• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.153-159
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• 2013

A mannitol dehydrogenase (MDH; EC 1.1.1.67) gene was cloned from the Sinorhizobium meliloti 1021 (KCTC 2353) genome and expressed in Escherichia coli. It was seen to have an open reading frame consisting of 1,485 bp encoding 494 amino acids (about 54 kDa), which shares approximately 35-55% of amino acid sequence identity with some known long-chain dehydrogenase/ reductase family enzymes. The recombinant S. meliloti MDH (SmMDH) showed the highest activity at $40^{\circ}C$, and pH 7.0 (D-fructose reduction) and pH 9.0 (D-mannitol oxidation), respectively. SmMDH could catalyze the oxidative/reductive reactions between D-mannitol and D-fructose in the presence of $NAD^+/NADH$ as a coenzyme, but not with NADP+/NADPH. These results indicate that SmMDH is a typical $NAD^+/NADH$-dependent mannitol dehydrogenase.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.156-162
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• 2012

A mannitol dehydrogenase (StMDH) gene was cloned from Salmonella typhimurium LT2 (KCTC 2421) and overexpressed in Escherichia coli. It has a 1,467 bp open reading frame encoding 488 amino acids with deduced molecular mass of 54 kDa, which shares approximately 36% of amino acid identity with known long-chain dehydrogenase/reductatse (LDR) family enzymes. The recombinant StMDH showed the highest activity at $30^{\circ}C$, and pH 5.0 and 10.0 for D-fructose reduction and D-mannitol oxidation, respectively. On the contrary, it has no activity on glucose, galactose, xylose, and arabinose. StMDH can catalyze the oxidative/reductive reactions between D-fructose and D-mannitol only in the presence of $NAD^+$/NADH as coenzymes. These results indicate that StMDH is a typical $NAD^+$/NADH-dependent mannitol dehydrogenase (E.C. 1.1.1.67).

• Journal of Microbiology and Biotechnology
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• v.21 no.9
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• pp.914-920
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• 2011

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a $6{\times}$His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a $K_m$ of $1.54{\pm}0.1$ mM and $V_{max}$ of $320.8{\pm}7.81\;{\mu}mol$/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and $37^{\circ}C$, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.

• Journal of Korean Neurosurgical Society
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• v.42 no.4
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• pp.337-341
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• 2007

Objective : Repeated administration of mannitol in the setting of large hemispheric infarction is a controversial and poorly defined therapeutic intervention. This study was performed to examine the effects of multiple-dose mannitol on a brain edema after large hemispheric infarction. Methods : A middle cerebral artery was occluded with the rat suture model for 6 hours and reperfused in 22 rats. The rats were randomly assigned to either control (n=10) or the mannitol-treated group (n=12) in which intravenous mannitol infusions (0.8 g/kg) were performed six times every four hours. After staining a brain slice with 2,3,5-triphenyltetrazolium chloride, the weight of hemispheres, infarcted (IH) and contralateral (CH), and the IH/CH weight ratio were examined, and then hemispheric accumulation of mannitol was photometrically evaluated based on formation of NADH catalyzed by mannitol dehydrogenase. Results : Mannitol administration produced changes in body weight of $-7.6{\pm}1.1%$, increased plasma osmolality to $312{\pm}8\;mOsm/L$. It remarkably increased weight of IH ($0.77{\pm}0.06\;gm$ versus $0.68{\pm}0.03\;gm$ : p<0.01) and the IH/CH weight ratio ($1.23{\pm}0.07$ versus $1.12{\pm}0.05$ : p<0.01). The photometric absorption at 340 nm of the cerebral tissue in the mannitol-treated group was increased to $0.375{\pm}0.071$ and $0.239{\pm}0.051$ in the IH and CH, respectively from $0.167{\pm}0.082$ and $0.162{\pm}0.091$ in the IH and CH of the control group (p<0.01). Conclusion : Multiple-dose mannitol is likely to aggravate cerebral edema due to parenchymal accumulation of mannitol in the infarcted brain tissue.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.780-787
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• 2023

Two mannitol producing lactic acid bacteria were isolated from pa (green onion)- kimchi, identified and named as Leuconostoc mesenteroides SKP 88 and Leuconostoc citreum SKP 92, respectively. Both isolates grew well at 25-30℃, initial pH 6-8, and 3% and lower NaCl concentration. Both isolates converted fructose into mannitol efficiently when grown on MRS broth containing fructose and glucose. Glucose was used as a carbon source and fructose was used as a precursor for mannitol. Mannitol yields were the highest in MRS broth with 3% fructose and 2% glucose. Shine muscat juice fermentation was done using each isolate as a starter. As fermentation progressed, decrease in pH and increases in titratable acidity and viable counts were observed. L. mesenteroides SKP 88 showed better mannitol conversion ability than L. citreum SKP 92, and shine muscat juice fermented with L. mesenteroides SKP 88 showed the mannitol production of 41.6 g/l at 48 h, and juice fermented with L. citreum SKP 92 showed 23.4 g/l at the same time. Yogurt fermentations showed similar patterns, and yogurt fermented with L. mesenteroides SKP 88 showed the mannitol production of 15.13 g/l. These results showed that both strains are useful as starters for healthy fermented foods with reduced fructose contents.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2009-2018
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• 2018

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study we modified the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains for mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deletion strain (${\Delta}pat::amy$), the fk gene (which encodes fructokinase) deletion strain (${\Delta}fk::amy$) and the stpk gene (which encodes serine-threonine protein kinase) deletion strain (${\Delta}stpk::amy$) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1%, respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (${\Delta}pat::mdh$, ${\Delta}fk::mdh$ and ${\Delta}stpk::mdh$) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ${\Delta}dts{\Delta}ldh{\Delta}pat::mdh{\Delta}stpk::mdh{\Delta}fk::mdh$ had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.

