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.

Miscibility, Crystallization, and Rheological Behavior of Solution Casting Poly（3-hydroxybutyrate）/poly（ethylene succinate） Blends Probed by Differential Scanning Calorimetry, Rheology, and Optical Microscope Techniques

The miscibility and crystallization of solution casting biodegradable poly（3-hydroxybuty- rate）/poly（ethylene succinate） （PHB/PES） blends was investigated by differential scanning calorimetry, rheology, and optical microscopy. The blends showed two glass transition temperatures and a depression of melting temperature of PHB with compositions in phase diagram, which indicated that the blend was partially miscible. The morphology observation supported this result. It was found that the PHB and PES can crystallize simultaneously or upon stepwise depending on the crystallization temperatures and compositions. The spherulite growth rate of PHB increased with increasing of PES content. The influence of compositions on the spherulitic growth rate for the partially miscible polymer blends was discussed.

γ-hydroxybutyrate protects the liver from warm ischemia-reperfusion injury in rat

BACKGROUND: Ischemia-reperfusion (I/R) syndrome remains an important clinical consideration in hepatic sur- gery, hemorrhagic shock, and liver transplantation, -y-hy- droxybutyrate (GHB) has been reported to exert protective effects against ischemia-reperfusion injury to various or- gans. To investigate whether GHB protects the liver from warm ischemia-reperfusion injury, we performed this study in rats. METHODS: Thirty male Wistar rats were randomly divided into a sham-operation group, a control group, and three I/R groups pretreated with GHB, GHB plus naloxone or naloxone. After 30 minutes of partial ischemia, followed by 60 minutes of reperfusion in the liver, histomorphological and enzymological changes, lipid peroxidation, apoptosis, and the plasma level of endothelin-1 were observed. RESULTS: I/R increased the serum levels of alanine ami- notransferase, aspartate aminotransferase and lactate dehy- drogenase and the plasma level of endothelin-1 significantly (P<0.01), in addition to increase of apoptotic index (AI) from 0.28%±0.25% to 17.68%±1.91%. The levels of he- patic malondialdehyde were markedly increased, whereas the activities of superoxide dismutase were markedly de- creased. GHB pretreatment prevented the liver from warm ischemia-reperfusion injury significantly, but naloxone par- tially blocked this effect. CONCLUSION: GHB may significantly protect the liver from hepatic warm ischemia-reperfusion injury via several different mechanisms.

D-β-hydroxybutyrate inhibits microglial activation in a cell activation model in vitro

Microglial activation plays an important role in a panel of neurological disorders such as multiple sclerosis(MS) and Parkinson's disease(PD),and is a key target for developing therapeutic strategies for these diseases.Ketogenic diet (KD),which is able to inhibit microglial activation in substantia nigra pars compacta of mice,has been shown effective in a mouse model of PD,possibly through increasing D-β-hydroxybutyrate(D-β-HB),a major component of ketone bodies.To verify this,we developed an in vitro model of microglia activation with a microglia line,BV-2,and investigated how D-β-HB have an effect on the LPS-stimulated BV-2 cells.We found D-β-HB is able to recover the cell viability,and inhibit the production of inflammatory mediators and cytokines such as ROS,nitrite,IL-1β,TNF-α,and IL-6,which otherwise were increased in LPS-stimulated BV-2 cells.We conclude that the LPS induced BV-2 cells activation is a valid in vitro model of microglia activation.D-β-HB is able to suppress the activation of BV-2 cells, which might account for one of the possible reasons of KD therapy on the PD model.

