• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1660-1669
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• 2015

The present study describes the gene cloning, overexpression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified enzyme were 45℃ and 7.5, respectively. The enzyme demonstrated good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75℃. The kinetic constants V_max and K_m of this nitrilase toward 3-cyanopyridine were 3.12 μmol/min/mg and 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency k_cat/K_m was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid.

• 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.

• Molecules and Cells
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• v.19 no.1
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• pp.97-103
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• 2005

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.

• Preventive Nutrition and Food Science
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• v.11 no.4
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• pp.298-304
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• 2006

This study was carried out to elucidate the relationship among exercise, bone mineral density and antioxidant enzyme activity of postmenopausal women. 60 women residing in the Iksan, Korea area were recruited. The questionnaires were designed to find out exercise habits. Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry. Parameters of antioxidative capacity, including the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (TA) were analyzed in fasting blood. The mean age, height, weight, and BMI of subjects were 65.0 years, 151.1 cm, 59.5 kg $26.0\;m/kg^2$, respectively. The mean BMDs of subjects were $0.85\;g/cm^2$ (lumbar spine), $0.6\;g/cm^2$ (Femoral neck), $0.49\;g/cm^2$ (trochanter), and $0.40\;g/cm^2$ (Ward's triangle). There was a significant difference in BMD among different age groups (50's, 60's and 70's) showing lower value with increasing age (p<0.05). The mean SOD, GPx, and CAT activities were 138.5 U/mL, 1,273.8 U/mL and 314.3 kU/L respectively, and TA was 1.16 mmol/L. TA of the group which exercised 3$\sim$4 times a week was significantly higher than those of the other exercise groups (p<0.05). The subjects with higher SOD activity also have a higher the T values in the lumbar spine, femoral neck, trochanter, and Ward's triangle. In conclusion, this study revealed that the levels of antioxidant enzyme activity were closely associated with the exercise status and bone mineral density in postmenopausal women.

• Applied Chemistry for Engineering
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• v.28 no.6
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• pp.720-725
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• 2017

M(10)-Ni(5)/SBA-15(M=Ce, Nd, Sm) catalysts were prepared for the partial oxidation of methane (POM) to syngas. The catalysts were characterized by BET, TEM, and XPS. The BET-specific surface area and average pore size for M(10)-Ni(5)/SBA-15(M=Ce, Nd, Sm) were 538.8, 504.3, and $447.3m^2/g$ and 6.4, 6.8, and 7.1 nm, respectively. TEM results showed that the mesoporous hexagonol structure was formed for SBA-15, while the homogeneous dispersion of Ni and Ce particles on the surface was formed for Ce(10)-Ni(5)/SBA-15 caused by the confinment effect of SBA-15. XPS data confirmed that $Ce^{4+}$ and $Ce^{3+}$ on the surface catalyst have two oxidation states due to the lattice oxygen species ($O^{2-}$, $O^-$). The yields of POM to syngas over Ce(10)-Ni(5)/SBA-15 were 52.9% $H_2$ and 21.7% CO at 1 atm, 973 K, $CH_4/O_2=2$, $GHSV=1.08{\times}10^5mL/g_{cat.}{\cdot}h$, and these values were kept constant even after 75 h on streams. The same tendency of syngas yields was observed for M(10)-Ni(5)/SBA-15(M=Ce, Nd, Sm). These results confirm that the redox reaction of promoters including Ce, Nd, and Sm enhanced the stability and yield of catalysts.

• KSBB Journal
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• v.27 no.4
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• pp.257-262
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• 2012

The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was $90^{\circ}C$ The half-life of heat inactivation was about 120 min at $90^{\circ}C$ The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest $K_m$ value being for 4-hydroxyindole, highest $k_{cat}$ value for syringaldazine, and highest $k_{cat}/K_m$ for guaiacol.

• Tuberculosis and Respiratory Diseases
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• v.80 no.1
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• pp.77-82
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• 2017

Background: Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Methods: Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. Results: CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-${\gamma}$ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor ${\alpha}$ and interleukin 10, respectively. Conclusion: ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.121-128
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• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• 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.14 no.2
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• pp.344-349
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• 2004

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.