Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association. Received 06 October 1998/ Accepted in revised form 05 February 1999     

Immunostimulatory effect on dendritic cells of the adjuvant-active exopolysaccharide from Leuconostoc mesenteroides strain NTM048

This study evaluated the immunostimulative effect on bone marrow-derived dendritic cells (DCs) of adjuvant-active exopolysaccharide (EPS) produced by Leuconostoc mesenteroides strain NTM048. EPS stimulation increased IL-6, IL-10, IL-12, and retinal dehydrogenase (RALDH) gene expression levels and induced retinoic acid-synthesizing RALDH-active DCs, which play a crucially important role in controlling adaptive immune responses in mucosa.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.     

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut

Abstract

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett–Burman (PB) experiment, sucrose, Ca²⁺, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87?g/L, Ca²⁺ 0.21?mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94?±?0.43?U/mL in 24?hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170?kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56?U/mg protein.     

Purification and characterization of a bacteriocin from an oenological strain of Leuconostoc mesenteroides subsp. cremoris

Malolactic fermentation (MLF), which improves organoleptic properties and biologic stability of some wines, may cause wine spoilage if uncontrolled. Bacteriocins were reported as efficient preservatives to control MLF through their bactericidal effect on malolactic bacteria. Leuconostoc mesenteroides subsp. cremoris W3 isolated from wine produces an inhibitory substance that is bactericidal against malolactic bacteria in model wine medium. Treatment of the culture supernatant of strain W3 with proteases eliminated the inhibitory activity, which proved that it is a true bacteriocin and we tentatively termed it mesentericin W3. The bacteriocin inhibited the growth of food-borne pathogenic bacteria such as Enterococcus faecalis, Listeria monocytogenes, and malolactic bacteria. It was active over a wide pH range and stable to organic solvents and heat. Mesentericin W3 was purified to homogeneity by a pH-mediated cell adsorption–desorption method, cation exchange, hydrophobic interaction, and reverse-phase chromatography. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy (MS) and partial amino acid sequence analysis revealed that mesentericin W3 was identical to mesentericin Y105.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

Leuconostoc mesenteroides growth kinetics with application to bacterial profile modification

Bacterial profile modification (BPM) is being developed as an oil recovery technique that uses bacteria to selectively plug oil depleted zones within a reservoir to divert displacing fluids (typically water) into oil-rich zones. Leuconostoc mesenteroides, which produces dextran when supplied with sucrose, is a bacterium that is technically feasible for use in profile modification. However, the technique requires controlled bacterial growth to produce selective plugging.A kinetic model for the production of cells and polysaccharides has been developed for L. mesenteroides bacteria. This model, based on data from batch growth experiments, predicts saccharide utilization, cell generation, and dextran production. The underlying mechanism is the extracellular breakdown of sucrose into glucose and fructose and the subsequent production of polysaccharide (dextran). The monosaccharides are then available for growth. Accompanying sucrose consumption is the utilization of yeast extract. The cell requires a complex media that is provided by yeast extract as a source of vitamins and amino acids. Varying the concentration ratio of yeast extract to sucrose in the growth media provides a means of controlling the amount of polymer produced per cell. Consequently, in situ bacteria growth can be controlled by the manipulation of nutrient media composition, thereby providing the ability to create an overall strategy for the use of L. mesenteroides bacteria for profile modification.     

Investigation of production of dextran and dextransucrase by Leuconostoc mesenteroides immobilized within porous stainless steel

Cells of Leuconostoc mesenteroides were immobilized within porus, stainless-steel (SS) supports and used for dextransucrase (DS) and dextran production. The pore size of the support significantly affected the dextran yields, which were greatest with average pore sizes of 2-5 mum. All immobilized-cell biocatalysts in porous stainless steel produced higher yields than free cells, with the exception of cells confined in submicrometer pores (0.5 mum). Coating supports of larger pore size (40 and 100 mum) with calcium alginate enhanced the cell-loading capacity of the supports and increased dextran and fructose yields in the cell-free broth. Controlled, fed-batch, DS production (activation), as a step preliminary to dextran production, significantly improved the subsequent dextran and fructose yields and shortened the time required to attain the maximum such yields. Scanning electron microscopy (SEM) of immobilized L. mesenteroides in stainless steel shows an irregular pattern of the microorganism inside the pores of the solid supports. Coating the porous solid supports with a cell-free calcium alginate layer led to an increase in the cell density inside the support. Cell growth inside the coated, porous stainless steel had no distinct growth form. (c) 1992 John Wiley & Sons, Inc.     

Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides

Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L. mesenteroides contain minute amounts of enolase, which separate as small crystals. Expression of L. mesenteroides enolase in Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing the conversion of 2-phsopho-D-glycerate to phosphoenolpyruvate. The pH optimum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s(-1), respectively. At phosphate concentrations of 1 mM and below, fluoride is a noncompetitive inhibitor with respect to 2-phospho-D-glycerate, but in the presence of 20 mM phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase significantly inhibits the activity of purified dextransucrase, and does not bind human plasminogen. Results here suggest that in some organisms enolase may participate in protein interactions that have no direct relevance to pathogenic invasion.     

Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.     

A mutant strain of Leuconostoc mesenteroides B-1355 producing a glucosyltransferase synthesizing α(1→2) glucosidic linkages

A mutant strain (R1510) of Leuconostoc mesenteroides B-1355 was isolated which synthesized primarily an insoluble polysaccharide and little soluble polysaccharide when grown in sucrose-containing medium. Glucose or sucrose cultures of this strain produced a single intense band of GTF-1 activity of 240 kDa on SDS gels, and a number of faint, smaller bands. Oligosaccharides synthesized by strain R1510 from methyl-α-D-glucoside and sucrose included a trisaccharide whose structure contained an α(1→2) glucosidic linkage. This type of linkage has not been seen before in any products from strain B-1355 or its mutant derivatives. The structure of the purified trisaccharide was confirmed by ¹³C-nuclear magnetic resonance. The insoluble polysaccharide also contained α(1→2) branch linkages, as determined by methylation analysis, showing that synthesis of the linkages was not peculiar to methyl-α-D-glucoside. GTF-1, which had been excised with a razor blade from an SDS gel of a culture of the parent strain B-1355, produced the same trisaccharides as strain R1510, showing that GTF-1 from the wild-type strain was the same as GTF-1 from strain R1510. Mutant strains resembling strain R1510, but producing a single intense band of alternansucrase (200 kDa) instead of GTF-1 were also isolated, suggesting that mutations may be generated which diminished the activities for any two of the three GTFs of strain B1355 relative to the third. Strain R1554 produced a soluble form of alternansucrase, while strain R1588 produced a cell-associated form. The mechanism(s) by which specific GTFs become associated with the cells of L. mesenteroides was not explored. Received 12 May 1998/ Accepted in revised form 16 July 1998     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

Alternansucrase mutants of Leuconostoc mesenteroides strain NRRL B-21138

Alternan is a unique α-D-glucan of potential commercial interest, produced by rare strains of Leuconostoc mesenteroides. Natural isolates that produce alternan, such as NRRL B-1355, also produce dextran as a troublesome contaminant. We previously isolated mutants of strain NRRL B-1355 that are deficient in dextran production, including the highly stable strain NRRL B-21138. In the current work, we mutagenized strain NRRL B-21138 and screened survivors for further alterations in production of alternansucrase, the enzyme that catalyzes the synthesis of alternan from sucrose. Second generation mutants included highly stable strain NRRL B-21297, which produced four-fold elevated levels of alternansucrase without an increase in the proportion of dextransucrase activity. Such alternansucrase overproducing strains will facilitate studies of this enzyme, and may become valuable for the enzymatic production of alternan. Another highly stable mutant strain, NRRL B-21414, grew slowly on sucrose with negligible production of glucan or extracellular glucansucrase activity. This strain may prove useful as an expression host for glucansucrase genes. Received 30 July 1996/ Accepted in revised form 15 December 1996     

Identification of a single and non-essential cysteine residue in dextransucrase of Leuconostoc mesenteroides NRRL B-512F

Amino acid analysis of purified dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was carried out. The enzyme is virtually devoid of cysteine residue there being only one cysteine residue in the whole enzyme molecule comprising over 1500 amino acid residues. The enzyme is rich in acidic amino acid residues. The number of amino acid residues was calculated based on the molecular weight of 188,000 (Goyal and Katiyar 1994). Amino sugars were not found, implying that the enzyme is not a glycoprotein. It has been shown earlier that the cysteine residue in dextransucrase is not essential for enzyme activity (Goyal and Katiyar 1998). The presence of only one cysteine residue per enzyme molecule illustrates that its tertiary structure is solely dependent on other types of non-covalent interactions such as hydrogen bonding, ionic and nonpolar hydrophobic interactions.     

一株高效解磷肠膜明串珠菌的分离鉴定及解磷能力研究

【背景】植物根际土壤含有多种溶磷微生物,但是具有溶磷能力的肠膜明串珠菌未见报道。【目的】从脐橙根际土壤分离高效解磷菌,研究其解磷应用。【方法】通过初筛和复筛从23株菌中筛选解磷能力较强的菌株,同时采用钼蓝比色法测定磷含量。通过测定发酵液中小分子有机酸含量、磷酸酯酶酶活及pH值的变化,探究菌株的解磷机理。【结果】经过筛选得到9株具有一定解磷能力的菌株。通过菌种16S rRNA基因序列分析和生理生化实验确定其中一株菌为肠膜明串珠菌,命名为肠膜明串珠菌G7。培养基初始pH6.0、碳源为葡萄糖、氮源为硫酸铵时G7的解磷能力较佳。G7发酵过程中产生大量有机酸,而其酸性磷酸酯酶活性高于碱性磷酸酯酶。【结论】碳源、氮源以及初始pH值都能影响G7的解磷能力,其解磷能力主要缘于在发酵过程中产生了大量小分子有机酸,关于G7的解磷机理还需要更深入的研究。     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides

For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides. All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside. In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst. The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for alpha-butylglucopyranoside and alpha-octylglucopyranoside, respectively. Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of alpha-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.