• Applied Biological Chemistry
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• v.48 no.3
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• pp.189-200
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• 2005

Biological nitrogen fixation is an important process for academic and industrial aspects. This review will briefly compare industrial and biological nitrogen fixation and cover the characteristics of biological nitrogen fixation studied in Azotobacter vinelandii. Various organisms can carry out biological nitrogen fixation and recently the researches on the reaction mechanism were concentrated on the free-living microorganism, A. vinelandii. Nitrogen fixation, which transforms atmospheric $N_2$ into ammonia, is chemically a reduction reaction requiring electron donation. Nitrogenase, the biological nitrgen fixer, accepts electrons from biological electron donors, and transfers them to the active site, FeMo-cofactor, through $Fe_4S_4$ cluster in Fe protein and P-cluster in MoFe protein. The electron transport and the proton transport are very important processes in the nitrogenase catalysis to understand its reaction mechanism, and the interactions between FeMo-cofactor and nitrogen molecule are at the center of biological nitrogen fixation mechanism. Spectroscopic studies including protein X-ray crystallography, EPR and $M{\ddot{o}}ssbauer$, biochemical approaches including substrate and inhibitor interactions as well as site-directed mutation study, and chemical approach to synthesize the FeMo-cofactor model compounds were used for biological nitrogen fixation study. Recent research results from these area were presented, and finally, a new nitrogenase reaction mechanism will be proposed based on the various research results.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.28-34
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• 2006

A native extracellular acid phosphatase, phytase (EC 3.1.3.8), from Bacillus coagulans IDCC 1201 (commercially known as Lactobacillus sporogenes) used as probiotics, was characterized. Though some strains of B. coagulans have been evaluated with regard to several health-promoting effects, it has not been reported to produce phytase. Partially purified phytase front the strain IDCC 1201 had a pH optimum of 4.0 and a temperature optimum of $50^{\circ}C$, respectively. The requirement for divalent cations was studied and cobalt ion remarkably increased the enzyme activity. The removal of metal ions from the enzyme by EDTA decreased activity below 50%. The enzyme activity depleted restored when the assay was performed in the presence of $Co^{2+}$. Also, $Co^{2+}$ is the most active stimulator and has unique activation effect at high temperature. The phytase was specific for sodium phytate and p-nitrophenylphosphate, which is different from other known Bacilli phytases. The putative amino acid sequences of the phytase from B. coagulans IDCC 1201 were very similar to that of the phytase from B. subtilis strain 168. Based on these data, we concluded that the phytase from B. coagulans IDCC 1201 is a $Co^{2+}$-dependent acid phosphatase. Therefore, the strain B. coagulans IDCC 1201 is thought to be a valuable addititive for livestocks as well as a beneficial probiotics for human.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.111-123
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• 1988

Neutrophil elastases were purified by a three step procedure consiting of one Sephadex G-75 and two HPLC elutions. The elastases cross-reacted with antibodies to human neutrophil elastase. Three bands with molecular weights between 26,000 and 29,700 were observed by gel electrophoresis. At each stage of purification the quantity of Zn increased, reaching molar ratio of 2:1 with elastase in the most purified samples. Calcium content. was seletively elevated during the earlier stages of purification but decreased to a ratio of 0.25 to 1 with elastase at the final step of purfication. Neutrophil elastase could be inhibited by EDTA, EGTA and 1,10-phenanthroline. EGTA inhbition was noncompetitive inhibition and reversible only if the time of preincubation was relatively short, indicating the instability of the apoenzyme. The concentration of chelator required to show significant inhibition of elastase was also dependent upon the stage of purity and the ionic strength of the reaction mixture. Inhibition by EGTA, followed by the removal of EGTA, could be reversed by Zn. In the presence of EGTA the enzyme could be returened to full activity by the addition of Zn, Mn and Ca, but not Mg or Na. All of the above evidence strongly supports human neturophil elastase could be a metalloenzyme as well as a serine protease.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.551-558
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• 1992

The extracellular endoinulase from Streptomyces sp. 556 was purified and characterized, The culture broth was fractionated by ammonium sulfate saturation followed by DEAE-cellulose column chromatography and 5ephadex G-200 gel filtration, The ultimately purified fraction revealed a single band in 7.5% polyacrylamide gel electropherogram. The purified enzyme showed the maximal activity at pH 5.5-6.0 and $50^{\circ}C$, but lost 93% of inulase activity after 30 min incubation at $55^{\circ}C$ . The essen.tial amino acid residue for catalytic activity appeared to be tryptophan. This endo inulase was activated by $Mn^{2+}$, whereas inactivated by $Ag^{+}$, $Hg^{+}$, $Cu^{2+}$, $Zn^{2+}$, $Fe^{3+}$ and $Mo^{6+}$ EDTA and 8-hydroxyquinoline inhibited the enzyme so that the enzyme was considered to be a metalloenzyme. The Km value for inulin was 0.287 mM, and no invertase or $\alpha$-glucosidase activity was found in the enzyme.

