M. Bekers

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.

Infrared spectra of some fructans

The FT–IR spectra of fructan – inulin (RAFTILINE), widely applied in the food industry and crystalline fructose as the main component of fructans, were studied. Special interest was to study the spectra of the levan precipitate and fructan syrup – produced by Zymomonas mobilis during the fermentation on sucrose–based medium.

Stabilization of anti-leukemic enzyme l-asparaginase by immobilization on polysaccharide levan

Abstract Biologically active fructose polymer levan from Zymomonas mobilis of different molecular mass (75 and 2000 kDa) was covalently coupled to anti-leukemic enzyme Erwinia carotovora l -asparaginase. The method used for the immobilization of the enzyme involved periodate oxidation of the polysaccharide, followed by reductive alkylation. A gentle periodate oxidation of levan (oxidation degree ≤24%) resulted in the highest residual enzyme activity (≥55%). The K m(app.) of glycoconjugates was higher than the K m of native l -asparaginase. The conjugation of l -asparaginase widened the optimum pH range of the enzyme. The electrophoretic mobility in polyacrylamide gel of glycoconjugates obtained was considerably reduced in comparison with native l -asparaginase. Immobilized l -asparaginase showed significantly higher stability in conditions of increased temperature (40°C and 50°C) and prolonged storage (1 month) in aqueous solutions as compared to the native enzyme. The results are discussed in relation to possible explanations of levan-induced enzyme stabilization, as well as to possible applications of immobilized l -asparaginase research.

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%).

Levan-ethanol biosynthesis using Zymomonas mobilis cells immobilized by attachment and entrapment

Fermentation of sucrose by Zymomonas mobilis cells attached to stainless steel wire spheres (WS) and to Al2O3 granules was compared with sucrose fermentation by cells entrapped in Ca-alginate. Similar amounts of cell biomass were applied at the beginning of the immobilized fermentation systems. The immobilization of Z. mobilis cells to the carrier surface was checked by scanning electron microscopy (SEM). Most cells were present in holes and pores of Al2O3 surfaces following colonization. Observation of the carrier after repeated fermentation cycles showed that a surface of Al2O3 granules was partly covered by levan. Alginate beads were extended in volume and partly disrupted. Changing the medium every 48 h established that free Z. mobilis cells in the culture liquid exceeded many times of cells attached to the carrier or incorporated in it. Ethanol and levan production did not differ significantly in these fermentation systems — maximal ethanol concentration at the end of second and third fermentation cycles reached 2.3–2.7% and levan 1.3–1.5%. Fermentation resulted in destruction of Ca-alginate beads. The conclusion was reached, that WS and Al2O3 with immobilized bacteria can be used as an inoculum generator for sucrose fermentation but that the general producer of levan and ethanol is the free suspended cell biomass.

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.

Carbohydrates in Jerusalem artichoke powder suspension

Purpose – The aim of this study was to evaluate the influence of temperature and action time on the extraction rate of carbohydrates of Jerusalem artichoke concentrate powder and inactivation of inulin during boiling and sterilization.Design/methodology/approach – Water suspension of Jerusalem artichoke concentrate (5g/100ml) at 25, 50 and 100○C was tested after 5, 15, 30 and 60min to determine the content of inulin, glucose, fructose and sucrose and evaluate the extraction rate. The stability of inulin was studied after boiling and sterilization at 120○C during 1, 2 and 3h. The extraction rate was evaluated by Fourier‐Transform Infrared (FT‐IR) spectroscopy as well.Findings – It was shown that extraction of soluble carbohydrates – fructose, glucose, sucrose and inulin, from water suspension of Jerusalem artichoke concentrate was practically completed in 5min at 25○C. The extraction rate was not significantly influenced by temperatures lower than 100○C. Inulin was stable during boiling for 1h but steriliz...

Attachment of yeast to modified stainless steel wire spheres, growth of cells and ethanol production

Abstract The immobilization of yeast Saccharomyces cerevisiae, their growth and ethanol production were investigated using untreated and modified stainless steel wire spheres (WS) as carriers. The carrier surface was modified by oxidation, by treatment with titanium (IV) chloride (TiCl4) or by γ-aminopropyltrietoxysilane (AS) in an attempt to raise the efficiency of the immobilization of the yeast cells. The influence of the cell fixation method on culture growth and ethanol synthesis was investigated. The immobilization of cells to carrier surface was checked by scanning electron microscopy (SEM). More closely attachment of yeast cells was seen on the aminated wire surface. It was established that during fermentation ethanol production by yeast was stimulated using immobilized cells in oxidized WS or treated with TiCl4. Aminated WS surface stimulated the culture growth but decreased ethanol synthesis. Free yeast cells located in the pores of WS increased the biomass concentration and ethanol production only during the first cycle of batch fermentation. Stable cell growth and ethanol production was observed during subsequent 4–5 repeated fermentation cycles using washing out of free cells from WS before fermentation. The system productivity Qeth for ethanol synthesis was 0.92–1.25 g/l per h. Cell fixation in WS by lyophilization or convective dehydration improved cell attachment to wire surface but did not influence positively culture growth and ethanol synthesis. The conclusion was made that stainless steel WS filled with paste-like yeast biomass can be used as inoculum for repeated batch ethanol production. The modification method of the stainless steel wire surface significantly influences the immobilization efficiency of yeasts. Oxidized or modified by TiCl4 wire surface and washing out free cells from WS can be recommended for ethanol production by immobilized yeasts.

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).

Fermentation of Jerusalem artichoke by Zymomonas and Saccharomyces

Purpose – The aim of this study is to find out the appropriate fermentation conditions of Jerusalem artichoke powder (JAP) based media to obtain light beverage containing inulin.Design/methodology/approach – JAP water suspension or filtrate were used for preparation of growth media with or without enzymatic hydrolysis of inulin for fermentation by Zymomonas mobilis 113 “S” or Saccharomyces cerevisiae.Findings – If enzymatic hydrolysis of inulin was not used significantly higher amount of inulin (7.42 per cent) was unconverted by Z. mobilis than by S. cerevisiae (2.22 per cent) while the ethanol concentration was much higher (2.86 per cent) after S. cerevisiae fermentation than after Z. mobilis fermentation (1.21 per cent). Considerably more ethanol was produced by Z. mobilis during co‐fermentation with Fructozyme L of JAP suspension filtrate (5.98 per cent) and suspension (4.96 per cent). Analyses of volatile components of fermentation broths showed that for production of inulin containing light beverages...