Yeong-Su Kim - Academia.edu (original) (raw)

Papers by Yeong-Su Kim

Journal of Biological Chemistry, Jun 1, 2009

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03... more Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme. Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals,

Biotechnology Letters, Sep 6, 2011

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones... more The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K (m) of 0.5 mM, and a k (cat) of 2532 s(-1). The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h(-1). Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k (cat), k (cat)/K (m), conversion yield, and productivity in the hydrolysis of daidzin.

Applied and Environmental Microbiology, Feb 1, 2011

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomer... more An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu 2؉. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k cat /K m) for L-ribulose using the R142N mutant were 1.4-and 1.6-fold higher than those of the wild-type enzyme, respectively. The k cat /K m of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k cat /K m. The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter ؊1 h ؊1 , which was 1.5-fold higher than that of the wild-type enzyme.

Journal of Agricultural and Food Chemistry, Oct 18, 2011

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 3... more A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.

Journal of Agricultural and Food Chemistry, Feb 1, 2012

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purifie... more The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.

Applied and Environmental Microbiology, Feb 1, 2013

Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monos... more Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphatebinding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a D-ribose isomerase, an L-ribose isomerase, and an L-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases.

한국생물공학회 학술대회, Apr 1, 2010

Antioxidants, Jun 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Plants, May 5, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Catalysts, Dec 4, 2019

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for ob... more Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant β-glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, β-glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

Plants

Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral... more Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral lipids, such as triacylglycerols (TAGs) and sterol esters, which are considered energy reserves. The metabolic pathways associated with LDs in eukaryotic species are involved in diverse cellular functions. TAG synthesis in plants is mediated by the sequential involvement of two subcellular organelles, i.e., plastids - plant-specific organelles, which serve as the site of lipid synthesis, and the ER. TAGs and sterol esters synthesized in the ER are sequestered to form LDs through the cooperative action of several proteins, such as SEIPINs, LD-associated proteins, LDAP-interacting proteins, and plant-specific proteins such as oleosins. The integrity and stability of LDs are highly dependent on oleosins, especially in the seeds, and oleosin degradation is critical for efficient mobilization of the TAGs of plant LDs. As the TAGs mobilize in LDs during germination and post-germinative growth...

Frontiers in Bioengineering and Biotechnology

Rare sugars are regarded as functional biological materials due to their potential applications a... more Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can ...

Journal of Microbiology, 2020

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate... more Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Catalysts, 2019

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for ob... more Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant β-glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, β-glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

World Journal of Microbiology and Biotechnology, 2010

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a ... more A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg-1 by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with L-lyxose, L-talose, D-gulose, D-ribose, L-mannose, D-allose, L-xylulose, L-tagatose, D-sorbose, D-ribulose, L-fructose, and D-psicose and exhibited particularly high activity with L-form monosaccharides such as L-lyxose, L-xylulose, L-talose, and L-tagatose. With L-xylulose (500 g l-1) and L-talose (500 g l-1) substrates, the optimum concentrations of RpiB were 300 and 600 U ml-1 , respectively. The enzyme converted 500 g l-1 L-xylulose to 350 g l-1 L-lyxose after 3 h, and yielded 450 g l-1 L-tagatose from 500 g l-1 L-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of L-form monosaccharides.

Journal of Biological Chemistry, 2009

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03... more Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme. Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals,

Journal of Agricultural and Food Chemistry, 2011

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 3... more A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.

Biotechnology Letters, 2010

For the removal of galactose inhibition, the predicted galactose binding residues, which were det... more For the removal of galactose inhibition, the predicted galactose binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The K (i) of the F349S mutant (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.

Biotechnology Letters, 2011

The gene encoding human β-carotene-9',10'-oxygenase, which cleave... more The gene encoding human β-carotene-9',10'-oxygenase, which cleaves the 9',10' double bond in β-carotene into β-apo-10'-carotenal, was cloned and expressed in Escherichia coli. Under aqueous conditions, the optimum organic solvent for the formation of detergent micelles was toluene. The optimum pH, temperature, detergent type, and the optimum concentrations of detergent, substrate, and enzyme for β-apo-10'-carotenal production were 8.0, 37°C, Tween 40, 2.4%, 300 mg β-carotene/l, and 0.25 U/ml, respectively. Under the optimum conditions, 43 mg β-apo-10'-carotenal/l was produced after 21 h with a conversion of 14%. This is the first report to describe the enzymatic production of β-apo-10'carotenal.

Biotechnology Letters, 2011

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones... more The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K (m) of 0.5 mM, and a k (cat) of 2532 s(-1). The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h(-1). Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k (cat), k (cat)/K (m), conversion yield, and productivity in the hydrolysis of daidzin.

