Blend miscibility of cellulose propionate with poly(N-vinyl pyrrolidone-co-methyl methacrylate)

The blend miscibility of cellulose propionate (CP) with poly(N-vinyl pyrrolidone-co-methyl methacrylate) (P(VP-co-MMA)) was investigated. The degree of substitution (DS) of CP used ranged from 1.6 to >2.9, and samples for the vinyl polymer component were prepared in a full range of VP:MMA compositions. Through DSC analysis and solid-state ¹³C NMR and FT-IR measurements, we revealed that CPs of DS < 2.7 were miscible with P(VP-co-MMA)s of VP ≥ ∼10 mol% on a scale within a few nanometers, in virtue of hydrogen-bonding interactions between CP-hydroxyls and VP-carbonyls. When the DS of CP exceeded 2.7, the miscibility was restricted to the polymer pairs using P(VP-co-MMA)s of VP = ca. 10–40 mol%; the scale of mixing in the blends concerned was somewhat larger (ca. 5–20 nm), however. The appearance of such a “miscibility window” was interpretable as an effect of intramolecular repulsion in the copolymer component. Results of DMA and birefringence measurements indicated that the miscible blending of CP with the vinyl polymer invited synergistic improvements in thermomechanical and optical properties of the respective constituent polymers. Additionally, it was found that the VP:MMA composition range corresponding to the miscibility window was expanded by modification of the CP component into cellulose acetate propionate.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer by the diazotrophic cyanobacterium Aulosira fertilissima CCC 444

Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), a well-known co-polymer of polyhydroxyalkanoates family, was investigated in a N₂-fixing cyanobacterium, Aulosira fertilissima CCC 444, in presence of propionate and valerate in the culture medium. The most significant rise in P(3HB-co-3HV) co-polymer content up to 77 % of dry cell weight was recorded under 0.5 % fructose?+?0.4 % valerate supplementation depicting a productivity of 38 mg L^?1 day^?1, which was further increased by 2.5-fold, i.e., up to 95 mg L^?1 day^?1 under P deficiency. Surface analysis revealed a regular and smooth surface for P(3HB-co-3HV) co-polymer, against rugged and porous surface of the homopolymer of poly-β-hydroxybutyrate. X-ray diffraction showed semi-crystalline nature of the P(3HB-co-3HV) co-polymer. The thermal and mechanical properties of the co-polymer are comparable with the chemoheterotrophic bacterial polymers, thus opens up possibilities of using cyanobacterial PHAs in various fields.     

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20 g/L of glucose as a sole carbon source, P(3HB-co-2.3 mol% 3HV) was produced up to the polymer content of 61.7 wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5 mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.     

Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate

Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, β-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, ¹H-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.     

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

     

Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli

In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers.     

Biosynthesis of diverse α,ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible plastics with potential to replace petroleum based plastics. Diversity of PHA monomer structures provides flexibility in material properties to suit more applications. In this study, 5-hydroxyvalerate (5HV) synthesis pathway was established based on intrinsic alcohol/aldehyde dehydrogenases. The PHA polymerase cloned from Cupriavidus necator functions to polymerize 5HV into its copolymers in ratios ranging from 8% to 32%. Elastic copolymer P(85% 3HB-co-15% 5HV) was generated with an elongation at break and a Young's modulus of 1283% and 73.1 MPa, respectively. The recombinant H. bluephagenesis was able to convert various diols including 1, 3-propanediol, 1, 4-butanediol and 1, 5-pentanediol into PHA, leading to 13 PHA polymers including transparent P(53% 3HB-co-20% 4HB-co-27% 5HV) and sticky P(3HB-co-3HP-co-4HB-co-5HV). The engineered H. bluephagenesis was successfully grown in a 7-L bioreactor to produce the highly elastic P(85% 3HB-co-15% 5HV) and the sticky P(3HB-co-3HP-co-4HB-co-5HV), demonstrating their potential for industrial scale-up.     

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.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli harboring propionyl-CoA synthase gene (prpE) or propionate permease gene (prpP)

In order to enhance 3-hydroxyvalerate (3HV) fraction in copolyesters of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the propionate permease gene prpP or the propionyl-CoA synthase gene prpE was transformed into Escherichia coli XL10-Gold with co-expression of PHB operon (phaCAB) from Ralstonia eutropha. The recombinant E. coli strains were cultured on mixed carbon sources composed of glucose and propionic acid to promote PHBV accumulation. It was shown that the over-expression of prpE suppressed 3HV incorporation into PHBV copolymer, which led to reduced 3HV fraction. In contrast, the over-expression of prpP improved the 3HV content from 5.6 to 14.3 mol%, followed by an increased PHBV accumulation up to 62 wt%. The results showed that the expression of prpP stimulated the uptake and utilization of propionic acid and increased the 3HV fraction in PHBV. However, the over-expression of prpE in E. coli did not affect 3HV content in PHBV. Surprisingly, co-expression of prpE and prpP did not lead to any 3HV formation. This study showed the possibility to change the PHBV composition without overdose of propionic acid which is expensive and toxic for the cells.     

Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) production from biodiesel by‐product and propionic acid by mutant strains of Pandoraea sp.

Pandoraea sp. MA03 wild type strain was subjected to UV mutation to obtain mutants unable to grow on propionic acid (PA) but still able to produce poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) [P(3HB‐co‐3HV)] from glycerol and PA at high 3HV yields. In shake flask experiments, mutant prp25 was selected from 52 mutants affected in the propionate metabolism exhibiting a conversion rate of PA into 3HV units of 0.78 g g^?1. The use of crude glycerol (CG) plus PA or valeric acid resulted in a copolymer with 3HV contents varying from 21.9 to 30 mol% and 22.2 to 36.7 mol%, respectively. Fed‐batch fermentations were performed using CG and PA and reached a 3HV yield of 1.16 g g^?1, which is 86% of the maximum theoretical yield. Nitrogen limitation was a key parameter for polymer accumulation reaching up to 63.7% content and 18.1 mol% of 3HV. Henceforth, mutant prp25 is revealed as an additional alternative to minimize costs and support the P(3HB‐co‐3HV) production from biodiesel by‐products. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1077–1084, 2017     

Quantification of cis and trans influences in [PtX(PPh3)3] complexes. A P NMR study

In [PtX(PPh₃)₃]⁺ complexes (X = F, Cl, Br, I, AcO, NO₃, NO₂, H, Me) the mutual cis and trans influences of the PPh₃ groups can be considered constants in the first place, therefore the one bond Pt-P coupling constants of P_(cis) and P_(trans) reflect the cis and trans influences of X. The compounds [PtBr(PPh₃)₃](BF₄) (2), [PtI(PPh₃)₃](BF₄) (3), [Pt(AcO)(PPh₃)₃](BF₄) (4), [Pt(NO₃)(PPh₃)₃](BF₄) (5), and the two isomers [Pt(NO₂-O)(PPh₃)₃](BF₄) (6a) and [Pt(NO₂-N)(PPh₃)₃](BF₄) (6b) have been newly synthesised and the crystal structures of 2 and 4·CH₂Cl₂·0.25C₃H₆O have been determined. From the ¹J_PtP values of all compounds we have deduced the series: I > Br > Cl > NO₃ > ONO > F > AcO > NO₂ > H > Me (cis influence) and Me > H > NO₂ > AcO > I > ONO > Br > Cl > F > NO₃ (trans influence). These sequences are like those obtained for the (neutral) cis- and trans-[PtClX(PPh₃)₂] derivatives, showing that there is no dependence on the charge of the complexes. On the contrary, the weights of both influences, relative to those of X = Cl, were found to depend on the charge and nature of the complex.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Exploiting metagenomic diversity for novel polyhydroxyalkanoate synthases: production of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) with a recombinant Pseudomonas putida strain

A metagenomic library of 2.1 × 10⁶ clones was constructed using oil-contaminated soil from Gujarat (India). One of the fosmid clones, 40N22, encodes a polyhydroxyalkanoate synthase showing 76% identity with an Alcaligenes sp. synthase. The corresponding gene was expressed in Pseudomonas putida KT2440 ΔphaC1 which is impaired in PHA production. The gene conferred the recombinant strain PpKT-40N22 with the ability to produce copolymers with up to 21% in medium-chain-length content. Thus, 37% and 45% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), respectively were obtained when using sodium heptanoate and oleic acid as carbon sources. These 3-hydroxybutyrate-(3HB)-based polymers are of interest since they incorporate the properties of medium chain length polymers and thus increase the range of applications of PHAs.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

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.     

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C₄ to C₆. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB-co-3HV).     

Triterpenoid saponins from a cytotoxic root extract of Sideroxylonfoetidissimum subsp. gaumeri

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylonfoetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D (¹H, ¹³C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, and the known compound, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3-O-(β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid and 3-O-(β-d-apiofuranosyl-(1 → 3)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC₅₀ = 11.9 ± 1.5 μg/ml) towards RAW 264.7 cells than the original extract (IC₅₀ = 39.5 ± 4.1 μg/ml), and the saponin-containing fraction derived from it (IC₅₀ = 33.7 ± 6.2 μg/ml).