Mutational analysis and characterization of dextran synthesizing enzyme from wild and mutant strain of Leuconostoc mesenteroides.

Dextransucrase producing Leuconostoc mesenteroides KIBGE IB-22 was subjected to mutagenesis by exposing the strain to UV irradiation. The dextransucrase produced by both the strains (wild and mutant) were characterized and the catalytic properties of both wild and mutant dextransucrase were compared. Among 42 mutants, KIBGE IB-22M20 exhibited 6.75 times increase in dextransucrase activity as compared to the wild one. Wild dextransucrase showed specific activity of 31.3 DSU/mg of protein with V(max) and K(m) of 18.84 DSU/ml/h and 77.09 mM, respectively at 30 °C in 0.3 M citrate buffer (pH 4.5) using sucrose as substrate. Whereas, mutant dextransucrase exhibited a specific activity of 173.2 DSU/mg with V(max) and K(m) values of 104.2DSU/ml/h and 101.7 mM, respectively at 35 °C in 0.3 M citrate buffer (pH 5.0) keeping the same substrate as for wild. Dextransucrase from both wild and mutant showed an approximate molecular weight of 221 kDa by SDS-PAGE.

Heterologous expression and secretion of Lactobacillus amylovorus alpha-amylase in Leuconostoc citreum.

To develop a gene expression system for Leuconostoc genus, construction of expression vector and expression of a heterologus protein in Leuconostoc was performed. Alpha-amylase gene from Lactobacillus amylovorus was cloned into a Leuconostoc cloning vector, pLeuCM, with its own signal peptide. pLeuCMamy was introduced into Leuconostoc citreum CB2567 and a successful expression of alpha-amy gene was confirmed by enzyme activity assays. About 90% of alpha-amylase activity was detected in the culture broth, revealing most of expressed alpha-amylase was secreted out cells. The signal sequence of alpha-amy gene is a good candidate for the secretion of heterologous protein by using Leuconostoc host-vector system.

Enzymatic polymerization of natural anacardic acid and antibiofouling effects of polyanacardic acid coatings.

Anacardic acid, separated from cashew nut shell liquid, is well known for its strong antibiotic and antioxidant activities. Recent findings indicate that phenolic compounds from plant sources have an effect on Gram-negative bacteria biofilm formation. In this work, a polyphenolic coating was prepared from anacardic acid using enzymatic synthesis and tested for its effects on biofilm formation of both Gram-negative and Gram-positive bacteria. Natural anacardic acid was enzymatically polymerized using soybean peroxidase. Hydrogen peroxide and phenothiazine-10-propionic acid were used as an oxidizing agent and redox mediator, respectively. Nuclear magnetic resonance and Fourier transform infrared (FTIR) analyses showed the formation of oxyphenylene and phenylene units through the phenol rings. No linkage through the alkyl chain was observed, which proved a high chemo-selectivity of the enzyme. Aqueous solvents turned out to play an important role in the polymer production yield and molecular weight. With 2-propanol, the highest production yield (61%) of polymer (molecular weight = 3,900) was observed, and with methanol, higher-molecular-weight polymers (5,000) were produced with lower production yields (43%). The resulting polyanacardic acid was cross-linked on a solid surface to form a permanent natural polymer coating. The FTIR analysis indicates that the cross-linking between the polymers took place through the unsaturated alkyl side chains. The polyanacardic acid coating was then tested for its antibiofouling effect against Gram-negative and Gram-positive bacteria and compared with the antibiofouling effects of polycardanol coatings reported in the literature. The polyanacardic acid coating showed more reduction in biofilm formation on its surface than polycardanol coatings in the case of Gram-positive bacteria, while in the case of Gram-negative bacteria, it showed a similar reduction in biofilm formation as polycardanol.

Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov.

