• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.881-888
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• 2010

solvent-tolerant bacterium strain, MH6, was isolated by hydrophilic organic solvent DMSO enrichment in the medium and identified as Serratia marcescens. The extracellular protease with novel organic-solvent-stable properties from strain MH6 was purified and characterized. The molecular mass of the purified protease was estimated to be 52 kDa on SDS-PAGE. The open reading frame (ORF) of the MH6 protease encoded 504 amino acids with 471 amino acid residues in the mature protease. Based on the inhibitory effects of EDTA and 1,10-phenathroline, the MH6 protease was characterized as a metalloproteinase. The enzyme activity was increased in the presence of $Ni^{2+}$, $Mg^{2+}$, and $Ca^{2+}$. The protease could also be activated by the nonionic surfactants Tween 80 (1.0%) and Triton X-100 (1.0%). The protease showed remarkable solvent stability in the presence of 50% (v/v) solutions of long-chain alkanes and long-chain alcohols. It was also fairly stable in the presence of 25% solutions of hydrophilic organic solvents. Owing to its high stability in solvents and surfactants, the MH6 protease is an ideal candidate for applications in organic catalysis and other related fields.

• BMB Reports
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• v.41 no.11
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• pp.808-813
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• 2008

Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin $H_2$ ($PGH_2$) into prostaglandin $E_2$ ($PGE_2$). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The $K_m$ and $V_{max}$ of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with $I_{C50}$ values of 100 and 200 ${\mu}M$, respectively. As these compounds competitively inhibited the catalysis of $PGH_2$, their binding sites appeared to be located near the $PGH_2$ binding pocket.

• Journal of Life Science
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• v.10 no.2
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.68-76
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• 2001

We have employed chemical modification to identify amino acids essential for the catalytic activity of alliinase (EC 4.4.1.4) from garlic (Allium sativum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the characteristic odor of garlic. The activity of alliinase was rapidly and completely inactivated by N-bromosuccinimide(NBS) and slightly decreased by succinic anhydride and N-acetylimidazole. These results indicate that tryptophanyl, lysyl, and tyrosyl residues play an important role in enzyme catalysis. The reaction of alliinase with NBA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with increasing reagent concentration of NBS, consistent with the oxidation of tryptophan residues. Kinetic analysis, fluorometric titration of tryptophans and correlation to residual alliinase activity showed that modification of only one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemical modification by NBS in the presence and absence of the protecting substrate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing residues 179-188 was isolated. We conclude that Trp182 is essential for alliinase activity.

• Analytical Science and Technology
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• v.19 no.1
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• pp.58-64
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• 2006

A mugwort-tissue-based modified carbon paste electrode was constructed for the amperometric detection of hydrogen peroxide and its electrochemical properties are described. Especially the amperometric signal was very stable and bigger than any other enzyme electrode studied in this lab. The effect of tissue composition on the response was linear within the wide range of experiment and the linearity of Lineweaver-Burk plot showed that the sensing process of the biosensor is by enzymatic catalysis. And pH dependent current profile connoted that two isozymes are active in this system.

• Journal of Life Science
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• v.19 no.2
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• pp.228-233
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• 2009

The purpose of this study is to evaluate the activities of five different antioxidant enzymes in various tissues of Papilio xuthus during pupal stage. Superoxide dismutase (SOD) activity in haemolymph was the highest just after pupation and then decreased gradually until 7 days after pupation but the activity in other tissue was constant during metamorphosis. This result indicates that primary antioxidant process of reactive oxygen species proceed in haemolymph. Catalase (CAT) activity in studied tissues was also the highest just after pupation and its relative activity was also high during pupal stage, suggesting that CAT is the primary enzyme in catalysis of hydrogen peroxide. Glutathion peroxidase (GPX) activity was constant and its relative activity was very low in all tested tissues. Glutathione S-transferase (GST) activity in haemolymph was high at 3 days and 5 days after pupation, and the activity in fat body was the highest at the 1 day after pupation and then decreased gradually for 7 days after pupation. Glutathion reductase (GR) activity in haemolymph and fat body was high at 1 day after pupation, but relatively low GR activity was detected in the rest tissues. Based on these results, GST activity was higher than that of GPX and GR, and it is also believed that GST was more involved in reduction process through lipid peroxidation than GPX.

• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.195-201
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• 2010

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.140-144
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• 2003

Type Ⅰ signal peptidase is an integral membrane protein that functions to cleave signal peptides from secreted and membrane proteins. The enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological properties. Despite being one of the best characterized enzymes, the catalysis of Type Ⅰ signal peptidase still remains controversy over the catalytic serine/lysine dyad mechanism. It appears that the dyad proteases are generally less efficient than the prototypical serine/histidine/aspartic acid triad found in most enzymes, although Type Ⅰ signal peptidase is an exception to this rule. In this paper, we have proposed that Type Ⅰ signal peptidase may act as the serine/lysine/aspartic acid triad cataltytic mechanism. Therefore, the aspartic acid 99 residue in the E. coli signal peptidase was chosen and mutated to an alanine to see if there is any possible role of the aspartic acid in the catalytic function. Type Ⅰ signal peptidase D99A protein was inactive in vitro assay using the procoat synthesized by in vitro transcription translation. However, the mutant was active using a highly sensitive in vivo assay. Pulse-chase experiments show that the replacement of aspartic acid 99 with alanine results in a very unstable signal peptidase molecule. Therefore, we conclude that it is unlikely that the residue is directly involved in catalysis, but rather plays an important role in stabilizing the protein structure.

• KSBB Journal
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• v.19 no.5
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• pp.368-373
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• 2004

The optimal condition for the production of a glucan and a fructan synthesizing enzymes from Leuconostoc mesenteroides NRRL B-1149 were studied based on the different medium compositions. Response surface methodology was applied to find the optimistic condition showing the relationship between the fermentation response (enzyme activities) and the fermentation variable concentrations of yeast extract, peptone concentration, K2HP04 concentration and sucrose. Optimum medium composition for both enzymes production was $0.75\%$ yeast extract, $0.72\%$ peptone, $1\%$ K2HP04 and $2.17\%$ sucrose. Using this medium, the activities produced in culture was 0.90 U/m~ for glucosyltransferase (GTase) and 0.96 U/ml for fructosyltransferase (FTase). After purification of 1149FTase by consecutive chromatographies using Sephadex G-150 and DEAE-Sepharose, a 1149FTase of 210 kDa on $7\%$ polyacrylamide gel was isolated and it synthesized soluble fructan. The 1149GTase showed a band of 180 kDa on $8\%$ polyacrylamide gel after purification using Bio-Gel P-100 gel chromatography and DEAE-Sepharose ion exchange chromatography and it synthesized insoluble glucan. The linkages of polymers were determined by methylation using Hakomori reagent and following NMR analysis. The glucan was composed of a(1~6) and a(1~3) linkages and the fructan was levan.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.10
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• pp.1612-1617
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• 2007

In this study, we have compared the skeletal muscle proteome at various stages of porcine postnatal development. Korean native pigs were divided into five postnatal stages of 30, 70, 130, 170 and 300 d and their loin muscles were analyzed for muscle proteome by using two-dimensional electrophoresis and mass spectrometry. We found 5 proteins showing a consistent pattern during skeletal muscle growth. Four proteins were identified as myosin light chain 1 slow-twitch (MLC1sa) isoform, troponin T, triosephosphate isomerase (TIP) and DJ-1 protein. The remaining protein was not identified. Two muscle fiber proteins of MLC1sa isoform and troponin T showed a high expression level at an early postnatal stage and then their levels were decreased markedly during growth stages. On the other hand, the expression of TIP and DJ-1 protein, which are well known as catalysis enzyme and antioxidant-related protein, respectively, were linearly increased during growth stages. Thus, the stage-related muscle proteins may be useful as parameters for understanding the developmental characteristics of biochemical and physiological properties in Korean native pig skeletal muscle.