• Korean Journal of Plant Resources
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• v.18 no.1
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• pp.15-21
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• 2005

Korean ginseng(Panax ginseng C.A. Meyer) is very difficult to obtain stable production of qualified ginseng roots because of variable stresses in soil environments. In transformation of ginseng with betain aldehyde dehydrogenase gene, compounds synthesized for controlling osmotic pressure such as proline, glycine, betaine, polyols and sugar were accumulated in cell for salt resistance in transgenic plants. 2 Agrobactgerium conjugants were acquired with bet A and bet B genes for solt resistant plants. A. tumefaciens MP90/pBetA and A. tumefaciens MP90/pBetB were recombined for increasing the tolerance to salt stress. To confirm the transformation of the binary vector, tobacco plant was transformed, and the transformant can grow on media containing high concentration of kanamycin. To identify NPT 11, BetA and BetB genes of the transformants, the band on the agarose was confirmed by PCR and RT-PCR techniques. The transformants of ginseng with bet A and bet B genes were acquired on the phytohormone free basic MS media containing only antibiotics and 1M mannitol used for selection of transgenic plant, but the transfomation efficiency for BetA and BetB was very low.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.415-418
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• 2000

Productivity of a rice plant is greatly influenced by salt stress. One of the ways to achieve tolerance to salinity is to transfer genes encoding protective enzymes from other organisms, such as microorganisms. The bacterial gene, mtlD, which encodes mannitol-1-phosphate dehydrogenase (Mtl-DH), was introduced to the cytosol of a rice plant by an imbibition technique to overproduce mannitol. The germination and survival rate of the imbibed rice seeds were markedly increased by transferring the mtlD gene when it was delivered in either a pBIN19 or pBmin binary vector. When a polymerase chain reaction was performed with the genomic DNAs of the imbibed rice leaves as a template and with mtlD-specific primers, several lines were shown to contain an exogenous mtlD DNA. However, a reverse transcription (RT)-PCR analysis revealed that not all of them showed an expression of this foreign gene. This paper demonstrates that the growth and germination of rice plants transiently transformed with the bacterial gene, mtlD, are enhanced and these enhancements may have resulted from the experssion of the mtlD gene. The imbibition method empolyed in this study fulfills the requirements for testing the function of such a putative gene in vivo prior to the production of a stable transgenic plant.

• Journal of the Korean Chemical Society
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• v.24 no.4
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• pp.315-321
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• 1980

A steady-state kinetic study on a dye-coupled cytoplasmic polyol dehydrogenase from G. melanogenus was carried by the initial velocity measurements in the direction of the polyol oxidation and the product inhibition by D-fructose. For the initial rate experiments, D-mannitol and D-sorbitol were employed as the specific polyol substrates and 2,6-dichlorophenolin-dophenol (DPIP) as the specific cofactor substrate for the enzyme. When the polyol and DPIP were examined by varying one of substrates and by fixing the second, the corresponding reciprocal plots showed the typical parallel pattern. This suggests that the enzyme from G. melanogenus proceeds by a Ping Pong Bi-Bi mechanism in which the polyol may account as the first reactant-in, and the ketose formed as the first product-out, respectively. The product inhibition patterns obtained by D-fructose (one no-inhibition, one non-competitive, and two competitive) may also provide an additional conformatory evidence for the above mechanism. Based on the kinetic parameters obtained, it was also suggested that the rate-limiting step in the direction of polyol oxidation is associated with the release of the ketose from the Enzyme${\cdot}$Polyol complex.

• Microbiology and Biotechnology Letters
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• v.46 no.2
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• pp.102-110
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• 2018

A bacterial strain capable of metabolizing myo-inositol (MI) and converting to other substances was isolated from soil of orchard. The isolate, named YB-46, was grown on minimal medium supplemented with MI as the sole carbon source and was presumed to belonging to genus Enterobacter according to the 16S rDNA sequence. Escherichia coli transformant converting MI into unknown metabolites was selected from a metagenomic library prepared with fosmid pCC1FOS vector. Plasmid was isolated from the transformant, and the inserted gene was partially sequenced. From the nucleotide sequence, an iolG gene was identified to encode myo-inositol dehydrogenase (IolG) consisting of 336 amino residues. The IolG showed amino acid sequence similarity of about 50% with IolG of Enterobacter aerogenes and Bacillus subtilis. The His-tagged IolG (HtIolG) fused with hexahistidine at C-terminus was produced and purified from cell extract of recombinant E. coli. The purified HtIolG showed maximal activity at $45^{\circ}C$ and pH 10.5 with the highest activity for MI and D-glucose, and more than 90% of maximal activity for D-chiro-inositol, D-mannitol and D-xylose. $K_m$ and $V_{max}$ values of the HtIolG for MI were 1.83 mM and $0.724{\mu}mol/min/mg$ under the optimal reaction condition, respectively. The activity of HtIolG was increased 1.7 folds by $Zn^{2+}$, but was significantly inhibited by $Co^{2+}$ and SDS.