• Applied Biological Chemistry
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• v.41 no.8
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• pp.608-610
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• 1998

To investigate the inhibitory activity of 2-hydroxy-3-amino-4-phenylbutyrate-harboring aminopeptidase N inhibitors, p-nitro-AHPA-peptide derivatives (1 and 2) and an AHPA-peptide derivative (3) were synthesized by chain elongation from C-terminal end using DCC/HOBt as a coupling reagent. The peptides $1{\sim}3$ exerted strong inhibitory activities against aminopeptidase N with $IC_{50}$ values of 1.8, 7.3 and $24.0\;{\mu}g/ml$, respectively, and cytotoxicity on cancer cell lines in vitro.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.119-125
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• 1994

Steroid $\Delta^1$-dehydrogenase purified from hydrocortisone-induced cells of Arthrobacter simplex converted various 3-ketosteroids into their corresponding $\Delta^1$-dehydrogenated products. The transformation efficiencies depend upon the chemical structure of the steroids, especially length of the side chain at 17 position and hydroxyl groups at 11 and 17 positions. The Km values for androstenedione, the most favorable substrate examined, and hydrocortisone were 74 ${\mu}M$ and 294 ${\mu}M$, respectively. The optimum temperature and pH of the enzyme reaction were 35$^{\circ}C$ and pH 9, respectively, and the enzyme was relatively stable at the range from 20 to 35$^{\circ}C$ and from pH 5 to 10 after one hour of incubation. The enzyme activity was markedly inhibited in the presence of $Cu^{2+},\;Fe^{3+},\;Hg^{2+},\;Mo^{6+}$ ions, and somewhat inhibited by $Zn^{2+}$ and $Fe^{2+}$. $\alpha,\alpha'$-Dipyridyl that inhibits 9$\alpha$-hydroxylase and accumulates 1,4-androstadiene-3,17-dione from sterols revealed no inhibitory effect on this enzyme. EGTA showed inhibitory effect. $\beta$-Estradiol competitively inhibited the enzyme activity. Chemical modifications of the enzyme were attempted with several reagents. p-Hydroxymer-curibenzoate showed inhibition of the enzyme activity and protection of the substrate. This suggests that cysteine residue may be involved in the active site of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.765-775
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• 2019

A new ${\alpha}$-amylase-encoding gene (amySL3) of glycoside hydrolase (GH) family 13 was identified in soda lake isolate Alkalibacterium sp. SL3. The deduced AmySL3 shares high identities (82-98%) with putative ${\alpha}$-amylases from the genus Alkalibacterium, but has low identities (<53%) with functionally characterized counterparts. amySL3 was successfully expressed in Escherichia coli, and the recombinant enzyme (rAmySL3) was purified to electrophoretic homogeneity. The optimal temperature and pH of the activity of the purified rAmySL3 were determined to be $45^{\circ}C$ and pH 7.5, respectively. rAmySL3 was found to be extremely halophilic, showing maximal enzyme activity at a nearly saturated concentration of NaCl. Its thermostability was greatly enhanced in the presence of 4 M NaCl, and it was highly stable in 5 M NaCl. Moreover, the enzyme did not require calcium ions for activity, and was strongly resistant to a range of surfactants and hydrophobic organic solvents. The major hydrolysis products of rAmySL3 from soluble starch were maltobiose and maltotriose. The high ratio of acidic amino acids and highly negative electrostatic potential surface might account for the halophilic nature of AmySL3. The extremely halophilic, calcium-independent, and surfactant-resistant properties make AmySL3 a promising candidate enzyme for both basic research and industrial applications.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.99-102
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• 2000

To establish optimal conditions for esterification by Candida cylindracea, lipase reactions were performed simultaneously, separately, or individually in the varying initial rates of $0.014-0.060\mu$mole free fatty acids consumed min-1g-1. The reactants which were conditioned at aw of 0.12 gave the highest initial rate of esterifying $0.060\mu$mole free fatty acids consumed min-1g-1. These results suggest that the esterifying activity of lipase in an organic system depends on the transfer of available water within the reaction system.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.829-835
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• 2004

A fibrinolytic enzyme has been found in several bacteria isolated from fermented food. This study was carried out to investigate the purification and characteristics of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 originated from Chungkookjang. The fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate fractionation and chromatographies on DEAE-cellulose and on Sephadex G-100. The final specific activity of the purified enzyme increased 11.0-fold, and the protein amount in the purified enzyme was about 16% of that in the culture supernatant. The molecular weight of the purified enzyme was estimated to be about 45,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 7.0 and $60^{\circ}C$, respectively. The enzyme activity was relatively stable up to $60^{\circ}C$ over the pH range of 7.0-10.0. The fibrinolytic enzyme activity increased by $Ca^{2+}$ and $Cu^{2+}$, whereas it was inhibited by $Hg^{2+}$ and $Ba^{2+}$. In addition, it was severely inhibited by PMSF and DFT. It is suggested that the purified enzyme was a serine protease for the fibrinolysis. The purified enzyme could completely hydrolyze fibrin in vitro within 8 h. Hence, it is suggested that the purified enzyme can be put into practice as an effective thrombolytic agent.

