Production of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) from glucose and valerate in Alcaligenes eutrophus

Production of poly(-hydroxybulyrate-co--hydroxyvalcrate) [poly(HB-co-HV)] in Alcaligenes eutrophus NCIMB 11599 was investigated at various concentrations of valerate. With valerate, higher specific production rate, yield, and HV content of the copolymer were obtained than those with propionate. By feeding valerate in the copolymer accumulation stage, a product titre as high as 90.4 g/l with HV content of 20.4 % could be obtained in 50 hr of fed-batch operation.     

Production of poly(3-hydroxyalkanoates) from α, ω-alkanedioic acids and hydroxylated fatty acids by Alcaligenes sp.

Summary Poly(3-hydroxybutyrate) (P(3HB)) was produced from a series of , -alkanedioic acids of both even and odd carbon numbers by the Alcaligenes sp. AK201. In contrast, copolymers of 3HB and 3-hydroxyvalerate(P(3HB-co-3HV)) were produced from hydroxylated fatty acids of even carbon numbers such as 12-hydroxystearate and 2-hydroxyoctanoate. The biosynthetic pathways to poly(3-hydroxyalkanoates)(P(3HA)) are discussed.     

Production of poly(β-hydroxybutyrate-β-hydroxyvalerate) copolymer from sugars by Azotobacter salinestris

Azotobacter salinestris, a sodium-dependent, microaerophilic N₂-fixing soil bacterium, formed polyhydroxyalkanoate copolymers comprised of β-hydroxybutyric acid and 9–12 mol% β-hydroxyvaleric acid (HV) during growth on sugars. Increased HV content was achieved by feeding valeric acid to the culture growing on glucose, but propionic acid could be directed to HV formation only when it served as the sole C source. Polymer production in nitrogen-fixing cells was increased at higher aeration, provided that a complex organic nitrogen source was also present, but there was no HV in the polymer. HV production was increased to 28 mol% in nitrogen-fixing cells when aeration was lower and acetate was provided with glucose in the medium. Enzymes leading to the production of polyhydroxyalkanoate copolymers were found to be similar in A. salinestris and Azotobacter vinelandii, but A. vinelandii is unable to form HV from propionate or from sugars without valeric acid addition. A biochemical scheme is proposed for the production of HV in A. salinestris, whereby the glyoxylate bypass assimilates acetate to generate succinate, which may be converted into propionyl-CoA for HV synthesis. The results suggest that it may be possible to control the molar yield of HV formed from sugars by A. salinestris. Received: 21 January 1997 / Received revision: 7 April 1997 / Accepted: 13 April 1997     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

Polymers from fatty acids: poly(ω-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies

This Article describes the synthesis and physicomechanical properties of bioplastics prepared from methyl ω-hydroxytetradecanoic acid (Me-ω-OHC14), a new monomer available by a fermentation process using an engineered Candida tropicalis strain. Melt-condensation experiments were conducted using titanium tetraisopropoxide (Ti[OiPr](4)) as a catalyst in a two-stage polymerization (2 h at 200 °C under N(2), 4 h at 220 °C under 0.1 mmHg). Poly(ω-hydroxytetradecanoate), P(ω-OHC14), M(w), determined by SEC-MALLS, increased from 53K to 110K as the Ti(OiPr)(4) concentration increased from 50 to 300 ppm. By varying the polymerization conditions (catalyst concentration, reaction time, second-stage reaction temperature) a series of P(ω-OHC14) samples were prepared with M(w) values from 53K to 140K. The synthesized polyesters with M(w) ranging from 53K to 140K were subjected to characterization by DSC, TGA, DMTA, and tensile testing. Influences of P(ω-OHC14) molecular weight, melting point, and enthalpies of melting/crystallization on material tensile properties were explored. Cold-drawing tensile tests at room temperature for P(ω-OHC14) with M(w) 53K-78K showed a brittle-to-ductile transition. In contrast, P(ω-OHC14) with M(w) 53K undergoes brittle fracture. Increasing P(ω-OHC14) M(w) above 78K resulted in a strain-hardening phenomena and tough properties with elongation at break ~700% and true tensile strength of ~50 MPa. Comparisons between high density polyethylene and P(ω-OHC14) mechanical and thermal properties as a function of their respective molecular weights are discussed.     

