PHB recovery by hypochlorite digestion of non-PHB biomass

Summary Hypochlorite digestion of bacterial biomass from intracellular poly--hydroxybutyrate (PHB) has not been used on a large scale since it has been reported to severely degrade PHB. In this study, to minimize degradation, the initial biomass concentration, digestion time and pH of the hypochlorite solution were optimized. Consequently, PHB of 95% purity with a weight average molecular weight (M_W) of 600,000 and a polydispersity index (PI) of 4.5 was recovered from biomass initially containing PHB with a M_W of 1,200,000 and a PI of 3.     

Cost-effective recovery and purification of polyhydroxyalkanoates by selective dissolution of cell mass

Highly efficient separation and purification of polyhydroxyalkanoates (PHAs) from PHA-containing cell mass is essential to production of the bioplastics from renewable resources in a cost-effective, environmentally friendly way. Based on selective dissolution of non-PHA cell mass (NPCM) by protons in aqueous solution and crystallization kinetics of PHA biopolymers, a simple process is developed and demonstrated to recover PHAs from cell mass to high purity (>97 wt %) with high yield (>95 wt %). The average molecular weight of biopolyesters is controlled, which follows an exponential function of process severity, a combined factor of processing conditions. Compared with conventional chemical treatment such as sequential surfactant and hypochlorite treatment, this new technology substantially reduces the chemical cost for PHA recovery and purification from PHA-containing cell mass.     

Effect of carbon source and concentration on the molecular mass of poly(3-hydroxybutyrate) produced by Methylobacterium extorquens and Alcaligenes eutrophus

In shake-flask culture, Methylobacterium extorquens accumulated poly(3-hydroxybutyrate) (PHB) possessing a substantially higher weight-average molecular mass (M_W) than previously reported for this organism. The M_W of PHB produced by M. extorquens was dependent on the initial concentration of methanol or sodium succinate, used as sole carbon sources. The highest M_W values (0.6 and 1.7 × 10⁶) were obtained with low initial concentrations of methanol or sodium succinate (4.0 and 3.0 g l^–1, respectively) and the latter substrate always yielded PHB of higher M_W than that produced from methanol. Thus PHB with an M_W in the range 0.2–1.7 × 10⁶ could be produced by selection of the carbon source and its concentration. In contrast to the findings with M. extorquens, the M_W of PHB produced by Alcaligenes eutrophus was high (1.1–1.6 × 10⁶) and generally unaffected by the choice or concentration of the carbon source. The use of glycerol as sole carbon source did, however, result in the accumulation of PHB with a markedly lower M_W (5.5–8.5 × 10⁵) than that produced from other sole carbon sources by this organism under similar conditions. Correspondence to: A. J. Anderson     

Production of medium-chain-length PHA in octanoate-fed enrichments dominated by Sphaerotilus sp.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher M_w and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower M_w. The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.     

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB_M) can compete with wild-type PLB (PLB_W) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB_W, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K_Ca, measured from the Ca²⁺-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB_W, while the potency of each of the LOF PLB_M was about one third that of PLB_W. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB_W and LOF PLB_M, inhibitory potency was reduced relative to that of PLB_W alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB_W was decreased by both (unlabeled) LOF PLB_M. These results show that LOF PLB_M can compete both physically and functionally with PLB_W, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB_M targeted for gene therapy of heart failure in humans.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Production of fructose syrup by selective removal of glucose from hydrolyzed Jerusalem artichoke juice

Summary The study shows that the yeastSaccharomyces cerevisiae ATCC 36859 can be successfully used for the production of fructose syrup from glucose-fructose mixtures or from Jerusalem artichoke juice by the conversion of glucose to ethanol. During these processes fructose concentration was unchanged.Ethanol yield (Y_P/S), based on glucose consumed in Jerusalem artichoke juice, and ethanol concentration were 0.428 g/g and 1.7% (w/v) respectively. When the juice was supplemented with glucose higher ethanol concentrations were attained but with lower ethanol yields.     

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (M_w: 480,000 g.mol^–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (M_w: 391,000 g mol^–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a K_m of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Recovery of megakaryocyte thromboxane production in vitro after aspirin inhibition

Megakaryocytes isolated in high purity from guinea pigs produced thromboxane B₂ in response to exogenously provided arachidonic acid. This production was inhibited by in vitro treatment with acetylsalicylic acid with a concentration response relationship similar to that seen in platelets. During in vitro culture, the aspirin-treated megakaryocytes recovered thromboxane synthetic ability. Following a lag of 12 hours, recovery of megakaryocyte thromboxane production resumed at a rate of 16% of control per day. This recovery was inhibited by the addition of cycloheximide to the culture medium.     