• Bulletin of the Korean Chemical Society
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• v.33 no.1
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• pp.48-54
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• 2012

The $[Fe_4S_4]^{2+}$ clusters with 2-, 3-, and 4-pyridinemethanethiolate (S2-Pic, S3-Pic, and S4-Pic, respectively) terminal ligands have been synthesized from the ligand substitution reaction of the $(^nBu_4N)_2[Fe_4S_4Cl_4]$ (I) cluster. The new $(^nBu_4N)_2[Fe_4S_4(SR)_4]$ (R = 2-Pic; II, 3-Pic; III, 4-Pic; IV) clusters were characterized by FTIR and UV-Vis spectroscopy. Cluster II was crystallized in the monoclinic space group C2/c with a = 24.530 (5) $\AA$, b = 24.636(4) $\AA$, c = 21.762(4) $\AA$, ${\beta}=103.253(3)^{\circ}$, and Z = 8. The X-ray structure of II showed two unique 2:2 site-differentiated $[Fe_4S_4]^{2+}$ clusters due to the bidentate-mode coordination by 2-pyridinemethanethiolate ligands. Cluster III was crystallized in the same monoclinic space group C2/c with a = 26.0740(18) $\AA$, b = 23.3195(16) $\AA$, c = 22.3720(15) $\AA$, ${\beta}=100.467(2)^{\circ}$, and Z = 8. The 3-pyridinemethanethiolate ligand of III was coordinated to the $[Fe_4S_4]^{2+}$ core as a terminal mode. Cluster IV with 4-pyridinemethanethiolate ligands was found to have a similar structure to the cluster III. Fully reversible $[Fe_4S_4]^{2+}/[Fe_4S_4]^+$ redox waves were observed from all three clusters by cyclic voltammetry measurement. The electrochemical potentials for the $[Fe_4S_4]^{2+}/[Fe_4S_4]^+$ transition decreased in the order of II, III and IV, and the reduction potential changes by the ligands were explained based on the structural differences among the complexes. The complex III was reacted with sulfonium salt of $[PhMeSCH_2-p-C_6H_4CN](BF_4)$ in MeCN to test possible radical-involving reaction as a functional model of the [$Fe_4S_4$]-SAM (S-adenosylmethionine) cofactor. However, the isolated reaction products of 3-pyridinemethanethiolate-p-cyanobenzylsulfide and thioanisole suggested that the reaction followed an ionic mechanism and the products formed from the terminal ligand attack to the sulfonium.

• Journal of Life Science
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• v.23 no.12
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• pp.1557-1561
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• 2013

Carbonic anhydrase isozymes are a widespread, zinc-containing metalloenzyme family. The enzyme catalyzes the reversible inter-conversion of $CO_2$ and $HCO_3$. This reaction is the main role played by CA enzymes in physiological conditions. This enzyme has been found in virtually all organisms, and at least 16 isozymes have been isolated in mammals. Unlike mammals, there is little information available regarding CA isozymes in the tissues of non-mammalian groups, such as fish. Carbonic anhydrase is very important in the osmotic and acid-base regulation in fish. It is well-known that the gills of fish play the most important role in acid-base relevant ion transfer, the transfer of $H^+$ and/or $HCO_3^-$, for the maintenance of systemic pH. Rainbow trout, Oncorhynchus mykiss, is the most important freshwater fish species in the aquaculture industry of Korea, with annual production increasing each year. In addition, environmental toxicology research has shown that rainbow trout is known to be the species that is most susceptible to environmental toxins. Consequently, carbonic anhydrase was detected in rainbow trout, Oncorhynchus mykiss. The isolated protein showed the specific band with a molecular weight of 30 kDa and pI of 7.0, and it was identified as being carbonic anhydrase. The immunohistochemical result demonstrated that the carbonic anhydrase was located in the epithelial cells of the gills.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.546-554
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• 2019