Journal of Biological Chemistry, Jun 1, 2009

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03... more Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme. Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals,

Biotechnology Letters, Sep 6, 2011

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones... more The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K (m) of 0.5 mM, and a k (cat) of 2532 s(-1). The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h(-1). Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k (cat), k (cat)/K (m), conversion yield, and productivity in the hydrolysis of daidzin.

Applied and Environmental Microbiology, Feb 1, 2011

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomer... more An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu 2؉. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k cat /K m) for L-ribulose using the R142N mutant were 1.4-and 1.6-fold higher than those of the wild-type enzyme, respectively. The k cat /K m of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k cat /K m. The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter ؊1 h ؊1 , which was 1.5-fold higher than that of the wild-type enzyme.

Journal of Agricultural and Food Chemistry, Oct 18, 2011

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 3... more A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.

Journal of Agricultural and Food Chemistry, Feb 1, 2012

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purifie... more The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.

Applied and Environmental Microbiology, Feb 1, 2013

Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monos... more Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphatebinding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a D-ribose isomerase, an L-ribose isomerase, and an L-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases.

한국생물공학회 학술대회, Apr 1, 2010

Antioxidants, Jun 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Plants, May 5, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Catalysts, Dec 4, 2019

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for ob... more Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant β-glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, β-glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

Plants

Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral... more Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral lipids, such as triacylglycerols (TAGs) and sterol esters, which are considered energy reserves. The metabolic pathways associated with LDs in eukaryotic species are involved in diverse cellular functions. TAG synthesis in plants is mediated by the sequential involvement of two subcellular organelles, i.e., plastids - plant-specific organelles, which serve as the site of lipid synthesis, and the ER. TAGs and sterol esters synthesized in the ER are sequestered to form LDs through the cooperative action of several proteins, such as SEIPINs, LD-associated proteins, LDAP-interacting proteins, and plant-specific proteins such as oleosins. The integrity and stability of LDs are highly dependent on oleosins, especially in the seeds, and oleosin degradation is critical for efficient mobilization of the TAGs of plant LDs. As the TAGs mobilize in LDs during germination and post-germinative growth...

Frontiers in Bioengineering and Biotechnology

Rare sugars are regarded as functional biological materials due to their potential applications a... more Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can ...

Journal of Microbiology, 2020

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate... more Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Catalysts, 2019

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for ob... more Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant β-glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, β-glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

World Journal of Microbiology and Biotechnology, 2010

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a ... more A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg-1 by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with L-lyxose, L-talose, D-gulose, D-ribose, L-mannose, D-allose, L-xylulose, L-tagatose, D-sorbose, D-ribulose, L-fructose, and D-psicose and exhibited particularly high activity with L-form monosaccharides such as L-lyxose, L-xylulose, L-talose, and L-tagatose. With L-xylulose (500 g l-1) and L-talose (500 g l-1) substrates, the optimum concentrations of RpiB were 300 and 600 U ml-1 , respectively. The enzyme converted 500 g l-1 L-xylulose to 350 g l-1 L-lyxose after 3 h, and yielded 450 g l-1 L-tagatose from 500 g l-1 L-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of L-form monosaccharides.

Journal of Biological Chemistry, 2009

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03... more Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme. Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals,

Journal of Agricultural and Food Chemistry, 2011

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 3... more A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.

Biotechnology Letters, 2010

For the removal of galactose inhibition, the predicted galactose binding residues, which were det... more For the removal of galactose inhibition, the predicted galactose binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The K (i) of the F349S mutant (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.

Biotechnology Letters, 2011

The gene encoding human β-carotene-9',10'-oxygenase, which cleave... more The gene encoding human β-carotene-9',10'-oxygenase, which cleaves the 9',10' double bond in β-carotene into β-apo-10'-carotenal, was cloned and expressed in Escherichia coli. Under aqueous conditions, the optimum organic solvent for the formation of detergent micelles was toluene. The optimum pH, temperature, detergent type, and the optimum concentrations of detergent, substrate, and enzyme for β-apo-10'-carotenal production were 8.0, 37°C, Tween 40, 2.4%, 300 mg β-carotene/l, and 0.25 U/ml, respectively. Under the optimum conditions, 43 mg β-apo-10'-carotenal/l was produced after 21 h with a conversion of 14%. This is the first report to describe the enzymatic production of β-apo-10'carotenal.

Biotechnology Letters, 2011

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones... more The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K (m) of 0.5 mM, and a k (cat) of 2532 s(-1). The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h(-1). Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k (cat), k (cat)/K (m), conversion yield, and productivity in the hydrolysis of daidzin.