem cells

Mesenchymal stem cells(MSCs)are stromal multipotent stem cells that can differentiate into multiple cell types,including fibroblasts,osteoblasts,chondrocytes,adipocytes,and myoblasts,thus allowing them to contribute to the regeneration of various tissues,especially bone tissue.MSCs are now considered one of the most promising cell types in the field of tissue engineering.Traditional petri dish-based culture of MSCs generate heterogeneity,which leads to inconsistent efficacy of MSC applications.Biodegradable and biocompatible polymers,poly(3-hydroxyalkanoates)(PHAs),are actively used for the manufacture of scaffolds that serve as carriers for MSC growth.The growth and differentiation of MSCs grown on PHA scaffolds depend on the physicochemical properties of the polymers,the 3D and surface microstructure of the scaffolds,and the biological activity of PHAs,which was discovered in a series of investigations.The mechanisms of the biological activity of PHAs in relation to MSCs remain insufficiently studied.We suggest that this effect on MSCs could be associated with the natural properties of bacteria-derived PHAs,especially the most widespread representative poly(3-hydroxybutyrate)(PHB).This biopolymer is present in the bacteria of mammalian microbiota,whereas endogenous poly(3-hydroxybutyrate)is found in mammalian tissues.The possible association of PHA effects on MSCs with various biological functions of poly(3-hydroxybutyrate)in bacteria and eukaryotes,including in humans,is discussed in this paper.

Syntheses of amphiphilic biodegradable copolymers of poly(ethyl ethylene phosphate) and poly(3-hydroxybutyrate) for drug delivery

Biodegradable and amphiphilic triblock copolymers poly(ethyl ethylene phosphate)-poly(3-hydroxybutyrate)-poly(ethyl ethylene phosphate) (PEEP-b-PHB-b-PEEP) have been successfully synthesized through ring-opening polymerization. The structures are confirmed by gel permeation chromatography and NMR analyses. Crystallization investigated by X-ray diffraction reveals that the block copolymer with higher content of poly(ethyl ethylene phosphate) (PEEP) is more amorphous, showing decreased crystallizability. The obtained copolymers self-assemble into biodegradable nanoparticles with a coreshell micellar structure in aqueous solution, verified by the probe-based fluorescence measurements and transmission electronic microscopy (TEM) observation. The hydrophobic poly(3-hydroxybutyrate) (PHB) block serves as the core of the micelles and the micelles are stabilized by the hydrophilic PEEP block. The size and size distribution are related to the compositions of the copolymers. Paclitaxel (PTX) has been encapsulated into the micelles as a model drug and a sustained drug release from the micelles is observed. MTT assay also demonstrates that the block copolymers are biocompatible, rendering these copolymers attractive for drug delivery.

Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis

Colitis is a common disease of the colon that is very difficult to treat.Probiotic bacteria could be an effective treatment.The probiotic Escherichia coli Nissle 1917(EcN)was engineered to synthesize the ketone body(R)-3-hydroxybutyrate(3HB)for sustainable production in the gut lumen of mice suffering from colitis.Components of heterologous 3HB synthesis routes were constructed,expressed,optimized,and inserted into the EcN genome,combined with deletions in competitive branch pathways.The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions.The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days,elevating gut 3HB and short-chain-length fatty acid(SCFA)levels 8.7-and 3.1-fold compared to those of wild-type EcN,respectively.The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria,especially Akkermansia spp.,to over 31%from the initial 2%of all the microbiome.As a result,the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium(DSS)in mice.Compared to wild-type EcN or oral administration of 3HB,oral EcNL4 uptake demonstrated better effects on mouse weights,colon lengths,occult blood levels,gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations.Thus,a promising live bacterium was developed to improve colonic microenvironments and further treat colitis.This proof-of-concept design can be employed to treat other diseases of the colon.