Extraction of poly-3-hydroxybutyrate using chlorinated solvents

Summary Recovery of poly-3-hydroxybutyrate (PHB) in three chlorinated solvents with or without acetone pretreatment and degradation of extracted PHB (99% pure) in hot chloroform were studied. When lyophilized Alcaligenes eutrophus biomass was used, the best results were obtained with acetone pretreatment and solvent reflux for 15 min in methylene chloride or chloroform. Recovered PHB had a 95% purity and molecular weights (M_w) of 1,050,000 and 930,000 g/mol respectively. Further heating resulted in a serious M_w, loss at reflux temperatures. Degradation of extracted PHB at 110°C in chloroform was due to random and chain-end scission, the former being predominant.     

Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines

Summary This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.     

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (K_s = 3.2 ± 1.6 μM, K_i = 38 ± 1.4 μM) and delF508 CFTR (K_s = 3 ± 1.8 μM, K_i = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (K_s = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (K_i = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Moisture adsorption capacity and surface area as deduced from vapour pressure isotherms in relation to hygroscopic water of soils

Six calcareous and alluvial soil profiles differing in their texture, CaCO₃ and salinity were chosen from west and middle Nile Delta for the present study. The 1^st and 2^nd profiles from Borg El-Arab area were sandy loam in texture and > 30% CaCO₃, while the 3^rd and 4^th profiles (from Nubaria area) were sandy clay loam and < 30% CaCO₃. The 2^nd and 4^th profiles were taken from cultivated area with maize. The 5^th profile from Epshan area was non-saline clay alluvial soil and the 6^th from El-Khamsen was saline clay alluvial soil. The relation between soil moisture content (W%) and water vapour pressure (P/P _o) was established for the mentioned soils. Data showed that the specific surface area (S) values were 34–53 and 44–60 m²/g for calcareous soils of Borg El-Arab and Nubaria areas, 206–219 and 206–249 m²/g for non-saline and saline clay alluvial soils of Epshan and El-Khamsen areas, respectively. The corresponding values of the external specific surface area (S _e) were 16–21, 14–22, 72–86 and 92–112 m²/g. Submitting W _m+W _me as an adsorption boundary of moisture films (W _c) (where W _m is mono-adsorbed layer of water vapour on soil particles and W _me is the external mono-adsorbed layer), the maximum water adsorption capacity (W _a) was found to be W _c + W _me or W _m + 2W _me. It was ranged from 1.88 to 2.70%, 1.97 to 2.95%, 9.70–10.70% and 10.80 to 13.12% while the maximum hygroscopic water (M _H) values were 2.43–3.78%, 2.91–4.65%, 16–17% and 18.30–21.9% for the studied soil profiles respectively. The residual moisture content (θ _r) at pF 7 and P/P _o = 0 was ranged from 0.0005–0.0010%, 0.0007–0.0019% and 0.0043–0.0048% in Borg El-Arab, Nubaria and Epshan soil profiles, respectively. The inter-relations between the surface area and the hygroscopic moisture parameters of the soils under investigation were as follows Calcareous soils; W _m = 0.40 M _H, W _c = 0.55 M _H, W _a = 0.70 M _H, S = 14 M _H Non-saline soil; W _m = 0.35 M _H, W _c = 0.49 M _H, W _a = 0.63 M _H, S = 13 M _H Saline soil; W _m = 031 M _H, W _c = 0.45 M _H, W _a = 0.59 M _H, S = 12 M _H These relations give possibility to deduce the soil moisture adsorption capacities and specific surface area via maximum hygroscopic water, which can be obtained through the experimental determination of water vapor adsorption isotherms.     

Production of 2,3-butanediol from HF-hydrolyzed aspen wood

Hydrolyzates from hydrogen fluoride (HF) treated aspenwood were predominantly composed of oligosaccharides which are not readily utilized by Klebsiella pneumoniae. Attempts at further hydrolyzing these oligosaccharides using a variety of glycolytic and xylanolytic enzymes (i.e., amylases, cellulases, and xylanases) were only partially successful. When a post-hydrolysis step was carried out using 3% H₂SO₄, significant amounts of the component monosaccharides were detected. Sugars released by acid or enzymatic hydrolysis of the HF treated aspenwood were utilized by K. pneumoniae for the production of butanediol and ethanol.     

