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.     

Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of Leuconostoc mesenteroides strain Lm 28

Aims:  To find different types of glucosyltransferases (GTFs) produced by Leuconostoc mesenteroides strain Lm 28 and its mutant forms, and to check the effectiveness of gluco-oligosaccharide synthesis using maltose as the acceptor.
Methods and Results:  Constitutive mutants were obtained after chemical mutagenesis by ethyl methane sulfonate. Lm M281 produced more active GTFs than that obtained by the parental strain cultivated on sucrose. GTF from Lm M286 produced a resistant glucan, based on endo-dextranase and amyloglucosidase hydrolysis. The extracellular enzymes from Lm M286 catalyse acceptor reactions and transfer the glucose unit from sucrose to maltose to produce gluco-oligosaccharides (GOS). By increasing the sucrose/maltose ratio, it was possible to catalyse the synthesis of oligosaccharides of increasing degree of polymerization (DP).
Conclusions:  Different types of GTFs (dextransucrase, alternansucrase and levansucrase) were produced from new constitutive mutants of Leuc. mesenteroides . GTFs from Lm M286 can catalyse the acceptor reaction in the presence of maltose, leading to the synthesis of branched oligosaccharides.
Significance and Impact of the Study:  Conditions were optimized to synthesize GOS by using GTFs from Lm M286, with the aim of producing maximum quantities of branched-chain oligosaccharides with DP 3–5. This would allow the use of the latter as prebiotics.     

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.     

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.     

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.     

Cloning of the D-lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. cremoris

Summary A genomic library from Leuconostoc mesenteroides subsp. cremoris was screened for D-lactate dehydrogenase activity using a stereospecific lactate detection test on agar plate. Among 3500 clones tested, six positive colonies were found on D-lactate detection plate, displaying significantly higher D-LDH activity than Escherichia coli host strain.     

Production of Bacteriocin ST33LD, Produced by Leuconostoc mesenteroides subsp. mesenteroides, as Recorded in the Presence of Different Medium Components

Summary Bacteriocin ST33LD, produced by Leuconostoc mesenteroides subsp. mesenteroides, is approximately 2.7 kDa in size and inhibits Enterococcus faecalis, Escherichia coli, Lactobacillus casei and Pseudomonas aeruginosa. Good growth was recorded in the presence of 10% (w/v) soy milk or 10% (w/v) molasses, but there was no bacteriocin production. Growth in MRS broth adjusted to pH 4.5 yielded low bacteriocin levels (800 AU/ml). However, the same medium adjusted to pH 5.0, 5.5 and 6.5, respectively, yielded 3200 AU/ml. Tween 80 decreased bacteriocin production by more than 50%. Growth in the presence of tryptone yielded maximal activity (12,800 AU/ml), whereas different combinations of tryptone, meat extract and yeast extract produced activity levels of 1600 AU/ml and less. Growth in the presence of 2.0% (w/v) sucrose, or maltose, yielded much higher levels of bacteriocin activity (12,800 AU/ml) compared to growth in the presence of 2.0% (w/v) glucose or lactose (6400 AU/ml). Lower yields were also recorded in the presence of fructose and mannose. KH₂PO₄ at 10.0% (w/v) stimulated bacteriocin production. Glycerol concentrations of 0.5% (w/v) and higher (up to 5.0%, w/v) repressed bacteriocin production by 50%. The addition of cyanocobalamin, thiamine and L-ascorbic acid to MRS broth (1.0 ppm) yielded 12,800 AU/ml bacteriocin, whereas the addition of DL-6,8-thioctic acid yielded only 6 400 AU/ml.     

Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides

An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract. The purified NADH oxidase was an acidic protein having an S0 20,W of 5.49S and a molecular weight of 104,000, consisting of a dimer with 53,000 subunit size. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide. The physiological substrate was beta-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward alpha-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with beta-NADH. alpha-NADPH and beta-NADPH were inert for the reaction. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45 degrees C. The activation energy was estimated to be 3.77 kcal/mol. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cu2+, but not by EDTA. Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.     

Co-metabolism of citrate and lactose by Leuconostoc mesenteroides subsp. cremoris

Citrate stimulated growth rate, increased the specific lactose consumption rate and enhanced the molar growth yield of Leuconostoc mesenteroides subsp. cremoris growing on lactose at pH 5.2 or 6.2 and at 22 or 30°C. As soon as citrate utilization began, diacetyl and acetoin were produced: 2,3-butylene glycol appeared later while acetoin decreased.     

Leuconostoc oenos and malolactic fermentation in wine: a review

This review article summarizes the state of the art on Leuconostoc oenos, the bacteria responsible for malolactic fermentation in wine. Both basic and practical aspects related to the metabolism of this microorganism and malolactic fermentation in general are critically reviewed. The former examines the role of genetics for the identification and classification of L. oenos and energetic mechanisms on solute transport (malic and lactic acid). The latter includes practical information on biomass production, optimal growth conditions and stress factors, which are important in growth optimization of malolactic starter cultures. Extensive data and references on the effect of malolactic fermentation on wine composition and sensory analysis are also included. Received 06 May 1999/ Accepted in revised form 13 July 1999     

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.     

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     

Molecular cloning and characterization of a novel glucansucrase from Leuconostoc mesenteroides subsp. mesenteroides LM34

Leuconostoc mesenteroides LM34 was isolated from kimchi, a traditional fermented Korean food. L. mesenteroides LM34 produced extracellular glucansucrase (DSRLM34), which is responsible for the synthesis of soluble glucan using sucrose. The DSRLM34 gene consists of a 4,503 bp open reading frame (ORF) and encodes an enzyme of 1,500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (98%) to that of glucansucrase of Lactobacillus lactis. The gene was over-expressed in Escherichia coli strain and the recombinant enzyme (rDSRLM34) was purified. Both DSRLM34 and rDSRLM34 synthesized glucan mainly containing α-1, 6 glucosidic linkage and branched α-1, 3 glucosidic linkages. The enzyme exhibited optimum activity at 30°C and pH 5.0. DSRLM34 has promising potential as a thickening agent in sucrose-supplemented milk.     

Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris

Summary The addition of citrate to glucose broth led to an increase in specific growth rate and glucose catabolism, but a decrease in molar growth yield from glucose, in Leuconostoc mesenteroides subsp. cremoris. Acetate and formate were produced during the stationary phase of growth. According to the fermentation balance, part of the acetate and lactate came from the pyruvate of citrate metabolism. L. mesenteroides subsp. cremoris incorporated radioactive metabolites from [1,5-¹⁴C] citrate into cell material, primarily into lipids. [U-¹⁴C] Glucose was not incorporated into cell material.     

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。     

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.