Weavability of β-Hydroxybutyrate and β-Hydroxyvalerate Copolymers( PHBV ) /PLA Used in Artificial Blood Vessels

The β-hydroxybutyrate and β-hydroxyvalerate copolymers( PHBV) /polylactic acid( PLA) is a new biocompatible material,which is developed through bacterial fermentation in vivo systems.The PHBV / PLA material could be used to make continuous filaments.However,features of artificial blood vessels,especially small diameter vascular grafts made of PHVB / PLA materials are not known.This research are to evaluate and improve weavability of the PHBV / PLA material, and to explore feasibility of using it in artificial blood vessels.Preliminary results showed that weavability of PHBV / PLV was not good,but its weavability could be improved by using methods of weak chemical,such as sizing.In this research,scanning electron microscope( SEM) was adopted to evaluate weavability of PHBV / PLV after sizing and observe surfaces of yarns and fabrics.Also,in order to set proper parameters in heat settings,differential scanning calorimetry( DSC) was used to identify glass transition temperature.

Analysis of γ-Hydroxybutyrate in Human Urine by LC-MSIMS Method and Its Forensic Application

GHB （γ-hydroxybutyrate） is becoming popular recreational drugs. As a result of its strong sedative and amnesiac effects, GHB has been implicated in a number of DFSA cases. The natural presence of GHB in the human body and its rapid elimination after ingestion make it difficult to detect and to evaluate its roles in suspected GHB-facilitated assaults. The paper describes an analytical method for the determination of GHB in urine using LC-MS/MS. Samples were acidified by ammonium chloride solution and extracted with ethyl acetate, and then the extracts were analysed by LC-MS/MS. The limit of detection was 0.05 p.g/mL （S/N = 3）. The intra- and inter-day precision was within 10.0% at three concentrations. The methods were found to be sensitive, accurate, rapid and suitable for the forensic toxicology to test GHB in real cases.

Disposable Electrochemical Biosensor for Amperometric Measurement of β-hydroxybutyrate and Total Cholesterol

A new type of disposable thin-film amperometric biosensor has been developed for measurement ofβ-hydroxybutyrate and total cholesterol in serum.The biosensor consists of two plain gold electrodes mounted on a Polyethylene terephthalate (PET) substrate.The reagent solution contains bienzymes (cholesterol oxidase and cholesterol esterase for total cholesterol,β-hydroxybutyrate dehydrogenase and diaphorase forβ-hydroxybutyrate respectively) with mediator (ferricyanide) were absorption at the surface of electrodes coated by electrodepositing platinum black.The presence of the mediator lowers the applied potential and eliminates the interference from other oxidizable species enhancing the sensitivity and selectivity of the biosensor without modifying the dynamic parameters of the response.The enzymes stably retains in the matrices of platinum black film improving the performance of the mediated sensor.The linearity is observed in the concentration range from 1.0×10^(-4) to 1.0×10^(-2) mM and 1.0×10^(-4) mM to 4.99×10^(-3) mM with sensitivity of 1.958μA/mM and 2.447μA/mM respectively.The optimized biosensor exhibits excellent reproducibility and stability retaining more than 90% of its original activity over a period of one month.The simple operation of the biosensor mass-produced at low cost is expected to find clinical application and homecare.

Study on Kinetics of Dissolving Biomass Using Sodium Hypochlorite in the Recovery of Poly-β-hydroxybutyrate

The effects of sodium hypochlorite concentration in the recovery of poly-β-hydroxybutyrate (PHB) are investigated. A simple reaction equation and a mathematical model describe the reaction kinetics. Results showed that the stoichiometric coefficient increases with increasing CAO/CBO, where CAO is the concentration of the active chlorine at t = 0 and CBO is the initial concentration of the non-PHB materials.

Production of Poly(3-Hydroxybutyrate)by Fed-Batch Culture of Alcaligenes Eutrophus at Phosphorus Limitation in 50 L fermenter

Poly(3-hydroxybutyrate) (PHB) is an intracellular carbon and energy storage material accumulated by many kinds of microorganism under unfavorable growth conditions. For the production of PHB, Alcaligenes eutrophus has been widely used because it is easy to grow, and its physiological and biochemical changes during the PHB synthesis is understood in details. A very high concentration and productivity of PHB could be obtained by fed-batch culture of Alcaligenes eutrophus with phosphate limitation in 50 L fermenter.

