• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

• Korean Chemical Engineering Research
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• v.54 no.3
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• pp.380-386
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• 2016

In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity ($R^2$ > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the $4^{\circ}C$. Finally, we demonstrated whether the Michaelis-Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions ($K_m=8.327{\times}10^{-3}M$), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.292-300
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• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.

• BMB Reports
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• v.39 no.2
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• pp.167-170
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• 2006

Bovine adenosine deaminase undergoes a nondenaturational conformational change at $29^{\circ}C$ upon heating which is characterized by a large increase in heat capacity. We have determined the transition state thermodynamics of the conformational change using a novel application of differential scanning calorimetry (DSC) which employs very slow scan rates. DSC scans at the conventional, and arbitrary, scan rate of $1^{\circ}C/min$ show no evidence of the transition. Scan rates from 0.030 to $0.20^{\circ}C/min$ reveal the transition indicating it is under kinetic control. The transition temperature $T_t$ and the transition temperature interval ${\Delta}T$ increase with scan rate. A first order rate constant $k_1$ is calculated at each $T_t$ from $k_1\;=\;r_{scan}/{\Delta}T$, where $r_{scan}$ is the scan rate, and an Arrhenius plot is constructed. Standard transition state analysis reveals an activation free energy ${\Delta}G^{\neq}$ of 88.1 kJ/mole and suggests that the conformational change has an unfolding quality that appears to be on the direct path to the physiological-temperature conformer.

• Journal of Nutrition and Health
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• v.10 no.4
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• pp.24-32
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• 1977

It was attempted in this study to assess the vitamin $B_{1},\;B_{2}$, and $B_6$ status in tissue by determination of erythrocyte transketolase (TK), glutathione reductase (GR), and aspartate aminotransferase (AST) activities, and their activation by their respective coenzymes, thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate. The activities of erythrocyte enzymes were stable for more than 30 days when erythrocyte had been stored at $-20^{\circ}C$ and affirmed that the enzyme activities were more stable in the case of deep frozen sotrage of erythrocytes rather than hemolysates. The assay procedures involving ultraviolet kinetic analysis with continuous monitoring for each of enzymes have good within-batch and between-batch precisions and will be avalable in the routine laboratories for the nutritional and clinical surveys. Activity coefficient of TK, GR, and AST was studied in healthy medical students (fifteen men and twelve women, between 21 and 30 years old) on an unrestricted diet. The mean activity coefficient of TK, GR, and AST were 1.18, 1.35, and 2.01 for men, and 1.14, 1.33, and 1.83 for women, respectively. And the upper limit of normal (mean+2SD) were 1.52, 1.69, and 2.61 for men, and 1.50, 1.61, and 2.37 for women, respectively.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.874-879
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• 2008

Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be reduced by treatment with depigmenting agents. The methanol extract of Lespedeza cyrtobotrya $M_{IQ}$ showed inhibitory activity against mushroom tyrosinase. The active compound was purified from the methanol extract of L. cyrtobotrya, followed by several chromatographic methods, and identified as dalbergioidin (DBG) by spectroscopic methods. The results showed that DBG exhibited tyrosinase inhibitory activity with an $IC_{50}$ of $20\;{\mu}M$. The kinetic analysis of tyrosinase inhibition revealed that DBG acted as a noncompetitive inhibitor. In addition, DBG showed a melanin biosynthesis inhibition zone in the culture plate of Streptomyces bikiniensis that has commonly been used as an indicator organism. Furthermore, $27\;{\mu}M$ DBG decreased more than 50% of melanin contents on the pigmentation using the immortalized mouse melanocyte, melan-a cell.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.230-237
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• 1991

Citrate synthase 1 (mitochondrial) and citrate synthase 2 (nonmitochondrial) were purified from Saccharomyces cerevisiae. The physical and enzymatic characteristics of citrate synthase 2 were ananlyzed in comparison with citrate synthase 1. Both isoenzymes were shown to be dimeric proteins of identical subunits, and the molecular weights of the subunits were estimated to be 48.3kDa for citrate synthase 1 and 47.0kDa for citrate synthase 2, respectively. The optimal pH value for enzyme activity was pH 7.5 for both isoenzymes. However, the optimal temperature for the activity was strikingly different; while the activity of citrate synthase 1 reached its peak at 65.deg.C, that of citrate synthase 2 was maximal at 40.deg.C. Citrate synthase 2 showed much lower thermal and pH stability than citrate synthase 1. In addition, citrate synthase 2 was affected much more by the metal ions such as $Zn^{2+}$ , $Mn^{2+ , and $Co^{2+} than citrate synthase 1. Among the several possible regulatory metabolites tested, ATP showed the strongest inhibitory effect on both enzymes. ADP and NADH were found to have greater effect on citrate synthase 2 than on citrate synthase 1. Kinetic analysis revealed that citrate synthase 2 has approximately 7- and 3.5-fold lower affinity to acetyl CoA and to oxaloacetate, respectively, than citrate synthase 1.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.1-6
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• 2023

Dendranthema zawadskii is a one of the popular plants as native in South Korea. In this study, linarin was isolated and purified using silica-gel, Diaion, and Sephadex LH-20 from the aerial parts of D. zawadskii. The chemical structure was completely identified through spectroscopic data including 1D, 2D nucleic magnetic resonance, and HRFABMS. Furthermore, linarin inhibited the bacterial neuraminidase (BNA) activity with 13.5 μM of IC₅₀ dose-dependently. Through the enzyme kinetic experiments, linarin as BNA inhibitor exhibited a typical noncompetitive inhibition mode which K_m was contestant and V_max decreased as the concentration of the inhibitor increased. It was further identified that the inhibition constant was 16.0 μM. Linarin was the most abundance metabolite in the aerial part of D. zawadskii extract by UHPLC-TOF/MS analysis. Therefore, D. zawadskii and its main component are expected that it can be effectively used for the infection and inflammation caused by bacteria.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.