Fructan Biosynthesis by Intra- and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

Summary The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra- and extracellular levansucrase produced by the Z. mobilis strain 113 ”S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that th…

Formation of Levan from Raffinose by Levansucrase ofZymomonas mobilis

Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non-catabolised melibiose into the medium. The kinetic parameters of the levansucrase-catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (Mn = 1693 kDa), an increased intrinsic viscosity (η = 49.47 cm3/g), and a diminished Huggin's constant (K' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtaine…

Hydrophobicity of bacteria Zymomonas mobilis under varied environmental conditions

Abstract Changes in the cell surface hydrophobicity (CSH) of bacteria Zymomonas mobilis 113S were examined in response to varied environmental conditions (temperature and phase of growth, concentration or type of carbon source, the presence of amphiphilic compounds). The values of CSH were elevated with a decreased growth rate over the time of cultivation up to 20–22% at the stationary phase. CSH values increased proportionally with the growth of cultivation temperature and concentration of carbon source (glucose or sucrose) or amphiphilic compound (aliphatic alcohols, Tween80) in the medium. Replacement of sucrose by glucose and the presence of Tween20 in the growth medium resulted in redu…

Discriminative features of type I and type III secreted proteins from Gram-negative bacteria

AbstractThe amino acid composition of sequences and structural attributes (α-helices, β-sheets) of C-and N-terminal fragments (50 amino acids) were compared to annotated (SWISS-PROT/ TrEMBL) type I (20 sequences) and type III (22 sequences) secreted proteins of Gram-negative bacteria.The discriminant analysis together with the stepwise forward and backward selection of variables revealed the frequencies of the residues Arg, Glu, Gly, Ile, Met, Pro, Ser, Tyr, Val as a set of strong (1-P < 0.001) predictor variables to discriminate between the sequences of type I and type III secreted proteins with a cross-validated accuracy of 98.6–100 %. The internal and external validity of discriminant…

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…

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters inZymomonas culture

This study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700-1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield). In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose-6-P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) act…

FT-IR spectroscopic investigation of bacterial cell envelopes from Zymomonas mobilis which have different surface hydrophobicities

Abstract Changes in the cell envelope composition of bacteria Zymomonas mobilis 113S which resulted in varied cell surface hydrophobicity (CSH) were examined using FT-IR-spectroscopy and conventional methods of biochemical analysis. Significant differences in all analyses were detected between hydrophilic and hydrophobic compounds in the envelopes of Z. mobilis . A significant positive linear relationship ( P Z. mobilis and the ratio of absorbance band intensities assigned to the CH 2 and CH 3 vibrations from lipids as well as the asymmetric and symmetric stretching vibrations of these methylene and methyl groups. Differences were also seen between the ratio of band absorbances arising from…

Relationships between metabolic fluxes and enzyme amino acid composition

AbstractMetabolic fluxes are a key parameter of metabolic pathways being closely related to the kinetic properties of enzymes and could be conditional on their sequence characteristics. This study examines possible relationships between the metabolic fluxes and the amino acid (AA) composition (AAC) for enzymes from the yeast Saccharomyces cerevisiae glycolysis pathway. Metabolic fluxes were quantified by the COPASI tool using the kinetic models of Hynne and Teusink at 25 mM, 50 mM, and 100 mM of external glucose or employing literature data for cognate kinetic or stoichiometric models. The enzyme sequences were taken from the UniProtKB, and the AAC computed by the ExPASy/ProtParam tool. Mul…

Search for a Minimal Set of Parameters by Assessing the Total Optimization Potential for a Dynamic Model of a Biochemical Network.

Selecting an efficient small set of adjustable parameters to improve metabolic features of an organism is important for a reduction of implementation costs and risks of unpredicted side effects. In practice, to avoid the analysis of a huge combinatorial space for the possible sets of adjustable parameters, experience-, and intuition-based subsets of parameters are often chosen, possibly leaving some interesting counter-intuitive combinations of parameters unrevealed. The combinatorial scan of possible adjustable parameter combinations at the model optimization level is possible; however, the number of analyzed combinations is still limited. The total optimization potential (TOP) approach is…

Production of extracellular fructans by Gluconobacter nephelii P1464.

