Copolymeric polythioesters by lipase-catalyzed thioesterification and transthioesterification of α,ω-alkanedithiols

Linear copolymeric polythioesters [PTE; poly(α,ω-alkanedioic acid-co-α,ω-alkanedithiols)] were formed in good yield (∼69%) by thioesterification of 1,12-dodecanedioic acid with 1,6-hexanedithiol and 1,8-octanedithiol, respectively, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) in vacuo without a solvent. Similarly, transthioesterification (thiolysis) of diethyl 1,12-dodecanedioate with 1,6-hexanedithiol led to the formation of ∼66% PTE. Poly (1,12-dodecanedioic acid-co-1,6-hexanedithiol) and poly (1,12-dodecanedioic acid-co-1,8-octanedithiol) were extracted from the reaction mixture using methyl-t-butylether, precipitated at −20°C and the precipitates extracted with boiling i-hexane to yield two fractions of PTE. The i-hexane-insoluble fraction of poly (1,12-dodecanedioic acid-co-1,6-hexanedithiol) shows an average molecular mass (M_w) of 1,212 Da, corresponding to a molecular weight range of up to 13,200 Da and a degree of polymerization of up to 38 monomer units. The i-hexane-insoluble fraction of poly (1,12-dodecanedioic acid-co-1,8-octanedithiol) shows a M_w of 2,360 Da, corresponding to a molecular weight range of up to 19,500 Da and a maximum degree of polymerization of up to 52 monomer units. The low-molecular weight (<800 Da) reaction products of thioesterification of 1,12-dodecanedioic acid with 1,6-hexanedithiol, elucidated by gas chromatography–mass spectroscopy, show the following intermediates: (1) 9,20-dioxo-1,8-dithiacycloeicosane; (2) 17,28-dioxo-1,8,9,16-tetrathiacyclooctacosane; (3) 1,12-dodecanedioic acid methyl(O)ester 6′-S-mercaptohexyl thio(S)ester; and (4) oligomeric linear thioester, formed by thioesterification of two molecules of 1,12-dodecanedioic acid with one molecule of 1,6-hexanedithiol.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

Hyaluronic acid depolymerization by ascorbate-redox effects on solid state cultivation of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> in cashew apple fruit bagasse

The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M_W) hyaluronic acid (HA) (10⁴–10⁵ Da). The cashew juice is a rich source of vitamin C containing, 1.2–2.0 g L⁻¹. This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M_W reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g⁻¹ h⁻¹ when the ascorbate concentration ranged from 1.7 to 10 mg mL⁻¹. These findings contribute to the controlled production of HA in a low M_W range, which is important in cell signalization, angiogenesis and nanoparticles production.     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

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).     

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     

Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by ¹³C NMR analysis. The number average molecular weight (M_n) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6–3.9.     

Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DO-stat mode were able to regulate the 4HB composition from 0–67 mol% by sequential feeding of γ-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy. Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M _n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.     

Synthesis, characterization and aqueous dispersion of dextran-g-poly(1,4-dioxan-2-one) copolymers

A novel biodegradable graft copolymer, dextran-g-poly(1,4-dioxan-2-one) (PODEX), was synthesized through the ring-opening polymerization (ROP) of 1,4-dioxan-2-one (PDO) onto a dextran backbone. Initially, dextran was silylated with 1,1,1,3,3,3-hexamethyldisilazane. The grafting from pathway was conducted with various (30–70) PDO/OH feed ratios to obtain PODEX copolymers with a various PPDO graft structures. Graft copolymers were characterized by FT-IR, ¹H and ¹³C NMR, DSC, TGA, and WAXD. Molecular weights of the PODEX copolymers (M_W: 94,700–117, 300 Da), glass transition temperature (−29 to −17 °C), melting temperature (82–100 °C), and crystallinity (32–40%) were increased with the content of PPDO. AFM observations revealed that polymeric micelles were spherical and uniform in morphology with around 30–58 nm diameter. Critical micelle concentration (CMC) of self-assembled system was significantly decreased from 3.2 to 1.09 mg/L with the increment of PPDO.     

Synthesis of biodegradable polyesters by Gram negative bacterium isolated from Malaysian environment

A locally isolated Gram negative bacterium, Cupriavidus sp. USMAA9-39 was able to produce various types of biodegradable polyesters through a two-step cultivation process. These are copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)]. These polymers were synthesized by this bacterium when grown with a combination of some carbon sources. The biosynthesis of P(3HB-co-4HB) was achieved by using carbon sources such as γ-butyrolactone or 1,4-butanediol or by a combination of oleic acid with either γ-butyrolactone or 1,4-butanediol. Meanwhile, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced using 1-pentanol or valeric acid or by a combination of oleic acid with either 1-pentanol or valeric acid. When γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol were used as mixed carbon sources, P(3HB-co-3HV-co-4HB) terpolymer were produced. The presence of 3HB, 3HV or/and 4HB monomers were confirmed by gas chromatography and nuclear magnetic resonance (NMR) spectroscopy.     