Preparation and characterization of biomimetic adsorbent from poly-3-hydroxybutyrate

Biomimetic adsorbent named as PHBBMA was prepared from lipophilic poly-3-hydroxybutyrate （PHB） by a modified double emulsion solvent evaporation method. PHBBMA, characterized by using scanning electron microscope and nitrogen adsorption/desorption measurements, is porous spherical particles. The characterization with the thermal gravimetric analysis and differential scanning calorimetry, 1 H nuclear magnetic resonance and Fourier transform infrared spectroscopy showed that PHBBMA preparation was a physical process without chemical reaction. The adsorption of PHBBMA for o-nitrochlorobenzene （o-NCB） was fitted better by Langmuir model than by Freundlich model, while the pseudo second-order model fitting was better than the pseudo first-order model fitting. The maximal adsorption capacity of PHBBMA for o-NCB was 57.83 mg/g at 30°C, although its specific surface area （S BET ） was only 8.45 m 2 /g. PHBBMA is a safe and environmental friendly adsorbent with high adsorption capacity because its component is innocuous and biodegradable PHB produced reusing wastes and contaminants, no byproduct can produced, and its ester and hydrocarbyl groups have strong affinity with organochlorine compounds. The further work will focus on the modification and improvement of PHBBMA in order to increase its S BET and adsorption capacity.

Structures and growth mechanisms of poly-(3-hydroxybutyrate) (PHB) crystallized from solution and thin melt film

The spherulitic structures and morphologies of poly-(3-hydroxybutyrate) (PHB) crystallized from a so- lution and a thin melt film were investigated in this study. The formation mechanisms of banded spherulites under different crystallization conditions are proposed. It was found that the formation of banded spherulites was caused by the rhythmic crystal growth of the spherulites and lamellar twisting growth for the polymer crystallization from a thin melt film and a solution, respectively.

功能化氧化石墨/聚羟基丁酸酯纳米复合材料的制备与性能研究

采用溶液插层技术制备功能化氧化石墨/聚羟基丁酸酯纳米复合材料(MGO/PHB),利用红外光谱分析(FTIR)和原子力显微镜(AFM)对MGO的表面结构进行表征,并用扫描电子显微镜(SEM)、X射线衍射(XRD)、示差扫描量热仪(DSC)及拉伸试验分别对MGO/PHB的微观形貌、结晶性能及力学性能进行分析测试。结果表明:MGO的加入加速了PHB基体的结晶并导致结晶度增大;氧化石墨经表面功能化处理后,改善了MGO片在PHB基体中的均匀分散以及其与PHB基体的界面相互作用;随着MGO质量分数的增加,复合材料的断裂强度先降低后增大,而与纯PHB相比,复合材料都表现出更高的断裂模量值,但复合材料的力学性能变化与MGO添加量的阀值有一定关系。

Comparative evaluation of physico-chemical characteristics of biopolyesters P（3HB） and P（3HB-co-3HV） produced by endophytic Bacillus cereus RCL 02

BACKGROUND： Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates （PHAs）. Bacillus cereus RCL 02 （MCC 3436）, a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly（3-hydroxybutyrate） [P（3HB）] of its cell dry biomass when grown in mineral salts （MS） medium. METHODS： The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P（3HB） isolated from RCL 02 in terms of thermal, mechanical and chemical properties. RESULTS： Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3- hydroxybutyrate （3HB） and 3-hydroxyvalerate （3HV） [P（3HB-co-3HV）] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction （XRD） analysis, Fourier transform infrared （FTIR） and nuclear magnetic resonance （NMR） spectroscopic studies. Thermogravimetric analysis （TGA） revealed that P（3HB） and P（3HB-co-3HV） films degraded at 278.66℃ and 273.49℃, respectively. The P（3HB-co-3HV） showed lower melting temperature （165.03℃） compared to P （3HB） （170.74℃） according to differential scanning calorimetry （DSC）. Incorporation of 3HV monomers decreased the tensile strength （21.52 MPa）, tensile modulus （0.93 GPa）, storage modulus （E＇） （0.99 GPa） and increased % elongation at break （12.2%） of the copolyester. However, P（3HB） showed better barrier properties with lower water vapor transmission rate （WVTR） of 0.55 g-mil/100 in2/24 h. CONCLUSION： These findings emphasized exploration of endophytic bacterial strain （RCL 02） to produce biodegradable polyesters which might have significant potential for industrial application.