• Journal of Applied Biological Chemistry
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• v.42 no.4
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• pp.169-174
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• 1999

Arabidopsis AHL gene contains 4 exons encoding a putative protein highly homologous to the yeast salt-sensitive enzyme HAL2, a 3'(2'),5'-bisphosphate nucleotidase involving in reductive sulfate assimilation. AHL cDNA complemented yeast met22 (hal2) mutant. AHL fusion protein expressed in E. coli exhibited $Mg^{2+}$-dependent, 3'-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase activity. $Li^+,\;Na^+,\;K^+$ and $Ca^{2+}$ ions inhibit the enzyme activity by competing with $Mg^{2+}$ for the active site of the enzyme. The enzyme activity was also sensitive to ${\mu}M$ concentrations of toxic heavy metal ions such as $Cd^{2+},\;Cu^{2+}$ and $Zn^{2+}$, but was not recovered by addition of more $Mg^{2+}$ ions, suggesting that these ions inactivate the enzyme with a mechanism other than competition with $Mg^{2+}$ ions. Inhibition of the AHL enzyme activity may result in accumulation of PAP, which is highly toxic to the cell. Thus, the AHL enzyme could be one of the intial targets of heavy metal toxicity in plants.

• BMB Reports
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• v.41 no.11
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• pp.790-795
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• 2008

An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of $NAD^+$, was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and $NAD^+$ served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The $K_m$ values for L-lysine and $NAD^+$ were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.

• Toxicological Research
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• v.26 no.3
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• pp.171-175
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• 2010

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme. The recombinant CYP2W1 protein containing a $6{\times}$(His)-tag at Nterminus has been expressed in Escherichia coli and purified. Expression level of CYP2W1 holoenzyme was around 500 nmol P450 holoenzyme per liter culture medium. The reduced CO difference spectrum of CYP2W1 showed a maximum absorption at 449 nm. CYP2W1 indicated the significant induction to bioactivate Trp-P-1, MeIQ, and IQ in E. coli DJ701 tester strain. However, the bioactivation of B[$\alpha$]P, and NNK by CYP2W1 was relatively low. The model structure of CYP2W1 suggested the characteristic P450 folds with the lengths and orientations of the individual secondary elements. The F-G loop is situated on the distal side of heme to accommodate the flexibility of active site of CYP2W1. These studies can provide useful information for the finding of its biological roles and structure-function relationships of an orphan CYP2W1 enzyme.

• BMB Reports
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• v.40 no.3
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• pp.412-418
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• 2007

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% $\alpha$-helix, 30.7%$\beta$-sheet, 18.5% $\delta$-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27$^{\circ}C$. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and $H_2O_2$. These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

• Journal of Soil and Groundwater Environment
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• v.26 no.5
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• pp.49-59
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• 2021

The effects of green manure crops, hairy vetch and sesban, supplemented with HS (humic substance) on biological soil health indicators was studied in a pot containing two kinds of reclaimed soil previously contaminated with petroleum hydrocarbons; a soil remediated by land-farming (DDC) and another soil by low-temperature thermal desorption (YJ). Treatments include no plant (C), plants only (H), and plants+2% HS (PH), which were evaluated in a pot containing respective soil. Biological indicators include microbial community analysis as well as soil enzyme activities of dehydrogenase, 𝛽-glucosidase, N-acetyl-𝛽-D-glucosaminidase (NAG), acid/alkaline phosphatase, arylsulfatase, and urease. Results showed an increase of enzyme activities in pot soils with plants and even greater in soils with plants+HS. The enzyme activities of DDC soil with plants (DDC_P) and with plants+HS (DDC_PH) increased 1.6 and 3.9 times on average, respectively than those in the control. The enzyme activities YJ soil with plants (YJ_P) and with plant+HS (YJ_PH) increased 1.8 and 3.8 times on average, respectively than those in the control. According to microbial community analysis, the relative abundance of nitrogen-fixing bacteria in DDC and YJ soil was increased from 1.5% to 7% and from 0 to 5%, respectively, after planting hairy vetch and sesban. This study showed that mixed planting of green manure crops with a supplement of humic substance is highly effective for the restoration of biological health indicators of reclaimed soils.