ABSTRACT
The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here, we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modelling and optimisation methods. Our model reveals the organism's potential for industrial application and metabolic engineering. Modelling results imply that the aeration regime can be used as a tool to optimise product yield and flux distribution in K. marxianus. Also rebalancing NADH and NADPH utilisation can be used to improve the efficiency of substrate conversion. Xylose is identified as a biotechnologically promising substrate for K. marxianus.
Subject(s)
Industrial Microbiology , Kluyveromyces/metabolism , Acetates/metabolism , Biomass , Calibration , Culture Media/chemistry , Ethanol/metabolism , Glucose/chemistry , Glutamic Acid/metabolism , Glycerol/metabolism , Inulin/chemistry , Kluyveromyces/genetics , Lactates/metabolism , Lactose/chemistry , Metabolic Engineering , Models, Molecular , Oxygen Consumption , Phenylalanine/metabolism , Phenylethyl Alcohol/metabolism , Reproducibility of Results , Succinic Acid/metabolism , Xylose/chemistryABSTRACT
The ethanol and temperature effects on the ratio between Zymomonas mobilis 113S extracellular levansucrase activities were studied using fermentation broth supernatant, "levan-levansucrase" sediment precipitated by ethanol and highly purified enzyme. The fructooligosaccharide (FOS) production at different temperatures in the presence of ethanol was investigated. An ethanol increases FOS biosynthesis activity part of levansucrase. Especially, this effect was pronounced at lower temperatures (35-40 °C) and using purified levansucrase. The inverse relationship between temperature and ratio synthetic activity/total activity of levansucrase was found. The FOS composition containing mostly 1-kestose, 6-kestose, and neokestose obtained in the presence of different ethanol concentrations was found relative constant, while the changes in the sucrose concentration and temperature gave slight changes in the ratio between 1-kestose and 6-kestose.
Subject(s)
Ethanol/pharmacology , Hexosyltransferases/metabolism , Zymomonas/enzymology , Hydrogen-Ion Concentration , Oligosaccharides/analysis , Oligosaccharides/biosynthesis , TemperatureABSTRACT
The fructan biosynthesis by ethanol sedimented "levan-levansucrase" complex from Zymomonas mobilis fermentation broth as well as purified levansucrase was investigated. The fructooligosaccharide (FOS) producing activity of "levan-levamsucrase" sediment was investigated in 55% sucrose syrup at 45 degrees C. It was shown that FOS in the syrup were presented by 1-kestose, 6-kestose, neokestose and nystose. The increase of gluconic acid concentration was observed in the reaction mixture during the incubation suggesting about presence of glucose/fructose oxidoreductase in "levan-levansucrase" sediment. The influence of ethanol, glycerol and NaCl on levan and fructooligosaccharide formation by "levan-levansucrase" complex and purified levansucrase was studied and the changes in the ratio between different activities of levansucrase (sucrose hydrolysis, levan biosynthesis and FOS formation) were observed. Ethanol increases the FOS biosynthesis part in total activity of purified levansucrase. The technology of the production of prebiotics containing food product--fructan syrup by "levan-levansucrase " sediment as biocatalyst was developed.
Subject(s)
Hexosyltransferases/metabolism , Zymomonas/enzymology , Catalysis , Culture Media , Extracellular Space/enzymology , Fructans/metabolism , Hexosyltransferases/isolation & purification , Kinetics , Oxidation-Reduction , Substrate Specificity , Sucrose , Zymomonas/growth & developmentABSTRACT
A new and efficient method for the purification of levansucrase from cell-free extracts of a flocculant mutant of Zymomonas mobilis ATCC 10988 was developed. Levansucrase activity was almost completely recovered and purified by a factor of 15 after precipitation with 0.1 m MnCl2 as a first capturing step. The enzyme was homogeneously purified by ultrafiltration and anion-exchange chromatography and exhibited a levan-forming activity of 39.2 U mg-1. The native enzyme formed large aggregates with an apparent molecular mass of more than 106 Da as determined by size-exclusion chromatography, whereas denaturing SDS-PAGE indicated an apparent molecular mass of 50 kDa for the subunits.
Subject(s)
Hexosyltransferases/isolation & purification , Zymomonas/enzymology , Cell Membrane/enzymology , Chromatography/methods , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel/methods , Kinetics , Zymomonas/physiologyABSTRACT
The intensification of biosynthesis of fructooligosaccharides in the presence of high salt concentrations was observed during sucrose (10%) fermentation by Zymomonas mobilis 113S. A 0.6 M NaCl concentration led to an increase of oligosaccharide productivity by 3.5-fold. Sorbitol formation was increased in the presence of 0.16 M NaCl and was inhibited at highest salt concentrations. In a medium with high (65%, w/w) sucrose content the salts gave inhibitory effects on fructooligosaccharide production by lyophilised Z. mobilis cells. Influence of salts on gluconic acid and sorbitol formation under these conditions was studied. The ratio of oligosaccharides and gluconic acid productivity (Qolig./Qglucon.) was increased approximately 2 times at 1% KCl. Sorbitol formation was not significantly influenced in the presence of KCl (up to 2%).
