• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.846-853
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• 2011

Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

• The Plant Pathology Journal
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• v.37 no.1
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• pp.36-46
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• 2021

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) in watermelon, a disease that poses a serious threat to watermelon production. Because of the lack of resistant cultivars against BFB, virulence factors or mechanisms need to be elucidated to control the disease. Glycerol-3-phosphate dehydrogenase is the enzyme involved in glycerol production from glucose during glycolysis. In this study, we report the functions of a putative glycerol-3-phosphate dehydrogenase in Ac (GlpdAc) using comparative proteomic analysis and phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in two pathogenicity tests. The putative 3D structure and amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, including carbohydrate metabolism, were affected by GlpdAc. Although AcΔglpdAc(EV) could not use glucose as a sole carbon source, it showed growth in the presence of glycerol, indicating that GlpdAc is involved in glycolysis. AcΔglpdAc(EV) also displayed higher cell-to-cell aggregation than the wild-type bacteria, and tolerance to osmotic stress and ciprofloxacin was reduced and enhanced in the mutant, respectively. These results indicate that GlpdAc is involved in glycerol metabolism and other mechanisms, including virulence, demonstrating that the protein has pleiotropic effects. Our study expands the understanding of the functions of proteins associated with virulence in Ac.

• The Korean Journal of Zoology
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• v.34 no.1
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• pp.123-130
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• 1991

Several parameters of u -glycerol-3-pholphate dehydrogenase (GPDH) such as activity, content and translatable mRNA levels were measured to elucidate mechanism underlving developmental and tissue specific regulation of 6PDH activity in Drosophila melonogastrr. In adult segments, most of total GPDH activity (62%1 Iwas detected in thorax where GPDH-1 resided, while 32% of total GPDH aUiviD was only detected in abdomen where GPDH-3 resided. The relative synthesis of GPDH was, however, similar in both tissues, although 58% of total GPDH was synthesized in abdomen. These results strongly suggest that the turnover rate of the abdominal enzyme (GPDH-3) was much more rapid than that of thoracic enzymes (GPDH-1). In nitro translation and immunoblotting experiments also indicate that GPDH-3 was arised by posttranslational modification from a single polypeptide (GPDH-1).

• Journal of the Korean Chemical Society
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• v.68 no.1
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• pp.32-39
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• 2024

Glycerol dehydrogenase (GlyDH) plays a crucial role in the glycerol metabolism pathway by catalyzing the oxidation of glycerol to dihydroxyacetone (DHA). Previous studies of GlyDH have predominantly focused on unraveling the structural features of the active site and its binding interactions with ligand. However, the structural details of GlyDH in complex with both NAD⁺ and the substrate bound have remained elusive. In this study, we present the crystal structures of Klebsiella pneumoniae GlyDH (KpGlyDH) in the absence and presence of NAD⁺ at a resolution of 2.1 Å. Notably, both structures reveal the binding of the substrate, ethylene glycol, to the zinc ion. Interestingly, a significant change in the coordination number of the zinc ion is observed, with three in the absence of NAD⁺ and four in its presence. These findings shed light on the structural aspects of GlyDH and its interactions with NAD⁺ and the substrate.

• BMB Reports
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• v.36 no.2
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• pp.159-166
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• 2003

Cytoplasmic $\alpha$-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was $59,500{\pm}650$ daltons; its subunit size was estimated to be $35,700{\pm}140$ by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were $3.9{\pm}0.7\;mM$, $0.65{\pm}0.05\;mM$, $0.26{\pm}0.06\;mM$, and $0.005{\pm}0.0004\;mM$ for L-glycerol-3-phosphate, $NAD^+$, DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were $2.30{\pm}0.21\;mM$ and $0.20{\pm}0.01\;mM$ for L-glycerol-3-phosphate and $NAD^+$, respectively. The turnover number, $k_{cat}$, of the forward reaction was $1.9{\pm}0.2{\times}10^4\;s^{-1}$. The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that $\alpha$-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.271-278
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• 2020

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD⁺ over NADP⁺. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest k_cat/K_m value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The k_cat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec^-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a T_m of 83.4℃, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1908-1914
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• 2008

An alkaliphilic bacterium, Bacillus clausii SKAL-16, was isolated from soil that had been contaminated with vegetable oil. The optimal pH and general pH range for bacterial growth was 8, and 7 to 10, respectively. The bacterium could grow on tributyrin and glycerol, but could not grow on acetate and butyrate. The SKAL-16 strain excreted butyric acid during growth on tributyrin, and selectively ingested glycerol during growth on a mixture of butyric acid and glycerol. The SKAL-16 generated intracellular lipase, but did not produce esterase and extracellular lipase. The DNA fragment amplified with the chromosomal DNA of SKAL-16 and primers designed on the basis of the esterase-coding gene of Bacillus clausii KSM-KI6 was not identical with the esterase-coding gene contained in the GenBank database. Pyruvate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase activities were detected in the cell-free extract (crude enzyme).

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.111-111
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• 2003

Green tea have been widely reported as functional foods because of their various bioactivities. In the present study, we used 3T3-Ll cells model of white adipocytes to clarify whether green tea and its main pharmaceutically effective compounds (EGCG, caffeine and theanine) prevent obesity. Cellular viability, glycerol-3-phosphate dehydrogenase activity, glycerol release and HSL mRNA levels were checked. Glycerol release into the medium was significantly increased by the cells treated with green tea extract. Glycerol release into the medium was significantly increased by the cells treated with green tea extract. Caffeine and theanine from green tea showed some level of lipolytic activity, and glycerol-3-phosphate dehydrogenase activity was remarkably decreased by EGCG. These results suggest that green tea has anti-obesity effect through inhibition of adipogenesis and stimulation of lipolysis. Catechins and theanine of green tea might be the factors responsible for the modulation of lipid metabolism and adipocyte differentiation.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1077-1086
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• 2016

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn²⁺ enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp¹⁷¹, His²⁵⁴, and His²⁷¹) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60℃ and pH 8.0 and towards DHA at 60℃ and pH 6.0. DHA reduction was the dominant reaction, with a lower K_m(DHA) of 1.08 ± 0.13 mM and V_max of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a K_m(glycerol) of 30.29 ± 3.42 mM and V_max of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60℃. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg⁴³, both related to dinucleotide binding.