Effect of Poly-β-hydroxybutyrate on Reproductive Performance and Larval Development of Fenneropenaeus chinensis

In the present study, Fenneropenaeus chinensis individuals were assigned into six groups, and fed with a diet supplemented with 0 （Control）, 0.5% （Group Eos）, 1.0% （Group E1.0）, 2.0% （Group E2.0）, 3.0% （Group E3.0） or 5.0% （Group E5.0） of poly-13-hydroxybutyrate （PHB） during their growth and spawning periods, to explore the effect of PHB on the reproductive performance of parents and the development of larvae. The experi- ment was lasted for 192 d to measure the mortality rate, relative percentage of survival（RPS）, weight gain rate（WGR）, specific growth rate （SGR） and cycle of gonadal development of parental shrimps, number of eggs, number of nauplii, stages of larval development and metamorphosis. The results indicated that PHB had an obvious effect on the fecundity of parental shrimps and growth performance of larva. Compared with control group, the mortality rate of broodstock in experimental groups decreased initially and increased subsequently with the increase of PHB concentration, while RPS showed an opposite pattern. The mortality rate of group E2.0 was the lowest among all groups, showing significant difference from that of other groups（P 〈 0.05） except group E1.0 （P 〉 0.05）. WGR of all experimental groups was higher than that of the control group, among which, group E2.0 and group E5.0 had significantly higher WGR than the control （P〈0.05）. However, there was no significant difference in the cycle of gonadal development between the experimental groups and the control group （P 〉 0.05）. The numbers of eggs and nauplii in the experimental groups were higher than those of the control group. And the numbers of eggs and nauplii in group E2.0 were significantly higher than those of the control （P 〈 0.05）, bud showed no significant difference from those of other groups （P 〉 0.05）. However, there was no significant difference in the duration of larval development and metamorphosis among all the groups. In summary, PHB is capable of improving the reproductive performance of parents and the survival and development of larva in F. chinensis, and 2.0% is the optimal concentration of PHB supplemented to the diet.

Complete Valorization of Olive Mill Wastewater through an Integrated Process for Poly-3-hydroxybutyrate Production

An economical and environmental sustainability of bioplastic production is dependent on the use of low cost and waste C-sources as raw materials. OMW （Olive Mill Wastewater） with its high organic load represents a dangerous polluting waste. Herein the authors present an integrated process for the simultaneous recovery of polyphenols, high value natural antioxidants, production of PHAs （polyhydroxyalkanotes）, thermoplastic bio-polymers, in particular of PHB （poly-3-hydroxybutyrate） starting from OMW. The combination of membrane filtration and bacterial digestion of OMW resulted in very high yields of polyphenols （3 2.5 g/L） and PHB （31.4 mg/L.h） if compared with the state of the art. These results make the technical approach described here effective for reducing the polluting effect of OMW and maximizing the valuable product yield. Moreover the process is readily suitable for an industrial scale PHB production from OMW.

Use of a Simple Fabrication Process to Produce a Biosensor： The 3-Hydroxybutyrate Dehydrogenase Case

This study was aimed to construct a biodegradable but reliable 3-β-hydroxybutymte biosensor. In this context a versatile paper based biosensor, quickly, easily and cheaply fabricated is reported. The procedure of fabrication is based on the assumption that the introduction of the enzyme in the carbon ink will allow enzyme stabilization and facilitate the study of the catalysis of enzymes and the detection of substrates. To prove this concept we use the enzyme 3-hydroxybutyrate dehydrogenase, in aqueous solution. This enzyme was chosen because it catalyzes the 3-β-hydroxybutyrate, which results from ketoacidosis. The quantification this substance in the diabetics＇ blood is very important as it can increase the reliability of the diagnosis of glycaemia. To prove the multi-use of this biosensor we not only study the redox process in steady state and during the catalytic process, but also detected and quantify the 3-β-hydroxybutyrate. Our results showed that it was possible to study the redox process that occurred during the catalysis and to confirm the amino acid residues that participate in it. It was also observed that glucose and ascorbic acid can interfere in the detection and quantification of the 3-β-hydroxybutyrate, what should be in mind when the quantification of the 3-β-hydroxybutyrate is made in blood samples.