A taxonomic study was made of the genus Leuconostoc. The species in the genus were divided into three subclusters by phylogenetic analysis based on the 16S rRNA gene sequences. The three subclusters were the Leuconostoc mesenteroides subcluster (comprising L. carnosum, L. citreum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis, L. mesenteroides and L. pseudomesenteroides), the L. fructosum subcluster (L. durionis, L. ficulneum, L. fructosum and L. pseudoficulneum) and the L. fallax subcluster (L. fallax). Phylogenetic trees based on the sequences of the 16S-23S rRNA gene intergenic spacer region, the rpoC gene or the recA gene indicated a good correlation with the phylogenetic tree based on 16S rRNA gene sequences. The species in the L. fructosum subcluster were morphologically distinguishable from the species in the L. mesenteroides subcluster and L. fallax as species in the L. fructosum subcluster had rod-shaped cells. In addition, the four species in the L. fructosum subcluster needed an electron acceptor for the dissimilation of d-glucose and produced acetic acid from d-glucose rather than ethanol. On the basis of evidence presented in this study, it is proposed that the four species in the L. fructosum subcluster, Leuconostoc durionis, Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc pseudoficulneum, should be transferred to a novel genus, Fructobacillus gen. nov., as Fructobacillus durionis comb. nov. (type strain D-24(T)=LMG 22556(T)=CCUG 49949(T)), Fructobacillus ficulneus comb. nov. (type strain FS-1(T)=DSM 13613(T)=JCM 12225(T)), Fructobacillus fructosus comb. nov. (type strain IFO 3516(T)=DSM 20349(T)=JCM 1119(T)=NRIC 1058(T)) and Fructobacillus pseudoficulneus comb. nov. (type strain LC-51(T)=DSM 15468(T)=CECT 5759(T)). The type species of the genus Fructobacillus is Fructobacillus fructosus gen. nov., comb. nov.. No significant physiological and biochemical differences were found between the species in the L. mesenteroides subcluster and L. fallax in the present study and thus L. fallax remains as a member of the genus Leuconostoc.

[Construction and culture conditions of dextransucrase-secreting engineered strain].

OBJECTIVE: Dextransucrase was a glucosyltransferases catalyzing the transfer of D-glucopyranosyl units from sucrose to synthesize alpha-glucans or oligosaccharides. METHODS: dexYG gene (GenBank Accession No. DQ345760), encoding the dextransucrase from Leuconstoc mesenteriodes 0326, was subcloned into expression plasmid pET28a(+). The recombinant plasmid was then transformed into E. coli BL21 (DE3). Kanamycin resistant transformants were selected and verified by restriction endonuclease assay. RESULTS: Dextransucrase could be efficiently expressed in engineered strain BL21 (DE3)/pET28-dexYG by Isopropyl beta-D-thiogalactopyranoside (IPTG) induction, although the growth of E. coli host was inhibited during induction. Recombinant enzyme producing conditions such as induction time, IPTG concentration, incubation temperature, cell density (OD(600)) and pH value were studied. The optimum conditions for producing dextransucrase were as follows: incubation at 25 degrees C, 0.5 mmol/L Isopropyl beta-D-thiogalactopyranoside (IPTG) induction at cell density (OD(600)) of 1.0 for 5h, pH 6.0. Under these conditions, the recombinant dextransucrase activity was increased from 5.39U/mL to 35.62 U/mL. The highest activity under the optimal culture conditions after 5h induction in medium with pH 6.0 was 3.5 times as that of in Luria-Bertani medium without pH-adjustment. Moreover, the pH value was one of the main reasons that caused the degradation of enzyme in the later stage of induction. CONCLUSION: These results showed that dextransucrase could be efficiently heterologous expressed in E. coli and a strong dextransucrase activity had been detected.

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine.

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.

Functional role of the additional domains in inulosucrase (IslA) from Leuconostoc citreum CW28.

BACKGROUND: Inulosucrase (IslA) from Leuconostoc citreum CW28 belongs to a new subfamily of multidomain fructosyltransferases (FTFs), containing additional domains from glucosyltransferases. It is not known what the function of the additional domains in this subfamily is. RESULTS: Through construction of truncated versions we demonstrate that the acquired regions are involved in anchoring IslA to the cell wall; they also confer stability to the enzyme, generating a larger structure that affects its kinetic properties and reaction specificity, particularly the hydrolysis and transglycosylase ratio. The accessibility of larger molecules such as EDTA to the catalytic domain (where a Ca2+ binding site is located) is also affected as demonstrated by the requirement of 100 times higher EDTA concentrations to inactivate IslA with respect to the smallest truncated form. CONCLUSION: The C-terminal domain may have been acquired to anchor inulosucrase to the cell surface. Furthermore, the acquired domains in IslA interact with the catalytic core resulting in a new conformation that renders the enzyme more stable and switch the specificity from a hydrolytic to a transglycosylase mechanism. Based on these results, chimeric constructions may become a strategy to stabilize and modulate biocatalysts based on FTF activity.