Production and characterization of terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB

Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB (phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB-co-3HV-co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43 g l⁻¹ to 3.29 g l⁻¹, 20.7% to 35.6%, 2.3 mol% to 7.1 mol%, respectively. Number average molecular (M_n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB-co-5 mol%3HV) or P(3HB-co-12 mol%3HHx). In addition, the terpolyester had lower melting (T_m) temperatures and enthalpy of fusions (ΔH_m) than PHB did.     

Netropsin binding in five duplex-dimer DNA constructs as a function of size and distance between binding sites: circular dichroism and absorption spectroscopy

The optical activity induced on binding the drug netrospin (NET) in the minor groove of DNA is studied in five oligonucleotides (OGNs) as a function of (1) the size of the binding site in (5′-(GC)₂AATT(GC)₂-3′)₂ (OGN 1a) versus (5′-(GC)₂AAATTT(GC)₂-3′)₂ (OGN 1b) and (2) the distance between two AATT binding sites in (5′-(GC)₂AATT(GC) _x AATT(GC)₂-3′)₂, with x = 1, 2, or 3 (OGNs 2a, b, c, respectively). NET binding is monitored via the induced circular dichroism (CD) at ~315 nm, where the nucleic acids are optically inactive. The CD titrations, fit to a tight binding model, yield lower limits for the binding constant, K_a, ≥8 × 10⁷ M⁻¹ for OGN 1a and ≥2 × 10⁸ M⁻¹ for OGNs 2a, b, c in 1 mM buffer. In 100 mM buffer, tight binding occurs in all five OGNs with K_a ≥ 8 × 10⁷ M⁻¹ for OGN 1a and ≥1 × 10⁸ M⁻¹ for OGNs 1b and 2a, b, c. In contrast, the elongated AAATTT binding site of OGN 1b results in weak binding of NET in 1 mM buffer, where competing electrostatic interactions with the solvent environment are lower. In the constructs with two binding sites, the increase in flexibility introduced by intervening GC base pairs does not induce co-operative binding, although differences in the number of binding sites, n (2.05–2.65), indicate that there may be differences in the way NET is bound in OGNs 2a, b, c. In addition, the large shifts in the absorption spectra induced in bound versus free NET, and effects on the CD spectral bands at higher energy, are discussed in terms of electrostatic and excitonic interactions.     

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.     

Evaluating the effective numbers of independent tests and significant <Emphasis Type="Italic">p</Emphasis>-value thresholds in commercial genotyping arrays and public imputation reference datasets

Current genome-wide association studies (GWAS) use commercial genotyping microarrays that can assay over a million single nucleotide polymorphisms (SNPs). The number of SNPs is further boosted by advanced statistical genotype-imputation algorithms and large SNP databases for reference human populations. The testing of a huge number of SNPs needs to be taken into account in the interpretation of statistical significance in such genome-wide studies, but this is complicated by the non-independence of SNPs because of linkage disequilibrium (LD). Several previous groups have proposed the use of the effective number of independent markers (M _e) for the adjustment of multiple testing, but current methods of calculation for M _e are limited in accuracy or computational speed. Here, we report a more robust and fast method to calculate M _e. Applying this efficient method [implemented in a free software tool named Genetic type 1 error calculator (GEC)], we systematically examined the M _e, and the corresponding p-value thresholds required to control the genome-wide type 1 error rate at 0.05, for 13 Illumina or Affymetrix genotyping arrays, as well as for HapMap Project and 1000 Genomes Project datasets which are widely used in genotype imputation as reference panels. Our results suggested the use of a p-value threshold of ~10⁻⁷ as the criterion for genome-wide significance for early commercial genotyping arrays, but slightly more stringent p-value thresholds ~5 × 10⁻⁸ for current or merged commercial genotyping arrays, ~10⁻⁸ for all common SNPs in the 1000 Genomes Project dataset and ~5 × 10⁻⁸ for the common SNPs only within genes.     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

Age and growth of the blackchin guitarfish Glaucostegus cemiculus (Geoffroy Saint-Hilaire, 1817) from Iskenderun Bay (Northeastern Mediterranean)

