Continuous Production of Dextran from Immobilized Cells of Leuconostoc mesenteroides KIBGE HA1 Using Acrylamide as a Support.
ABSTRACT: The cells of L. mesenteroides KIBGE HA1 were immobilized for the production of dextran on acrylamide gel and gel concentration was optimized for maximum entrapment. Sucrose at substrate concentration of 10% produced high yield of dextran at 25°C with a percent conversion of 5.82 while at 35°C it was 3.5. However, increasing levels of sucrose diminished dextran yields. The free cells stopped producing dextran after 144 h, while immobilized cells continued to produce dextran even after 480 h. Molecular mass distribution of dextran from free cells indicate that it is identical to that of blue dextran while the molecular mass of dextran from immobilized cells is lower than that of free cells.
Project description:On the basis of high enzyme activity a newly isolated strain of L. mesenteroides CMG713 was selected for dextran production. For maximum yield of dextran, effects of various parameters such as pH, temperature, sucrose concentration and incubation period were studied. L. mesenteroides CMG713 produced maximum dextran after 20 hours of incubation at 30 masculineC with 15% sucrose at pH 7.0. The molecular mass distribution of dextran produced by this strain showed that its molecular mass was about 2.0 million Da. Dextran analysis by (13)C-NMR spectrometry showed no signals corresponding to any other linkages except alpha-(1-->6) glycosidic linkage in the main chain, which has not been reported before. Physico-chemical properties of this unique dextran were also studied. These optimised conditions could be used for the commercial production of this unique high molecular weight dextran, which have significant industrial perspectives.
Project description:Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3' end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K(m) for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.
Project description:Dextransucrase production by Leuconostoc mesenteroides NRRL B-512F in media containing carbon sources other than sucrose is reported for the first time. Dextransucrases were analyzed by gel electrophoresis and by an in situ activity assay. Their polymers and acceptor reaction products were also compared by (13)C nuclear magnetic resonance and high-performance liquid chromatography techniques, respectively. From these analyses, it was found that, independently of the carbon source, L. mesenteroides NRRL B-512F produced dextransucrases of the same size and product specificity. The 5' ends of dextransucrase mRNAs isolated from cells grown under different culture conditions were identical. Based on this evidence, we conclude that dextransucrases obtained from cells grown on the various carbon sources result from the transcription of the same gene. The control of expression occurs at this level. The low dextransucrase yields from cultures in D-glucose or D-fructose and the enhancement of dextransucrase gene transcription in the presence of sucrose suggest that an activating phenomenon may be involved in the expression mechanism. Dextransucrase mRNA has a size of approximately 4.8 kb, indicating that the gene is located in a monocistronic operon. The transcription start point was localized 34 bp upstream from the ATG start codon. The -10 and -35 sequences found, TATAAT and TTTACA, were highly homologous to the only glycosyltransferase promoter sequence reported for lactic acid bacteria.
Project description:Leuconostoc mesenteroides DRP105 isolated from Chinese sauerkraut juice is an intensive producer of dextran. We report the complete genome sequence of Leu. mesenteroides DRP105. This strain contains a dextransucrase gene (dsr) involved in the production of dextran, possibly composed of glucose monomers. To explore the dextran synthesis mechanism of Leu. mesenteroides DRP105, we constructed a dsr-deficient strain derived from Leu. mesenteroides DRP105 using the Cre-loxP recombination system. The secondary structure prediction results showed that Leu. mesenteroides DRP105 dextransucrase (Dsr) was coded by dsr and contained 17.07% ?-helices, 29.55% ?-sheets, 10.18% ?-turns, and 43.20% random coils. We also analyzed the dextran yield, monosaccharide change, organic acid, and amino-acid content of Leu. mesenteroides DRP105 and Leu. mesenteroides DRP105-?dsr. The result showed that the lack of dsr changed the Leu. mesenteroides DRP105 sugar metabolism pathway, which in turn affected the production of metabolites.
Project description:During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8?h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.
Project description:1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.
Project description:Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme-substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in Vmax of immobilized enzyme from 8411.0 to 4919.0 U ml-1 min-1 whereas, Km apparently increased from 1.71 to 3.17 mM ml-1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.
Project description:Leuconostoc mesenteroides is a member of lactic acid bacteria (LAB) with wide applications in the food and medical industries. Species in the genus Leuconostoc are catalase-negative and generally regarded as facultative anaerobic or aerotolerant organisms. Despite their extensive use in industry, certain issues concerning the aerobic life of L. mesenteroides, e.g., the mechanism involved in the tolerance to oxygen, remain to be addressed. In this manuscript, a survival strategy employed by L. mesenteroides BD3749 in response to oxidative stress was elucidated. BD3749 cells cultivated in medium with sucrose available synthesized large amounts of exopolysaccharides, mostly consisting of insoluble EPS. When BD3749 cells were challenged with oxidative stress, the amount of insoluble EPS was greatly enhanced. The synthesized EPSs reduced the accumulation of reactive oxygen species (ROS) in bacterial cells and improved their survival during chronic oxidative stress. Another study showed that Gsy, a novel glucansucrase in the GH70 family that is induced by sucrose and up-regulated following exposure to oxygen, was responsible for the synthesis of insoluble EPS. Gsy was subsequently demonstrated to play pivotal roles in the formation of aggregates to alleviate the detrimental effects on BD3749 cells exerted by oxygen.
Project description:Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.
Project description:Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter P dsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the P dsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the P dsrLL -mrfp fusion or the P dsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from P dsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from P dsrLL . A different behavior was detected, when expression from P dsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.