Armands Vigants

Fructooligosaccharide and levan producing activity of Zymomonas mobilis extracellular levansucrase.

Abstract The present work was devoted to investigations of the fructooligosaccharide (FOS) and levan forming activity of ethanol producing bacteria Zymomonas mobilis and their extracellular levansucrase. After cell separation the culture liquid was treated with ethanol to precipitate levan together with extracellular levansucrase. Levan–levansucrase sediment can be used as biocatalyst for fructooligosaccharide (FOS) production in sucrose syrup and levan sediment as soluble fibre source. The dynamics of sucrose conversion and glucose, fructose, and FOS formation by extracellular levansucrase showed that the fructose content increased only during the first 6 h, while the glucose content continued to increase during all 24 h of incubation. The glucose content exceeded the fructose approximately four times. 1-Kestose, 6-kestose, neokestose and nystose, as well as other non identified fructooligosaccharides, was found in fructan syrup. Native or lyophilized Z. mobilis biomass added to levan-levansucrase sediment showed small changes in the activity of the biocatalyst. Z. mobilis biomass sedimented by ethanol together with levan and used as biocatalyst together with levansucrase did not increase the FOS forming activity of the biocatalyst . The presence of ethanol (7.0%) in sucrose syrup decreased the enzyme FOS forming activity on 24% during the first 24 h of incubation. Fructan syrup obtained from sucrose syrup by levan–levansucrase sediment as biocatalyst had a satisfactory taste, reduced energetic value and can be used as source of prebiotics—fructooligosaccharides and soluble fibre—levan as cholesterol lowering factor.

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation.

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.

OATS AND FAT-FREE MILK BASED FUNCTIONAL FOOD PRODUCT

The present study addresses problems and solutions related to new functional bio-active food product creation. Commercial rolled oats and fat-free milk were used as raw materials. Limited enzymatic hydrolysis of oat starch was carried out by α-amylase. The lactic acid bacteria strains Lactobacillus acidophilus, Bifidobacterium sp., and commercial starter cultures ABT-1 were cultivated in enzymatically hydrolysed oat mash. Lactobacillus acidophilus was a more active producer of lactic acid, while Bifidobacterium sp. improved the taste properties. A fat-free milk additive was applied to stabilise the texture of the oat mash, and increase the protein content of the final product. All procedures ensured the preservation of beta-glucan, and the number of active lactic acid bacteria in the final product reached 109 per ml. A yogurt-like oat based product “Oat bio Lacto” supports oat beta-glucan, oat lipid unsaturated fatty acids and living probiotic lactic acid bacteria, and can be considered as polyfunctional nutrition.

The effect of osmo-induced stress on product formation by Zymomonas mobilis on sucrose

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%).

Response of Zymomonas mobilis levansucrase activity to sodium chloride

An activation of levansucrase-catalysed levan formation by NaCl, KCl and Na2 SO4 (0.03–0.7 M) was observed using cell-free extract of Zymomonas mobilis. A sigmoidal response of the rate of levansucrase-catalysed reaction to the sucrose concentration was significantly reduced in the presence of salts the Hill coefficient 2.10 and 1.0–1.2 respectively), possibly, due to the heterotropic activation of levansucrase as an allosteric enzyme.

A Simple and Efficient Method for the Purification of Membrane-Bound Levansucrase from Zymomonas mobilis

A new and efficient method for the purification of levansucrase from cell-free extracts of a flocculant mutant of Zymomonas mobilisATCC 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.

The effect of mixing on glucose fermentation by Zymomonas mobilis continuous culture

Abstract The influence of mixing (in the range from 300 to 1100 rpm) on the performance of Zymomonas mobilis anaerobic continuous culture was studied. Biomass yield and ethanol productivity were improved at higher stirring intensities, in parallel with a decrease in byproduct synthesis. A mixing-dependent formation of minor amounts of fructose was first demonstrated in Z. mobilis anaerobic culture on glucose. The rate of fructose synthesis and the specific activity of the key enzyme in the pathway to fructose, phosphoglucose isomerase (EC 5.3.1.9), had a maximum at 700 rpm.

A Novel and Simple Method for the Purification of Extracellular Levansucrase from Zymomonas mobilis

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/mgprotein. 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).

Sugar beet diffusion juice and syrup as media for ethanol and levan production by Zymomonas mobilis

Abstract Sugar beet is an important crop cultivated widely in Europe. Roots contain up to 6–9 t/ha of fermentable sugar. Our laboratory is targeting to investigate the production possibilities of ethanol and levan by Zymomonas mobilis bacteria, using sugar beet juice or their syrup as raw material. The Z.mobilis strain 113 “S”;, selected in our institute, produces simultaneously with ethanol up to 40–70 g/1 fructose polymer levan in sucrose medium. It was established that Z.mobilis can convert sucrose into ethanol and levan in batch fermentation using natural sugar beet diffusion juice, without additives of mineral salts and growth factors. Syrup obtained in sugar industrial production by conventional technology from diffusion juice after evaporation with 65% sucrose can be used also as raw material for medium preparation. Additives of N, P, K, Mg, as well as yeast extract, were shown to stimulate the culture growth and product synthesis on such media. Reuse of liquid fermentation products after separatio...

Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism

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.