UNLABELLED Bacterial extracellular fructans, known as levans, have potential applications in food, pharmaceutical and cosmetic industries and high fructan producing strains could contribute into the cost reduction and more extensive commercial usage of them. An acetic acid bacteria (AAB) isolate P1464 was obtained from the Microbial Strain Collection of Institute of Microbiology and Biotechnology, University of Latvia and identified as Gluconobacter nephelii by DNA-DNA hybridization and the formation of extracellular fructans by this strain was confirmed. Isolated extracellular fructose polymers were characterized using FT-IR spectroscopy and the structural features of fructan appeared as s…

Distinctive attributes for predicted secondary structures at terminal sequences of non-classically secreted proteins from proteobacteria

Abstract C- and N-terminal sequences (64 amino acid residues each) of 89 non-classically secreted type I, type III and type IV proteins (Swiss-Prot/TrEMBL) from proteobacteria were transformed into predicted secondary structures. Multivariate analysis of variance (MANOVA) confirmed the significance of location (C- or N-termini) and secretion type as essential factors in respect of quantitative representations of structured (a-helices, b-strands) and unstructured (coils) elements. The profiles of secondary structures were transcripted using unequal property values for helices, strands and coils and corresponding numerical vectors (independent variables) were subjected to multiple discriminan…

The effect of amphiphilic compounds on the secretion of levansucrase by Zymomonas mobilis

Abstract The effect of some aliphatic (n-butanol to n-hexadecanol) and aromatic (benzyl and phenethyl alcohols), anesthetics (procaine) and surfactants (Tween 20 to Tween 80) on the secretion of levansucrase by the levan-producing strain of Gram-negative ethanologenic bacteria Zymomonas mobilis 113S were examined in this study. During incubation of Z. mobilis cells with sucrose (10 mM) a decrease of the levansucrase activity was observed in the presence of these amphiphilic compounds concomitantly with an increase of a total amount of protein in the medium. Since none of the compounds under study had any effect on enzyme activity in vitro observed structure- and concentration-dependent rela…

Relationship between Metabolic Fluxes and Sequence-Derived Properties of Enzymes

Metabolic fluxes are key parameters of metabolic pathways being closely related to the kinetic properties of enzymes, thereby could be dependent on. This study examines possible relationships between the metabolic fluxes and the physical-chemical/structural features of enzymes from the yeast Saccharomyces cerevisiae glycolysis pathway. Metabolic fluxes were quantified by the COPASI tool using the kinetic models of Hynne and Teusink at varied concentrations of external glucose. The enzyme sequences were taken from the UniProtKB and the average amino acid (AA) properties were computed using the set of Georgiev’s uncorrelated scales that satisfy the VARIMAX criterion and specific AA indices th…

Interrelationships between Growth Yield, ATPase and Adenylate Kinase Activities inZymomonas mobilis

The presence of cytoplasmic and membrane-bound adenylate kinase (EC 2.7,4.3) as well as inorganic pyrophosphatase (EC 3.6.1.1) was detected in Zymomonas mobilis ATCC 29191. An increase in the molar growth yield (Y X/S ) of Z. mobilis under aerobic growth conditions appeared to be in proportion to a reduction of membrane-bound adenylate kinase (mAK) and ATPase activities and to an increase in cytoplasmic adenylate kinase (AK) activity. Significant (1 - P < 0.01) multiple regressions were observed between the values of Y x (dependent variable), ATPase and AK or AK and mAK as independent variables, suggesting that a combined operation of these phosphohydrolases and phosphotransferases would be…

Molar growth yields ofZymomonas mobilis on glucose after the transition from anaerobic to aerobic continuous growth