Purification of recombinant salmon growth hormone expressed inEscherichia coli

Summary Recombinant salmon growth hormone (SGH) expressed inEscherichia coli was refolded and purified. Native form SGH with a purity of 98% was obtained with a recovery of 9%. We found that purified SGH in reduced form under denaturing conditions efficiently formed correct disulfide bonds.     

Biosynthesis and characterization of copolymer poly(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) from saponified <Emphasis Type="Italic">Jatropha curcas</Emphasis> oil by <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polymers with promising application in the formulation of plastic materials. PHAs are produced by numerous bacteria as energy/carbon storage materials from various substrates, including sugars and plant oils. Since these substrates compete as food sources, their use as raw material for industrial-scale production of PHA is limited. Therefore, efforts have been focused on seeking alternative sources for bacterial production of PHA. One substrate that seems to have great potential is the seed oil of Jatropha curcas plant. Among other favorable properties, J. curcas seed oil is non-edible, widely available, and can be cheaply produced. In this study, Pseudomonas oleovorans (ATCC 29347) was grown in a mineral salt medium supplemented with saponified J. curcas seed oil as the only carbon source under batch fermentation. Optimum PHA yield of 26.06% cell dry weight was achieved after 72 h. The PHA had a melting point (T _m) between 150 and 160°C. Results of polymer analyses by gas chromatography/mass spectrometry (GC/MS) identified only the methyl 3-hydroxybutanoate monomeric unit. However, electrospray ionization–time of flight mass spectroscopy (ESI–TOF MS) confirmed that the PHA was a copolymer with the characteristic HB/HV peaks at m/z 1155.49 (HB) and 1,169, 1,184–1,194 (HV). The data were further supported by¹H and ¹³C NMR analysis. Polymer analysis by gel permeation chromatography (GPC) indicated a peak molecular weight (MP) of 179,797, molecular weight (M _W) of 166,838, weight number average mass (M _n) of 131,847, and polydispersity (M _w/M _n) of 1.3. The data from this study indicate that J. curcas seed oil can be used as a substrate to produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV).     

枸橼酸咖啡因联合肺泡表面活性物质治疗新生儿呼吸窘迫综合征的疗效及对血清BMP-7、CC16、SF水平的影响

目的:探讨枸橼酸咖啡因联合肺泡表面活性物质治疗新生儿呼吸窘迫综合征患儿的疗效及对血清骨形态发生蛋白-7(BMP-7)、Clara细胞分泌蛋白(CC16)、铁蛋白(SF)水平的影响。方法:选择2016年3月到2018年3月我院接诊的新生儿呼吸窘迫综合征患儿90例作为研究对象,以随机数表法分为观察组(n=48)和对照组(n=42)。对照组使用肺泡表面活性物质进行治疗,观察组在对照组的基础上加用枸橼酸咖啡因进行治疗。比较两组治疗后的疗效,治疗前后血清BMP-7、CC16、SF水平、血气指标[氢离子浓度指数(p H)、二氧化碳分压(PCO_2)、氧合指数(PaO_2/Fi O_2)]的变化,通气时间及支气管肺发育不良(BPD)的发生率。结果:治疗后,观察组总有效率为95.83%,明显高于对照组(71.43%,P0.05);两组患儿血清BMP-7、CC16、SF水平较治疗前均显著降低(P0.05),且观察组以上指标均明显低于对照组(P0.05);两组患儿pH、PaO_2/Fi O_2均较治疗前明显升高,而PCO_2较治疗前显著降低(P0.05),且观察组患儿p H、PaO_2/Fi O_2显著高于对照组,而PCO_2明显低于对照组(P0.05)。观察组患儿通气时间明显短于对照组,BPD发生率显著低于对照组(P0.05)。结论:枸橼酸咖啡因联合肺泡表面活性物质治疗新生儿呼吸窘迫综合征的临床效果显著优于单用肺泡表面活性物质治疗,其可有效改善患儿血清BMP-7、CC16、SF水平、缩短机械通气时间,降低支气管肺发育不良发生率。     

Laccase fromPleurotus ostreatus

Summary Laccase was purified from culture broth ofPleurotus ostreatus mycelium. The enzyme was a single protein of M_r 59000, pI 2.9 and was active on o-diphenyl substrates. Amino acid composition and N-terminal sequence (15 residues) were determined. Polyclonal anti-laccase antibodies were obtained.