Between September 2010 and June 2012, a total 291 (166 females and 125 males) blackchin guitarfish Glaucostegus cemiculus were captured by a commercial bottom trawler (F/V Coşkun Reis) in Iskenderun Bay, Turkey (northeastern Mediterranean Sea). The total length (L) and total weight (W) of the female and male guitarfish ranged between 32.0–165.0 cm and 88 g–16.68 kg, and 34.3–128.3 cm and 112 g–6.00 kg, respectively. Vertebral age estimates ranged from 0 to 8 years for females and 0 to 5 years for males. The growth models of von Bertanlanffy and Gompertz were fitted to the length at age data using the nonlinear regression method. Model selection was based on the values of the residual standard error and the Akaike's information criterion corrected for small sample size (AIC_C) associated with each fit. The von Bertalanffy growth model provided the best fitting growth curves for each sex with parameters reaching L_∞ = 187.17 cm, K = 0.195 year^-1, t₀ = −1.38 year for females, and L_∞ = 144.85 cm, K = 0.321 year^-1, t₀ = −1.13 year^-1 for males. The W–L relationship parameters did not differ significantly between sexes, the estimated values of a and b were 0.0018 and 3.11, respectively. By using these values of a and b, and also respective estimates of L_∞, the values of W_∞ were obtained as 20.53 kg for females and 9.25 kg for males. The overall percentage ratios of females and males in the samples were 57% and 43% respectively.     

Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

Length–weight and otolith size to standard length relationships in 12 species of Southern Ocean Myctophidae: A tool for predator diet studies

Fish morphometric relationships are key tools for fisheries science and studies of food web dynamics and predator foraging behaviour, but parameterisations are limited for Southern Ocean myctophids (Family Myctophidae). New standard length (L_S) to total mass (M_T) relationships are therefore described for the 12 biomass-dominant myctophid fish species living in the Scotia Sea, Southern Ocean, using the most comprehensive data collected in the region to date. New linear regressions for otolith size (length; O_L and width; O_W) and L_S are also described. Significant (p < .01) L_S–M_T relationships were established for all species using simple non-linear regression. Significant (p < .01) relationships between L_S and both O_L and O_W were also determined for all species, with O_W being the best predictor of L_S in all but one species. Our study provides a comprehensive tool for reconstructing the myctophid component of marine predator diets that will improve future food web, predator behaviour and ecosystem studies in the Scotia Sea.     

Heterologous expression of <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 PHA synthase gene in PHB<Superscript>−</Superscript>4 mutant for the production of poly(3-hydroxybutyrate) and its copolymers

Ecological deterioration and human health concerns arising from the usage of non-biodegradable plastics have prompted mankind to search for greener alternatives which are biodegradable, biocompatible and easily produced from renewable sources. Polyhydroxyalkanoates (PHA), among other biopolymers, are emerging as a viable replacement for fossil fuel-based synthetic plastics. A PHA-producing strain, identified as Cupriavidus sp. (designated Cupriavidus sp. USMAA2-4) was isolated from a soil sample from western peninsular Malaysia. Heterologous expression of the PHA synthase gene (phaC _USMAA2-4) in mutant C. necator PHB⁻4 complemented its PHA-producing ability. More than 60 wt% of P(3HB) was synthesized from various plant oils. The highest P(3HB) production of 2.38 g/l at 68 wt% was attained when crude palm kernel oil was fed as the sole carbon source. The 3HV molar fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was significantly affected by the type of the precursor used and their respective feeding time. The 3HV molar fraction ranged from 4 to 31 mol% when sodium propionate/valerate was fed at different cultivation times. In addition, with the supplementation of 4HB-monomer precursors, approximately 67 wt% P(3HB-co-4HB) with 4–5 mol% of 4-hydroxybutyrate monomer was synthesized, regardless of the precursor feeding time used. Variation in the molar fraction of the second monomer along with its biodegradability and biocompatibility characteristics promotes the potential of these copolymers as replacements for traditional commodity plastics.     

State of aggregation of recombinant hirudin in solution under physiological conditions

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight ¯M _w determined by sedimentation equilibrium was found to be 6914±76 Da compared to 6964 Da expected from the amino acid sequence. The ¯M _z /¯M _w ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M _r ) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991),Eur. J. Biochem. 202, 67–73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konnoet al. (1988),Arch. Biochem. Biophys. 267, 158–166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.Abbreviations ¯M _w weight-average molecular weight - ¯M _z Z-average molecular weight - M _r relative molecular weight - NTSB 2-nitro-5-thiosulfobenzoic acid - Tris Tris(hydroxymethyl)aminomethane - r-HV1 recombinant desulfatohirudin - _M molar extinction coefficient