An increase in the molar growth yield (Y X/S = 14.3-20.3 g/mol) on glucose (25 mM) was achieved after the transition of Zymomonas mobilis ATCC 29191 from anaerobic to aerobic steady state growth at dilution rates of D = 0.31-0.40 1/h and under oxygen-unlimited conditions. The transfer of anaerobically or aerobically grown steady state cells into a fresh medium resulted in the higher values of Y X/S . A positive correlation was established between biomass and acetaldehyde yield within the range of 5-9 mM acetaldehyde in the medium. An inhibitory effect of the exogenously added acetaldehyde (Kj = 16.7 ± 2.8 mM) on the ATPase activity was observed in vitro, using cell-free extracts of anaerobi…

Ethanol production improvement in Zymomonas mobilis by supplementation of fructan-cleaving enzymes on sucrose containing substrates

Distinctive amino acid residue periodicities in terminal sequences of type III and type I secreted proteins from proteobacteria

AbstractThe Fourier transform (FT) method was applied to specify the distribution of 14 predefined groups of amino acids (64 residues) at both termini of annotated type III and type I secreted proteins from proteobacteria. Type I proteins displayed a higher occurrence of significant periodicities at both C-and N-termini, indicating potent features to discriminate between secretion types, particularly by the use of variables selected from the full periodicity profiles at 19 orders of FT. The Fishers linear discriminant analysis, together with the stepwise selection of variables throughout equal pairs of combinations for all predefined groups of residues, revealed the C-terminal harmonics of …

An Influence of Fructan Containing Concentrate from Jerusalem Artichoke Tubers on the Development of Probiotic Dairy Starters on Milk and Oat-based Substrates

Supplementation of milk and oat hydrolysate containing medium with Jerusalem artichoke concentrate (JAC) and subsequent fermentation with probiotic dairy starters resulted in substantial stimulation of probiotics Bifidobacterium lactis and Lactobacillus acidophilus as well as yogurt starter culture Lactobacillus bulgaricus development and acidification rate. The strain-specific responses of the general yogurt cultures, as well as probiotics to the addition of JAC, should be considered to achieve optimal composition of probiotic strains and conformable fermentation conditions. JAC is suggested to be perspective prebiotic additive for fermented synbiotic milks or oat-hydrolysate-based product…

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 cell surface hydrophobicity and survival of the cells under adverse environmental conditions.

Abstract Changes in the cell surface hydrophobicity (CSH) of probiotic bacteria Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 and the survival of these cells were examined in response to varied cultivation conditions and adverse environmental conditions. An inverse linear relationship (P &amp;lt; 0.01) was detected between the CSH of intact L. acidophilus La5 and B. lactis Bb12 and survival of cells subjected to subsequent freezing/thawing, long-term storage or exposure to mineral and bile acids. The observed relationships were supported by significant correlations between the CSH and changes in composition of the cell envelopes (proteins, lipids and carbohydrates) of L. aci…

Relationships between kinetic constants and the amino acid composition of enzymes from the yeast Saccharomyces cerevisiae glycolysis pathway

The kinetic models of metabolic pathways represent a system of biochemical reactions in terms of metabolic fluxes and enzyme kinetics. Therefore, the apparent differences of metabolic fluxes might reflect distinctive kinetic characteristics, as well as sequence-dependent properties of the employed enzymes. This study aims to examine possible linkages between kinetic constants and the amino acid (AA) composition (AAC) for enzymes from the yeast Saccharomyces cerevisiae glycolytic pathway. The values of Michaelis-Menten constant (K M), turnover number (k cat), and specificity constant (k sp = k cat/K M) were taken from BRENDA (15, 17, and 16 values, respectively) and protein sequences of nine…

Development ofBifidobacterium lactis Bb 12 onβ-(2,6)-Linked Fructan-Containing Substrate

β-(2,1)-linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non-digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related (3-(2,6)-linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the prefe…

Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism

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 engi…