Production of poly(β-hydroxybutyric acid) and exopolysaccharide by Azotobacter beijerinckii WDN-01

The physico-chemical factors influencing the production of poly(-hydroxybutyric acid) [PHB] and exopolysaccharide (EPS) by a yellow pigmented Azotobacter beijerinckii strain WDN-01 were investigated. Under N-free condition with excess carbon, PHB accumulation attained its maximum at the late exponential phase followed by a sharp decline while EPS production was more or less parallel with growth. Polymer synthesis, however, was carbon-source-specific, the highest yield of PHB (2.73 g/l) and EPS (1.5 g/l) was obtained with 3% (w/v) glucose and mannitol respectively. Organic N-sources enhanced PHB production significantly, but inorganic nitrogenous compounds were inhibitory to both PHB and EPS synthesis. At optimum K₂HPO₄ concentration, the polymer yield was attributed to biomass yield. Oxygen-limiting conditions, irrespective of carbon sources favoured production of PHB and EPS.     

Interaction of 9-O-(ω-amino) alkyl ether berberine analogs with poly(dT)·poly(dA)*poly(dT) triplex and poly(dA)·poly(dT) duplex: a comparative study

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson–Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.     

Purification and propertes of poly(β-d-mannuronate) lyase from Azotobacter chroococcum

A bacterium, Azotobacter chroococcum 4A1M, isolated from a soil sample, produced an alginate-decomposing enzyme in the culture broth. The enzyme was purified to an electrophoretically homogeneous state. The purified enzyme showed maximum activity at pH 6.0 and 60°C;it was stable up to 60°C at pH 6.0 and activated by Ca²⁺ and inhibited strongly by Hg²⁺. The molecular mass of the enzyme was estimated to be 23 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 24 kDa by gel filtration. Therefore, the enzyme was considered to be monomeric. The NH₂-terminal amino acid sequence was determined to be H₂N-Ala-Ser-Ile-Ala-Ile-Thr-Asn-Pro-Gly-Phe. The enzyme reacted only on the polymannuronate block of alginic acid, and two main reaction products were obtained when short-chain polymannuronate was used as a substrate. The degrees of polymerization of the two products were three and two respectively.     

Production of ethanol from molasses at 45 °C using Kluyveromyces marxianus IMB3 immobilized in calcium alginate gels and poly(vinyl alcohol) cryogel

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 has been immobilized in calcium alginate gel and poly(vinyl alcohol) cryogel (PVAC) beads. The immobilized preparations were used as biocatalyst in fed-batch reactor systems for prolonged periods. The substrate utilized in each case consisted of sugar cane molasses diluted to yield a sugar load of 140?g/l. During the first cycle the maximum ethanol concentration produced by the alginate system was 57?g/l, representing 80% of the maximum theoretical yield. In the system employing the PVAC-immobilized biocatalyst, ethanol production increased to a maximum of 52–53?g/l, representing 73% of the maximum theoretical yield. In both cases, maximum ethanol concentration was achieved within a 72-hour period. When each system was operated on a fed-batch basis for a prolonged period of time the average ethanol concentrations produced in the alginate- and the PVAC-immobilized systems were 21 and 45?g/l, respectively. The results suggest that the PVAC-based immobilization system may provide a more practical alternative to alginate for the production of ethanol by K. marxianus IMB3 in continuous or semi-continuous fermentation systems.     

Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima

Biopolymers such as polyhydroxyalkanoates (PHAs) are a class of secondary metabolites with promising importance in the field of environmental, agricultural, and biomedical sciences. To date, high-cost commercial production of PHAs is being carried out with heterotrophic bacterial species. In this study, a photoautotrophic N₂-fixing cyanobacterium, Aulosira fertilissima, has been identified as a potential source for the production of poly-β-hydroxybutyrate (PHB). An accumulation up to 66% dry cell weight (dcw) was recorded when the cyanobacterium was cultured in acetate (0.3%) + citrate (0.3%)-supplemented medium against 6% control. Aulosira culture supplemented with 0.5% citrate under P deficiency followed by 5?days of dark incubation also depicted a PHB accumulation of 51% (dcw). PHB content of A. fertilissima reached up to 77% (dcw) under P deficiency with 0.5% acetate supplementation. Optimization of process parameters by response surface methodology resulted into polymer accumulation up to 85% (dcw) at 0.26% citrate, 0.28% acetate, and 5.58?mg?L^?1 K₂HPO₄ for an incubation period of 5?days. In the A. fertilissima cultures pre-grown in fructose (1.0%)-supplemented BG 11 medium, when subjected to the optimized condition, the PHB pool boosted up to 1.59?g?L^?1, a value ～50-fold higher than the control. A. fertilissima is the first cyanobacterium where PHB accumulation reached up to 85% (dcw) by manipulating the nutrient status of the culture medium. The polymer extracted from A. fertilissima exhibited comparable material properties with the commercial polymer. As compared with heterotrophic bacteria, carbon requirement in A. fertilissima for PHB production is lower by one order magnitude; thus, low-cost PHB production can be envisaged.     

Lipase-catalyzed copolymerization of dialkyl carbonate with 1,4-butanediol and ω-pentadecalactone: synthesis of poly(ω-pentadecalactone-co-butylene-co-carbonate)

Candida antarctica lipase B (CALB) was successfully used to promote synthesis of aliphatic poly(carbonate-co-ester) copolymers from dialkyl carbonate, diol, and lactone monomers. The polymerization reactions were carried out in two stages: first-stage oligomerization under low vacuum, followed by second-stage polymerization under high vacuum. Therefore, copolymerization of ω-pentadecalactone (PDL), diethyl carbonate (DEC), and 1,4-butanediol (BD) yielded PDL-DEC-BD copolymers with a M(w) of whole product (nonfractionated) up to 33?000 and M(w)/M(n) between 1.2 and 2.3. Desirable reaction temperature for the copolymerization was found to be ～80 °C. The copolymer compositions, in the range from 10 to 80 mol % PDL unit content versus total (PDL + carbonate) units, were effectively controlled by adjusting the monomer feed ratio. Reprecipitation in chloroform/methanol mixture allowed isolation of the purified copolymers in up to 92% yield. (1)H and (13)C NMR analyses, including statistical analysis on repeat unit sequence distribution, were used to determine the polymer microstructures. The synthesized PDL-DEC-BD copolymers possessed near random structures with all possible combinations of PDL, carbonate, and butylene units via either ester or carbonate linkages in the polymer chains and are more appropriately named as poly(PDL-co-butylene-co-carbonate).     

Structures for the polynucleotide complexes poly(dA) · poly(dT) and poly(dT) · poly(dA) · poly(dT)

X-ray diffraction analyses of fibers of polydeoxyadenylic acid · polydeoxythymidylic acid show that this molecule exists as a 10-fold double-helix with axial rise per nucleotide $\text{h = 3.24}\text{to}\text{3.29}\text{A}\text{?}$. The structure is very similar to B-DNA ($\text{h = 3.37}\text{A}\text{?}$) in having C3-exo furanose rings and base-pairs positioned centrally on the helix axis, but distinctive enough to have two packing modes, neither of which has been observed for B-DNA. Although the triple-stranded poly(dT) · poly(dA) · poly(dT) also has a large value of h(3.26 Å), each of the chains is a 12-fold helix of the A-genus with C3-endo furanose rings and bases displaced several Angstrom units from the helix axis.     