玄参药材中多类型成分的定量分析方法建立及其在脉络宁口服液中的应用

目的建立超高效液相色谱-串联三重四极杆质谱(UPLC-QqQ-MS/MS)方法同时测定玄参Scrophulariae Radix中多类型成分的含量,并将此方法应用于脉络宁口服液(Mailuoning Oral Liquid,MOL),为玄参药材和MOL的质量评价提供依据。方法采用Waters HSS T3色谱柱(100 mm×2.1 mm,1.8μm),以0.1%甲酸水溶液和乙腈为流动相进行梯度洗脱。质谱采用电喷雾离子源(electrospray ionization,ESI),在多反应监测模式(multiple reaction monitoring,MRM)下对环烯醚萜苷、苯丙素苷、有机酸和核苷4类成分进行定量分析,结合化学计量学分析评价不同批次样品的质量。结果化学计量学结果表明,不同地区的玄参药材得到了有效区分,其质量差异可能与其生长环境相关。此外,4个年份(2019、2020、2021和2022年)的MOL各自聚成1类,可能是当年使用的药材质量相对稳定使得同一年份的MOL质量更相近;通过正交偏最小二乘-判别分析(orthogonal partial least squares-discriminate analysis,OPLS-DA)进一步筛选出不同年份MOL中异毛蕊花糖苷、对甲氧基肉桂酸、腺苷、1-(3-hydroxy-4-methoxyphenyl)ethyl-3-α-L-rhamnopyranosyl-6-caffeoyl-β-D-glucopyranoside、对羟基苯甲酸、阿魏酸和原儿茶酸7个差异性化学成分。结论该方法特异性强、准确度高且稳定性好,可为MOL中臣药玄参的药材选择及复方整体质量评价提供科学依据。

Expressing PHB synthetic genes through chloroplast genetic engineering

Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.

Cloning and sequencing of phbA gene of poly-β-hydroxybuty rate synthesis and its expression analysis

The gene of the first key enzyme of poly-β-hydroxybutyrate synthesis, 3-ketothiolase, has been amplified and cloned from chromosomal DNA of Alcaligenes eutrophus H16 by PCR. DNA sequencing results show that phbA cloned in pBluescriptSK^+ has an identical sequence with that reported previously except for one base pair. The plant constitutive expression vector has been constructed and tobacco has been transformed in order to examine the phbA gene function and the efficiency of ctp gene product. SDS-polyacrylamide gel electrophoresis result shows that the ctp gene product could direct foreign protein into plastid efficiently and phbA gene could be translated into corresponding protein with correct size. The enzyme activity analysis of 3-ketothiolase shows that the enzyme could catalyze acetyl-CoA to form acetoacetylCoA.

Chiral derivatization coupled with liquid chromatography/mass spectrometry for determining ketone metabolites of hydroxybutyrate enantiomers

Ketone bodies are small lipid-derived molecules and the metabolism of ketone bodies interfaces with various physiological processes. 3-Hydroxybutyric acid （3HB） is the most stable ketone body and can be employed to supply energy source. 2-Hydroxybutyric acid （2HB）, an isomer of 3HB, was demonstrated to be an early biomarker for both insulin resistance and impaired glucose regulation. Both 2HB and 3HB are chiral carboxylic acids and exist in two steric configurations （D/L-2HB and D/L-3HB）. It is difficult for these enantiomers to be differentiated by routine analytical methods In the current study, we developed a strategy by chiral derivatization coupled with liquid chromatography/mass spectrometry （LC-ESI-MS） analysis for simultaneous determination of D-2HB, L-2HB, D-3HB and L-3HB enantiomers. （5）-（＋）-l-（2- Pyrrolidinylmethyl）-pyrrolidine （PMP） was used for efficient labeling of HBs. Our results showed that the retention behavior of D/L-2HB and D/L-3HB enantiomers was greatly improved after labeling by PMP and the four derivatives can be distinctly separated on C18 reversed-phase column. Moreover, PMP chiral derivatization greatly enhanced the detection sensitivities of HBs up to 55.3 folds because of the introduction of easily ionizable tertiary amino group. Using this method, we simultaneously quantified D-2HB, L-2HB, D-3HB, and L-3HB enantiomers in human renal cell carcinoma （RCC） tissues and the tumor adjacent normal tissues. The result demonstrated that both D-3HB and L-3HB can be detected in human renal tissues, however, only L-2HB was detected in human renal tissues. In addition, the quantification results showed that the contents of D-3HB were approximate 10 folds higher than L-3HB. Taken together, the developed method offered an efficient approach for the sensitive analysis of D/L-2HB and D/L-3HB enantiomers, which may facilitate the in-depth study of the functions of HBs.