The current research is devoted to the investigation of the plasticization of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV) with triethyl citrate (TEC). Three different PHB or PHBV-based systems with 10, 20, and 30 wt.% of TEC were prepared by two-roll milling. The effect of TEC on the rheological, thermal, mechanical, and calorimetric properties of the developed compression-molded PHB and PHBV-based systems was determined. It was revealed that the addition of TEC significantly influenced the melting behavior of both polyhydroxyalkanoates (PHA), reducing their melting temperatures and decreasing viscosities. It was also revealed that all the investigated systems demonstrated less than 2% weight loss until 200 °C and rapid degradation did not occur until 240-260 °C in an oxidative environment. Apart from this, a remarkable increase (ca 2.5 times) in ultimate tensile deformation εB was observed by increasing the amount of TEC in either PHB or PHBV. A concomitant, considerable drop in ultimate strength σB and modulus of elasticity E was observed. Comparatively, the plasticization efficiency of TEC was greater in the case of PHBV.
Metabolically active cells produce a wide array of metabolites that can inhibit their growth. Acetate is a widely known preservative, and it is also produced by yeast cells during their growth. Kluyveromyces marxianus DSM 5422 is a promising yeast strain that could be employed in biotechnological processes, but the knowledge of its stress physiology is scarce. Here, we investigate the effects of acetate on growth and changes in cell population structure during adaptation to elevated concentrations of acetate in K. marxianus DSM 5422. Our results indicate that acetate inhibits growth in a pH-dependent manner and has pronounced effects if yeast is grown on lactose or galactose. When challenged with acetate, culture extends lag phase, during which cells adapt to elevated acetate concentrations, and growth reoccurs, albeit at a slower rate, when majority of the population is acetate resistant. Acetate resistance is maintained only if acetate is present in the media or if the culture has reached end of active growth phase. This study shows possible caveats in lactose fermentation with K. marxianus and gives a further perspective in non-conventional yeast applications in biotechnology.
Acetates/chemistry , Industrial Microbiology , Kluyveromyces/drug effects , Kluyveromyces/growth & development , Kluyveromyces/metabolism , Adaptation, Physiological/drug effects , Bioreactors , Culture Media/chemistry , Fermentation , Galactose/chemistry , Hydrogen-Ion Concentration , Lactose/chemistry
Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate D-xylose, D-fucose, L- and D-arabinose, D-ribose, D-sorbitol, xylitol, xylobiose, D-mannitol, D-galacturonic acid and methyl-α-D-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of D-sorbitol, xylobiose, D-galacturonic acid, D-mannitol, xylitol and methyl-α-D-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Fructose/metabolism , Hexosyltransferases/chemistry , Hexosyltransferases/metabolism , Pseudomonas/enzymology , Amino Acid Sequence , Bacterial Proteins/genetics , Chromatography, Thin Layer , Fructans/metabolism , Hexosyltransferases/genetics , Histidine , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides , Pseudomonas/genetics , Pseudomonas syringae/enzymology , Pseudomonas syringae/genetics , Raffinose/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Substrate Specificity/genetics , Sucrose/metabolism
A new and simple method for the purification of extracellular levansucrase from Zymomonas mobilis from highly viscous fermentation broth was developed. After incubation of the fermentation broth with a fructose-polymer cleaving enzyme preparation (Fructozyme, Novozymes, DK) for 48 h, levansucrase precipitated as aggregates and was redissolved in a 3 M urea solution. By ongoing size-exclusion chromatography on Sephacryl S-300 the final levansucrase preparation was purified 100-fold and exhibited a specific activity of 25-35 U/mg(protein). The levansucrase was stable in 3 M urea solution for at least four months without inactivation. To maximize the enzyme yield the dynamic changes of extracellular levansucrase activity during fermentation were investigated. The highest levansucrase activity was observed during the logarithmic phase of growth (15-19 h of fermentation).
Hexosyltransferases/isolation & purification , Zymomonas/enzymology , Centrifugation , Chemical Precipitation , Chromatography, Gel , Culture Media/metabolism , Electrophoresis, Polyacrylamide Gel , Fermentation , Fructans/metabolism , Hydrogen-Ion Concentration , Molecular Weight , Solubility , Sucrose/metabolism , Urea , Viscosity
