Enhanced production of poly(3-hydroxybutyrate) by filamentation-suppressed recombinantEscherichia coli in a defined medium

Fed-batch cultures of recombinantEscherichia coli strains were carried out for the production of poly(3-hydroxybutyric acid) (PHB) in a chemically defined medium. TheE. coli strains used were XL1-Blue, harboring pSYL105, a stable high-copy number plasmid containing theAlcaligenes eutrophus polyhydroxyalkanoate (PHA) genes, and XL1-Blue, harboring pSYL107, which is pSYL105 containing theE. coli ftsZ gene to suppress filamentation. With XL1-Blue(pSYL105) the final cell mass and PHB concentration obtained in 62 h were 102 and 22.5 g/L, respectively. Fed-batch culture of XL1-Blue(pSYL107) under identical conditions resulted in a final cell mass and PHB concentration of 127.5 and 48.2 g/L, respectively. The PHB contents obtained with XL1-Blue(pSYL105) and XL1-Blue(pSYL107) were 22.1 and 37.8%, respectively. Therefore, PHB was more efficiently produced in a defined medium by employing filamentation-suppressed recombinantE. coli.     

含硫化合物对Methylosinus trichosporium OB3b生长和合成聚-β-羟基丁酸酯的影响

为了将人类活动排放的含有甲烷的废气用于聚-β-羟基丁酸酯(PHB)合成过程,以M.trichosporium OB3b为菌种,考察硫化氢(H2S)和甲硫醚(DMS)对甲烷氧化菌生长和PHB合成的影响。实验结果表明,H2S浓度高达2 500μmol/mol时对甲烷氧化菌生长及甲烷单加氧酶(MMO)活性基本无影响,含H2S时的PHB含量和产率比无H2S时高,甲烷氧化菌经H2S培养后PHB合成能力增强。然而,DMS浓度为200μmol/mol时即可明显抑制甲烷氧化菌的生长及MMO活性。在DMS存在条件下,PHB含量和PHB产率都明显降低,经DMS培养后甲烷氧化菌的PHB含量进一步降低。     

Effect of ammonium on nitrous oxide emission during denitrification with different electron donors

Nitrous oxide(N2O) emission during denitrification is receiving intensive attention due to its high potential to cause greenhouse effects.In this study,denitrifiers were acclimated in sequencing batch reactors with methanol or acetate as the electron donor and nitrate as the electron acceptor.The effects of ammonium on N2O emission were examined in batch experiments with various electron donors.With the addition of ammonium,N2O emission increased under all the examined conditions compared to experiments without ammonium addition.With different electron donors,the highest ratio of N2O emission to the removed oxidized nitrogen was 0.70% for methanol,5.34% for acetate,and 34.79% for polyhydroxybutyrate.     

单一好氧环境下的强化生物除磷研究

将乙酸钠为单一碳源、厌氧/好氧交替、具有较好除磷效果的传统生物除磷SBR系统,改为单一的好氧SBR运行方式,发现改变后的SBR系统仍可取得较好的除磷效果,除磷率最高达73.9%,最低约40%,平均维持在50%左右.这种现象可以维持长达80个周期.污泥含磷率由最初的1.43%增加到6.56%.对污泥微生物胞内PHB和糖原进行测定,结果表明此系统中微生物PHB和糖原在VSS中含量分别约为27 mg/g和26 mg/g,二者含量在好氧过程中都基本保持不变.通过对反应过程中碳源消耗与磷吸收关系的分析,认为该单一好氧条件下的生物除磷机制是由于长期以乙酸钠为唯一碳源下,试验系统中活性污泥被驯化,在胞内聚磷颗粒含量容纳能力范围内还可以在好氧环境下以乙酸钠氧化产生的ATP为能量进行磷吸收所致.     

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHA_m). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHA_m and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHA_m and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHA_m and PHB accumulating strains have shown that PHA_m and PHB granules can be distinguished from each other: PHA_m granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHA_m and PHB chains were stored in separate granules. To be able to determine whether the separation of PHA_m and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHA_m granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHA_m and PHB, the resulting polymer chains are segregated completely and stored in separate granules.     

Purification and Properties of a Poly (β-hydroxybutyrate) Depolymerase From Penicillium sp.

An extracellular poly (β-hydroxybutyrate) (PHB) depolymerase was purified from a Penicillium sp. DS9701-09a by centrifugation, ultrafiltration, precipitation and gel filtration chromatography. The specific activity of the purified enzyme was 37.9-folds higher than that of the culture supernatant and the recovery yield was 11.8%. The PHB deploymerase molecular mass was 44.8 kDa from analysis of both Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. The isoelectric point of 6.7 for the enzyme was determined by a two-dimensional electrophoresis. The optimum enzyme activity was observed at a temperature of 50 °C and pH 5.0. The apparent K _m of the enzyme was found to be 1.35 mg/mL. The PHB depolymerase consisted of 16 kinds of normal amino acids. The secondary structure of the enzyme was determined by CD spectrum. α-helix and β-turn were found to be 66% and 34% for the enzyme without ammonium sulphite. Chemical inhibition on the PHB depolymerase activity was examined and EDTA was found to have a significantly inhibitory effect.     

慢生型大豆根瘤菌(Bradyrhizobium japonicum)聚羟丁酸合成酶基因(phbC)突变体的构建及其特性

通过转座子Tn5诱变和同源重组，构建了Bradyrhizobium japonicum USDA110聚羟丁酸合成酶基因(phbC)突变体．序列测定确定了转座子插入的精确位置，所获得的4个转座子诱变的质粒其Tn5插在phbC基因内两个相距仅9bp的位点．被Southern和PCR证实的突变体菌株仍能产生相当于野生型菌株12．97％—25．10％的PHB，并且在突变体和野生型菌株总DNA杂交图上都呈现出一条约5kb的阳性带，推测在B.japonicum基因组中存在不止一个聚羟丁酸合成酶基因．图3表4参17     

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a lipase box sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.     

球衣细菌胞内PHB的提取和精制

报道了一种从球衣细菌胞内提取和精制PHB的有效方法。通过红外光谱、紫外光谱及1H谱分析得知 ,利用该方法制得的PHB精制品在结构上与标准品 (美国Sigma公司提供 )一致 ,纯度达 99 4 %以上。     

聚糖菌颗粒污泥的有机物吸收及胞内储存特性

采用特殊运行方式的厌氧-好氧SBR系统(厌氧后排水),在乙酸钠、葡萄糖及葡萄糖-乙酸钠混合基质条件下均培养出了稳定的聚糖菌颗粒污泥.通过对典型周期有机物、磷酸盐、胞内糖原及聚β-羟基丁酸(PHB)变化的测定分析,证明有机基质的种类对于聚糖菌能量利用模式、有机物吸收速率及胞内储存物质种类具有显著的影响.污泥初始胞内糖原水平是有机物吸收数量及吸收速率的关键影响因素,当糖原水平低于0.05g/gSS时,厌氧有机物去除率与糖原水平直接相关:而糖原水平高于0.05g/gSS时,厌氧有机物去除率趋于稳定.不同糖原水平污泥厌氧吸收有机物的速率具有明显差异,在厌氧初期有机物快速吸收阶段,有机物厌氧吸收速率随胞内糖原水平升高而增加.