Yeong-Su Kim

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

ABSTRACT The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia.

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.

Lactulose production from lactose as a single substrate by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

The conditions for maximum lactulose production from lactose, as a single substrate, by a thermostable recombinant cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus were determined to be pH 7.5, 80 °C, 700 g l(-1) lactose, and 150 U ml(-1) of enzyme. Under the conditions, the enzyme produced the two bifidus factors lactulose at 408 g l(-1) and epilactose at 107 g l(-1) after 2 h. The yields of lactulose and epilactose from lactose and the productivities of lactulose and epilactose were 58%, 15%, 204 g l(-1) h(-1), and 54 g l(-1) h(-1), respectively. The yield and productivity of both lactulose and epilactose from lactose were 74% and 258 g l(-1) h(-1), respectively. The yield, concentration, and productivity of lactulose in the present study are the highest among enzymatic syntheses. This is the first trial of enzymatic synthesis of lactulose using the single substrate lactose.

Characterization of a recombinant β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus

A recombinant beta-glucosidase from Caldicellulosiruptor saccharolyticus DSM 8903 with a specific activity of 13 U/mg was purified by heat treatment and His-Trap affinity chromatography and identified as a single 54 kDa band on SDS-PAGE. The molecular mass of the native enzyme was 108 kDa as a dimer by gel filtration. beta-Glucosidase showed optimum activity at pH 5.5 and 70 degrees C for p-nitrophenyl (pNP)-beta-d-glucopyranoside. The half-lives of the enzyme at 60, 70, and 80 degrees C were 250, 24.3, and 0.4 h, respectively. The enzyme exhibited catalytic efficiency and specific activity for pNP-beta-d-fucopyranoside, pNP-beta-d-glucopyranoside, and pNP-beta-d-galactopyranoside in decreasing order among aryl-beta-glycosides, but not for aryl-alpha-glycosides. Cello-oligosaccharides from n = 2 to 5 as substrates using 4 mM each sugar and 3 U/mg of enzyme were completely hydrolyzed to glucose at 70 degrees C within 16 h.

Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus.

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.

Characterization of a GH3 Family β-Glucosidase from Dictyoglomus turgidum and Its Application to the Hydrolysis of Isoflavone Glycosides in Spent Coffee Grounds

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.

Borate enhances the production of lactulose from lactose by cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus was used in the presence of borate to increase the production of lactulose from lactose. Maximum production of lactulose occurred using a 1:1M ratio of borate-lactose. Under this condition, the enzyme produced 614 g l(-1) lactulose from 700 g l(-1) lactose after incubation at pH 7.5 and 80 °C for 3h, with a conversion yield of 88% and a productivity of 205 g l(-1) h(-1). The yield and productivity of lactulose production obtained in the present study are among the highest achieved through chemical or biological synthesis.

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant.

ABSTRACT 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 Cu2+. 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/Km ) 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/Km 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/Km . 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.

Hydrolytic properties of a thermostable α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims:  To characterize of a thermostable recombinant α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l‐arabinose.

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The Km, kcat, and kcat/Km values for β-carotene as substrate were 37 μm, 3.6 min−1, and 97 mm−1 min−1, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C35 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 β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-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 β-carotene-cleaving enzyme.