This study, for the first time, reports the functional expression of lipase B derived from the yeast Candida antarctica (CALB) in Corynebacterium strain using the Escherichia coli plasmid PK18. The CALB gene fragment encoding a 317-amino-acid protein was successfully obtained from the total RNA of C. antarctica. CALB was readily produced in the Corynebacterium strain without the use of induction methods described in previous studies. This demonstrated the extracellular production of CALB in the Corynebacterium strain. CALB produced in the Corynebacterium MB001 strain transformed with pEC-CALB recombinant plasmid exhibited maximum extracellular enzymatic activity and high substrate affinity. The optimal pH and temperature for the hydrolysis of 4-nitrophenyl laurate by CALB were 9.0 and 40℃, respectively. The enzyme was stable at pH 10.7 in the glycine-KOH buffer and functioned as an alkaline lipase. The CALB activity was inhibited in the presence of high concentration of Mg²⁺, which indicated that CALB is not a metalloenzyme. These properties are key for the industrial application of the enzyme.

• Journal of Applied Biological Chemistry
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• v.51 no.6
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• pp.253-261
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• 2008

Isoflavone daidzein is a phytoestrogen widely distributed in Leguminosae and is especially rich in the soybean. The C6-C3 (rings B and C) unit of isoflavones is derived from the phenylpropanoid pathway and the remaining C6 (ring A) unit is from the polyketide pathway. This unique carbon skeleton is the result of isomerization of the flavone catalyzed by the isoflavone synthase, a cytochrome P450 enzyme. The isoflavones daidzein and genistein are present in the plant mostly in the glucosylated forms. However, in the human intestine, the glycosidic linkage is broken, and the free form is uptaked into blood stream. The free form is further metabolized into various reduction products to end up at the equol, which is known to have the most potent estrogenic effect among the metabolites. Several human intestinal bacteria that can convert daidzein into equol have been described, and the study into the chemistry and biochemistry of the daizein reduction would be rewarding to the improvement of the human health.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.2
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• pp.33-42
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• 2011

Zinc plays a key role in genetic expression, cell division, and cell growth and is essential for the functions of more than 450 metalloenzyme. There are high concentrations of zinc in pigment epithelium in bullfrog eye. Zinc deficiency causes night blindness and abnormal dark adaptation. The purpose of this study was to identify ERG (electroretinogram) b-wave sensitivity during light and dark adaptation in bullfrog retina after zinc and zinc chelators treatment such as histidine and TSQ (N-(6-methoxy-8-qunolyl)-p-toluenesulfon amide). Especially, we focused whether histidine act as a zinc chelator in the Muller cell. The results of our study are summarized as follows: 1) Both zinc and histidine elevated ERG b-wave amplitude and threshold in Muller cells by accelerating rhodopsin regeneration time and increased a-peak absorbance during light adaptation. 2) TSQ reduced those by prolonging rhodopsin regeneration time and decrement of a-peak absorbance during light adaptation. 3) Zinc shortened rhodopsin regeneration time and prolonged a-peak absorbance. These results suggested that histidine may act as a zinc-mediated transporter in presynaptic Muller cell membrane rather than zinc chelator and acts as a GABA-receptor inhibitor which blocks $Cl^-$ influx to the postsynapse.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.579-587
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• 2018

For biotechnological production of high-valued ${\beta}-{\text\tiny{D}}$-hexyl glucoside, the catalytic properties of Hanseniaspora thailandica BC9 ${\beta}$-glucosidase purified from the periplasmic fraction were studied, and the transglycosylation activity for the production of ${\beta}-{\text\tiny{D}}$-hexyl glucoside was optimized. The constitutive BC9 ${\beta}$-glucosidase exhibited maximum specific activity at pH 6.0 and $40^{\circ}C$, and the activity of BC9 ${\beta}$-glucosidase was not significantly inhibited by various metal ions. BC9 ${\beta}$-glucosidase did not show a significant activity of cellobiose hydrolysis, but the activity was rather enhanced in the presence of sucrose and medium-chain alcohols. BC9 ${\beta}$-glucosidase exhibited enhanced production of ${\beta}-{\text\tiny{D}}$-hexyl glucoside in the presence of DMSO, and 62% of ${\beta}-{\text\tiny{D}}$-hexyl glucoside conversion was recorded in 4 h in the presence of 5% 1-hexanol and 15% DMSO.