Production of Poly-b-hydroxybutyrate by Activated Sludge in Sequencing Batch Reactor under Aerobic Conditions

In order to improve poly-β-hydroxybutyrate（PHB） production in activated sludge, the anaerobic/aerobic alternative operating sequencing batch reactor（SBR） process was applied in this paper to accumulate PHB. Effects of nutritional conditions and carbon concentration on PHB accumulation were studied. Results indicated that PHB accumulation reached the highest level and accounted for 11.2 % under anaerobic condition for phosphate limitation and 20.84 % under aerobic condition for nitrogen and phosphate limitation of mixed liquor suspended solid（MLSS）, respectively. In addition, 4 g/L was proved to be the optimum carbon concentration in both anaerobic and aerobic experiments, and the PHB accumulation reached 17.1 %（anaerobic, phosphorus limitation） and 60.4 %（aerobic, nitrogen and phosphorus limitation） of MLSS, respectively. PHB could be successfully extracted with sodium hypochlorite and chloroform method from the activated sludge. In addition, the infrared spectrum showed that the PHB sample extracted was of high purity.

Effect of Poly-β-Hydroxybutyrate on the Activity of Nonspecific Immunity Related Enzymes in Fenne rope naeus chinensis

[Objective] This study was carried out to explore the effect of poly-β-hydroxybutyrate（PHB） on the performance of the nonspecific immune system in Fenneropenaeus chinensis. [Method] F. chinensis individuals were assigned into six groups, and each group was fed with a diet containing 0（Control）, 0.5%（Group E0.5）, 1.0%（Group E1.0）, 2.5%（Group E2.5）, 5.0%（Group E5.0） or 10.0% PHB（Group E10.0）. The mortality rate and relative percent of survival（RPS） of each group were calculated after 6 weeks. Meanwhile, the total antioxidant capacity（T-AOC）, the activity of acid phosphatase（ACP）, peroxidase（POD） and catalase（CAT）, and the content of malondialdehyde（MDA） in hepatopancreas and serum were measured,and their correlation with PHB concentration was analyzed. [Result] The RPS in PHB treated shrimps increased initially and decreased subsequently with increasing PHB concentration. RPS of Group E1.0 was the highest, showing significant difference from that of other groups（ P 〈0.05）.With the increase in PHB concentration, the activity of immunity related enzymes changed in a similar pattern with RPS： increasing at first and decreasing subsequently. In addition, the activity of the enzymes was elevated in the 2nd and 3rd weeks of PHB administration. Among them, T-AOC in serum of groups E1.0 and E2.5, T-AOC in hepatopancreas of Group E1.0, ACP activity in serum of groups E1.0 and E2.5, ACP activity in hepatopancreas of Group E1.0, CAT activity in groups E0.5, E1.0 and E2.5, CAT activity in hepatopancreas of groups E0.5, E1.0 and E10.0, POD activity in both serum and hepatopancreas of groups E0.5, E1.0 and E2.5, SOD activity in both serum and hepatopancreas of Group E1.0, MDA content in serum of Group E1.0 and MDA content in hepatopancreas of groups E0.5 and E1.0 showed significant difference from those of other groups（P〈0.05）. [Conclusion] PHB can improve the immunity of F. chinensis, 1.0% in feed has the best effect, and the total enzyme activity reaches the highest level in the 2nd and 3rd weeks of PHB administration.