Leucocins 4010 from Leuconostoc carnosum cause a matrix related decrease in intracellular pH of Listeria monocytogenes.

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L. monocytogenes was exposed to the bacteriocins leucocins 4010 and nisin either in a liquid filled chamber or on the surface of an agar containing bacteriocins. Both bacteriocins caused dissipation of the pH gradient in L. monocytogenes and the effect was clearly dependent on the matrix, as the decrease in pHi occurred much more rapidly in liquid than in agar.

Effect of phosphate concentration on the production of dextransucrase by Leuconostoc mesenteroides NRRL B512F.

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K(2)HPO(4). In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.

Production of insoluble dextran using cell-bound dextransucrase of Leuconostoc mesenteroides NRRL B-523.

Water-insoluble, cell-free dextran biosynthesis from Leuconostoc mesenteroides NRRL B-523 has been examined. Cell-bound dextransucrase is used to produce cell-free dextran in a sucrose-rich acetate buffer medium. A comparison between the soluble and insoluble dextrans is made for various sucrose concentrations, and 15% sucrose gave the highest amount of cell-free dextran for a given time. L. mesenteroides B-523 produces more insoluble dextran than soluble dextran. The near cell-free synthesis was validated in a batch reactor, by monitoring the cell growth which is a small (10(6)-10(7) CFU/mL) and constant value throughout the synthesis.

High-level production of D-mannitol with membrane cell-recycle bioreactor.

Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2 g l(-1) h(-1)) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l(-1) resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters.

Biomass plug development and propagation in porous media.

Exopolymer-producing bacteria can be used to modify soil profiles for enhanced oil recovery or bioremediation. Understanding the mechanisms associated with biomass plug development and propagation is needed for successful application of this technology. These mechanisms were determined from packed-bed and micromodel experiments that simulate plugging in porous media. Leuconostoc mesenteroides was used, because production of dextran, a water-insoluble exopolymer, can be controlled by using different carbon sources. As dextran was produced, the pressure drop across the porous media increased and began to oscillate. Three pressure phases were identified under exopolymer-producing conditions: the exopolymer-induction phase, the plugging phase, and the plug-propagation phase. The exopolymer-induction phase extended from the time that exopolymer-producing conditions were induced until there was a measurable increase in pressure drop across the porous media. The plugging phase extended from the first increase in pressure drop until a maximum pressure drop was reached. Changes in pressure drop in these two phases were directly related to biomass distribution. Specifically, flow channels within the porous media filled with biomass creating a plugged region where convective flow occurred only in water channels within the biofilm. These water channels were more restrictive to flow causing the pressure drop to increase. At a maximum pressure drop across the porous media, the biomass yielded much like a Bingham plastic, and a flow channel was formed. This behavior marked the onset of the plug-propagation phase which was characterized by sequential development and breakthrough of biomass plugs. This development and breakthrough propagated the biomass plug in the direction of nutrient flow. The dominant mechanism associated with all three phases of plugging in porous media was exopolymer production; yield stress is an additional mechanism in the plug-propagation phase.

The effects of exopolymers on cell morphology and culturability of Leuconostoc mesenteroides during starvation.

Biofilm formation by bacterial cells can be used to modify the subsurface permeability for the purpose of microbial enhanced oil recovery, bio-barrier formation, and in situ bioremediation. Once injected into the subsurface, the bacteria undergo starvation due to a decrease in nutrient supply and diffusion limitations in biofilms. To help understand the starvation response of bacteria in biofilms, the relationship between exopolymer formation and cell culturability was examined in a batch culture. The average cell diameter was observed to decrease from 0.8 micron to 0.35 micron 3 days after starvation began. Cell chain fragmentation was also observed during starvation. Cells that underwent starvation in the presence of insoluble exopolymers showed a slower rate of decrease in cell diameter and in cell chain length than cells without insoluble exopolymers. The rate of decrease in the average cell diameter and cell chain length were determined using a first order decay model. Cells starved in the presence of exopolymers showed greater culturability than cells starved without exopolymers. After 200 days starvation, 2.5 x 10(-3)% cells were culturable, but no increase in cell number was observed. During starvation, the exopolymer concentration remained constant, an indication that the exopolymer was not consumed by the starving bacteria as an alternative carbon or energy source.

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice.

A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos. This is the first report of L. oenos as a spoilage organism in fruit products other than wine.

Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus.

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.