Helix-coil equilibria of poly (rA) · poly (rU)

Absorbance melting curves of the double-stranded (rA) · (rU) helix, made with fractionated homopolynucleotides of matched length, have been obtained over a 15-fold range of [Na⁺] and 30° range of temperature. An excellent fit of the observed profiles was obtained with theoretical curves calculated on the basis of the simplest interpretation for the occurrence of particular equilibria [1–3]; the complete molecular partition function being evaluated by the power series method developed by Applequist [4–6]. The stability constant was evaluated from literature values for the calorimetric enthalpy. The loop closure exponent was best represented by 2.22 ± 0.04 for the mismatching loop mode of melting and 1.22 for the matching mode and was independent of [Na⁺] and temperature. Assuming the applicability of the nonintersecting random walk value of 1.9 ± 0.1, these results would suggest a slight bias toward matched loop formation during melting of homopolynucleotides that might be expected to form only mismatched loops. The value of the stacking parameter at 60°C was only ~6% higher than that at 30°C, 0.0221 (0.0184 for the matching case). Calculated melting curves indicate the occurrence of a fifth-order phase transition when the mean helix length is only ~13 base-pairs, or about one full turn of the helix.     

Interpretation of DNA vibration modes: I-The guanosine and cytidine residues involved in poly(dG-dC)·poly(dG-dC) and d(CG)3·d(CG)3

Abstract

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5′-dGMP and 2′-deoxycytidine molecules to be reproduced. The role of both C2′-endo and C3′-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (χ), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm^?1 and 785-500 cm^?1 regions, when the right → left-handed conformational transition occurs.     

Production of poly(β-hydroxybutyrate) by Comamonas testosteroni during growth on naphthalene

Comamonas testosteroni has been found to produce poly(-hydroxybutyrate) (PHB) during its growth on naphthalene. Fourier transform infrared spectroscopy (FTIR) and ¹³C nuclear magnetic resonance (NMR) analysis confirmed it as a homopolymer of 3-hydroxybutyrate. Oxygen and essential nutrient limitation other than carbon source play a major role in maximum PHB production. Nitrogen limitation was found to have a profound effect, with 0.2 g ammonium nitrate/l optimum for PHB production. Both aeration and iron were found to be essential for growth and PHB accumulation. Ferric chloride at 0.04 g/l concentration was found to be optimum for PHB accumulation. Phosphate source variation showed no significant effect. Using naphthalene as a sole carbon source in optimized Bushnell Haas medium, 85% of the dry cell mass was extracted as chloroform-soluble PHB.     

Oxidation of solid paraffin (C11−40) byPseudomonas aeruginosa MGP-1

Isolated from oil-contaminated soil,Pseudomonas aeruginosa strain MGP-1 was identified by 16S rDNA sequence analysis and phenotypic characterisation. This strain was highly competent in medium and in soils, grew and survived well under adverse conditions. MGP-1 did not produce the pathogenic factor pyocyanin and used paraffin (C_11?40) as sole carbon source. It degraded eicosane (C₂₀) more efficiently than other n-alkanes, removing 60% within 16 days. AnalkB gene and intermediate metabolites of mono-alcohol and lipid acid corresponding to the oxidised paraffin were detected in the cell-free extracts of MGP-1, indicating that it possessed a mono-terminal oxidation. This is the first report to demonstrate that aPseudomonas strain is able to degrade paraffin with more than 18 carbon atoms by mono-terminal oxidation. Considering all the results, MGP-1 could be designed as a soil-born, highly competent, long chain n-alkane degrading bacterium with low or perhaps without pathogenicity.     

Destabilization and stabilization of poly(A) · poly(U) structure by platinum(II) compounds

A methodology for analyzing the intramolecular structural order of the polynucleotide duplex poly(A) · poly(U) has been developed on the basis of molecular biophysics. The combination of circular dichroism spectroscopy and differential scanning calorimetry was shown to be an optimal approach. It ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) capable of stabilizing the structure and increasing the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral drug.     

Conformational transitions and hydration of poly d (A-T) · poly d (A-T) in fibers

Conformational transitions of poly d(A-T) · poly d (A-T) have been studied by fiber X-ray diffraction and measurement of fiber dimensions. Results obtained for the D-A-B and D-B transitions are presented and analyzed. For all these form transitions, cooperativity effects are observed for the variation of the rise per nucleotide versus the relative humidity. Detailed information about hydration of the polynucleotide during form transitions and the numbers of water molecules per nucleotide necessary to stabilize the different helical conformations are presented.Offprint requests to: S. Premilat