棉花bZIP转录因子基因GhbZIP15的克隆与表达分析

b ZIP转录因子在植物抵御各种逆境胁迫中有重要作用。本研究依据生物信息学分析,采用RT-PCR(Reverse transcription polymerase chain reaction)方法从棉花中克隆了一个b ZIP转录因子基因,命名为Ghb ZIP15(Gen Bank登录号为KP938299)。该基因c DNA全长1980 bp,其开放阅读框为966 bp,编码321个氨基酸的蛋白,预测分子量为37.1 k D,等电点为7.44。SMART蛋白结构预测发现,该蛋白含有b ZIP家族典型的BRLZ碱性结构域和亮氨酸拉链,属于B-zip1家族。系统进化树分析表明Ghb ZIP15属于b ZIP转录因子AREB亚家族,和拟南芥A亚家族的Atb ZIP12,At ABF1和At ABF2亲缘关系最近。洋葱表皮细胞中瞬时表达分析表明,Ghb ZIP15主要定位于细胞核中。RT-PCR分析表明Ghb ZIP15对干旱、高盐、低温等处理都有一定程度的响应,推测该基因对棉花非生物胁迫的调控起重要作用。

毛发中GHB的检测及评价

目的探索毛发中外源性GHB的检测及判断的可行性,为涉GHB的鉴定提供方法和依据。方法建立毛发中GHB的GC/MS分析方法,并通过动物实验,考察毛发中内源性GHB的质量分数范围、外源性GHB在毛发中的时间过程以及给药剂量、毛发颜色与毛发中GHB的质量分数关系。结果豚鼠和中国人黑色毛发中内源性GHB质量分数分别为(3.01±1.41)ng/mg(n=28)和(1.02±0.27)ng/mg(n=20);摄GHB后毛发中GHB质量分数明显增加且与给药剂量呈正相关性;GHB在毛干中呈窄带分布;深色毛发中GHB质量分数高于浅色毛发。结论毛发中GHB的检测适用于GHB滥用和中毒的法医毒物学鉴定;根据毛发中的GHB质量分数和毛发分段分析可判断GHB的来源。

新型迷奸药γ-羟基丁酸(GHB)及相关物质

γ-羟基丁酸及相关物质的滥用日渐流行,同时由于其强烈的镇静及健忘效果常被用作迷奸药。在人体内γ-羟基丁酸的天然存在和摄入后的迅速消除,使得体内γ-羟基丁酸及相关物质的检测和浓度评价变得困难。本文就γ-羟基丁酸及相关物质的理化性质、合成、滥用、内源性产生、药理学、药效学、中毒与死亡、临床应用、在DFSA案件中的使用、检测方法等诸多问题作一综述,以期为法医毒物分析等相关领域的实际检案和研究提供参考。

GHB光学性质的DFT理论计算研究

采用密度泛函理论(DFT)方法对γ-羟基丁酸(GHB)进行了B3LYP/6-31G水平上的分子结构优化、红外光谱、Raman光谱、紫外-可见光谱及分子前线轨道理论等计算。研究结果表明:理论计算结果与实验数据吻合得较好,对IR、THz、UV-Vis吸收光谱和Raman散射光谱中的特征峰进行了归属,发现GHB在0.1～10 THz波谱范围内有四个明显的吸收峰,分别位于0.85、2.89、7.21以及8.61 THz,GHB在紫外波段的235.50、190.23及169.79 nm有3个吸收峰。基于GHB具有特征性的IR、Raman、THz、UV-Vis光谱性质,可以使用光谱技术对GHB进行探测、识别、分析和检测。

棉花抗枯萎病相关GhB301基因对烟草的遗传转化及功能分析

【目的】ERF是乙烯信号传导途径中的重要转录因子,在植物与病原菌互作中发挥着重要的调控作用。棉花抗枯萎病相关Gh B301基因是从棉花中获得的ERF-B3亚组基因,探索该基因在烟草异位表达后的功能,为进一步研究其在棉花抗枯萎病中的功能奠定基础。【方法】以p PZP35S表达载体为基础,构建Gh B301的过表达载体;采用农杆菌介导法转化烟草并获得转基因植株。【结果】经卡那霉素筛选、PCR和RT-PCR检测,获得3株转基因阳性植株;q PCR分析显示,转基因阳性植株中部分病程相关蛋白基因的表达量较野生型明显升高。【结论】棉花抗枯萎病相关基因Gh B301在烟草中异位表达后可能会通过增强部分病程相关蛋白基因的表达来提高植株抗病性。