• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.532-541
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• 2010

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1653-1663
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• 2010

The first gene (${\alpha}$-gal1) encoding an extracellular ${\alpha}$-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The ${\alpha}$-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal ${\alpha}$-galactosidases belonging to glycosyl hydrolase family 27. The ${\alpha}$-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant ${\alpha}$-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at $70^{\circ}C$, pH 4.5, and lost no activity over 10 days at $50^{\circ}C$. ${\alpha}$-Gal1 followed Michaelis-Menten kinetics ($V_{max}\;of\;240.3{\mu}M/min/mg,\;K_m\;of\;0.294 mM$) and was inhibited competitively by galactose ($K_m{^{obs}}$ of 0.57 mM, $K_i$ of 2.77 mM). The recombinant T. emersonii ${\alpha}$-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the ${\alpha}$-galactosidic linkage. Owing to its substrate preference and noteworthy stability, ${\alpha}$-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.

• Mycobiology
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• v.43 no.3
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• pp.280-287
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• 2015

In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at $15^{\circ}C$ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and $5^{\circ}C$higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and $50^{\circ}C$, which may reflect the physiological conditions at the primordiation stage.

• Applied Biological Chemistry
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• v.31 no.2
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• pp.137-142
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• 1988

The analysis of condensation and cleavage reaction was carried out at $30^{\circ}C$ and pH 7.0 with purified isocitrate lyase from Saccharomycopsis lipolytica ATCC 44601. The Km values for condensation reaction of glyoxylate and succinate were 0.06 and 0.21 mM, respectively. In the cleavage reaction, glyoxylate was a linear competitive inhibitor with a Ki of 0.22 mM and succinate was a linear noncompetitive inhibitor with a Ki of 0.82 mM. Therefore, these kinetic analyses showed that the enzyme functioned in a ordered reaction with glyoxylate binding before succinate in the condensation reaction. 3-Bromopyruvate(BrP) was found to be irreversibly inactivation showing saturation kinetics, the inactivation half-time was 0.15 min and $K_{BrP}$ was 0.032 mM, and substrate or reactant protected against the inactivation.

• Toxicological Research
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• v.12 no.2
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Molecules and Cells
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• v.43 no.9
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• pp.784-792
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• 2020

Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK.

• Archives of Pharmacal Research
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• v.22 no.2
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• pp.219-224
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• 1999

In our search for the anticonvulsant consitutent of Gastrodia elata repeated column chromatographies guided by activity assay led to isolation of an active compound, which was identified as gastrodin on the basis of spectral data. Brain succinic semialdehyde dehydrogenase (SSADH) was inactivated by preincubation with gastrodin in a time-dependent manner and the reaction was monitored by absorption and fluorescene spectroscopic methods. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of $1.2{\times}10^{3} M^{-1} min^{-1}$. The time course of the reaction was significantly affected by the coenzyme NAD^{+}$, which affected complete protection against the loss of the catalytic activity, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme. It is postulated that the gastrodin is able to elevate the neurotransmitter GABA levels in central nervous system by inhibitory action on one of the GABA degradative enzymes, SSADH.

• Journal of Life Science
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• v.22 no.2
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• pp.209-219
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• 2012

The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific $C_4$ isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific $C_4$ isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the $A_4$ than the $B_4$ isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific $C_4$ isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific $C_4$ isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The ${K_m}^{PYR}$ of eye-specific $C_4$ was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific $C_4$ isozymes were high in ${\alpha}$-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific $C_4$ isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific $C_4$ isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific $C_4$ isozyme in M. salmoides was used at the first stage of predatory behavior.

• Journal of Sensor Science and Technology
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• v.6 no.1
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• pp.35-42
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• 1997

The optical biosensor using the electrically controlled release of reactive reagent is developed for the detection of peroxide. Rapid degradation of polymer complex of PEOx and PMAA occurs as the applied current increases and thus released amount of HPA increases. The degradation velocity of polymer and the amount of HPA released are linearly proportional to the applied current. Peroxide is reacted with the released reagent by peroxidase and then the product, a fluorescent dimer DBDA, is formed. The monochromic light from light source (150W Xe arc ramp) excites the DBDA and the excited light is transmitted through an optical fiber to be detected by a photodiode array. The change of fluorescence intensity is related to the change of peroxide concentration. The peroxidase is entrapped in Ca-alginate get on the inner surface. The biosensor has the linear signal range of 0.025mM-10.mM peroxide. By applying the step function of peroxide, reproducibility of biosensor has been investigated. The mathematical model is constructed by the combination of enzyme kinetics with reactor flow model. Good agreement is obtained between the experimental result and model prediction in the sensor signal.

• The Journal of the Korean Society for Microbiology
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• v.34 no.6
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• pp.533-542
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• 1999

H. pylori produces urease abundantly amounting to 6% of total protein of bacterial mass. Urease genes are composed of a cluster of 9 genes of ureC, ureD, ureA, ureB, ureI, ureE, ureF, ureG, ureH. Production of H. pylori urease in E. coli was studied with genetic cotransformation. Structural genes ureA and ureB produce urease apoprotein in E. coli but the apoprotein has no enzymatic activity. ureC and ureD do not affect urease production nor enzyme activity ureF, ureG, and ureH are essential to produce the catalytically active H. pylori urease of structural genes (ureA and ureB) in E.coli. The kinetics of activation of H. pylori urease apoprotein were examined to understand the production of active H. pylori urease. Activation of H. pylori urease apoprotein, pH dependency, reversibility of $CO_2$ binding, irreversibility of $CO_2$ and $Ni^{2+}$ incorporation, and $CO_2$ dependency of initial rate of urease activity have been observed in vitro. The intrinsic reactivity (ko) for carbamylation of urease apoprotein co expressed with accessory genes was 17-fold greater than that of urease apoprotein expressed without accessory genes. It is concluded that accessory genes function in maximizing the carbamylating deprotonated ${\varepsilon}$-amino group of Lys 219 of urease B subunit and metallocenter of urease apoprotein is supposed to be assembled by reaction of a deprotonated protein side chain with an activating $CO_2$ molecule to generate ligands that facilitate productive nickel binding.