• 미생물학회지
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• 제18권2호
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• pp.59-66
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• 1980

Two strains S-P and S-P-2, both Streptomyces sp., have been isolated and were found to have relatively high specific enzyme activity compared to other organisms reported. The specific activity of the enzyme produced from these two strains were 0.25 and 0.2 international units respectively. The productivity of the enzyme achieved was about 50 IU/l/hr. Glucose isomerase form these strains was found to be stable under the temperature of heat treatment (at $65^{\circ}C$) for fixation of enzyme inside the dell. This organism has an advantage in that it did not require toxic metalic ion for enzyme activity and could utilize xylan in leu of xylose as an inducer. The optimal temperature and pH of enzymatic reaction purpose of using these data for the optimal operation and designing of enzyme reactor system. The reaction mechanism was found to follow the single substrate reversible reaction kinetics. The kinetic constants determined experimentally are : $K_{mf}=0.33M,\;K_{mb}=1.0M,\;V_{mf}=0.88{\mu}mole\;per\;min.,\;V_{mb}= 2.96{\mu}mole\;per\;min.\;and\;K_{eq}=0.74.

• BMB Reports
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• 제33권1호
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• pp.69-74
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• 2000

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned, then the AdoMetDC protein was expressed and purified. The purified AdoMetDC was inactivated by salicylaldehyde in a pseudo first- order kinetics. The secondorder rate constant for inactivation was 126 $M^{-1}min^{-1}$ with the slope of n=0.73, suggesting that inactivation is the result of the reaction of one lysine residue in the active site of AdoMetDC. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations in conserved $lysine^{81}$ residues. These were chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. Changing $Lys^{81}$ to alanine showed an altered optimal pH. The substrate also provided protection against inactivation by salicylaldehyde. Considering these results, we suggest that the $lysine^{81}$ residue may be involved in catalytic activity.

• Fisheries and Aquatic Sciences
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• 제3권3_4호
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• pp.188-194
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• 2000

A protease purified from hepatopancreas of shrimp, Penaeus japonicus, had maximum activity at $70^{\circ}C$ and in neutral and alkaline pH ranges. Specific activity at optimum reaction condition of the protease was estimated to be approximately 12 U/mg/min. The protease was stable in neutral and alkaline pH ranges and activity was retained after heat treatment at $50^{\circ}C$ for 30 min. Apparent $K_m$ and $V_{max}$ value against casein substrate were estimated to be $0.29\%$ and $7.8see^{-1}$, respectively, and those against N-CBZ-L-tyrosine p-nitropheny1 ester (CBZ­Tyr-NE) were 0.38 mM and $2,400 see^{-1}$, respectively. The N-termina1 sequence of the protease showed high homology to the trypsin from same species and the proteases from shrimp. Myosin heavy chain (MHC) from shrimp tail meat was the most susceptible to the protease and actin/tropomyosin were degraded progressively during 4 hr incubation, but to a lesser degree than MHC.

• 한국응용과학기술학회지
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• 제9권1호
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• pp.7-13
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• 1992

Lipases catalyzed the transesterification reaction between esters and various primary and secondary alcohols in a 99% organic medium, porcine pancreatic, yeast, mold lipases can vigorously act as catalysts in a number of nearly anhydrous organic solvents. Various transesterification reactions catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The dependence of the catalytic activity of the enzyme in organic media on the pH of the aqueous solution from which it was recovered is bell-shaped, with the maximum coinciding with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases Can catalyze several other processes in organic media.

• 정보과학회지
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• 제18권8호
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• pp.78-89
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• 2000

Today's computers are built up from a minimal set of standard pattern recognition operations. Logic gates, such as NAND, are common examples. Biomolecular materials offer an alternative approach, both in terms of variety and context sensitivity. Enzymes, the basic switching elements in biological cells, are notable for their ability to discriminate specific molecules in a complex background and to do so in a manner that is sensitive to particular milieu features and indifferent to others, The enzyme, in effect, is a powerful context sensitivity pattern processor that in a rough way can be analogized to a neuron whose input-output behavior is controlled by enzymatic dynamics.

• Journal of Pharmaceutical Investigation
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• 제26권3호
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• pp.175-185
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• 1996

To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

• BMB Reports
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• 제28권2호
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• pp.112-117
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• 1995

Succinic semialdehyde reductase was inactivated by o-phthalaldehyde. The inactivation followed pseudo-first order kinetics, and the second-order rate constant for the inactivation process was 28 $M^{-1}s^{-1}$ at pH 7.4 and $25^{\circ}C$. The absorption spectrum ($\lambda_{max}$ 337 nm) and fluorescence excitation ($\lambda_{max}$ 340 nm) and fluorescence emission spectra ($\lambda_{max}$ 409 nm) were consistent with the formation of an isoindole derivative in the catalytic site between a cysteine and a lysine residue approximately about 3 $\AA$ apart. The substrate, succinic semialdehyde, did not protect enzymatic activity against inactivation, whereas the coenzyme NADPH protected against o-phthaladehyde induced inactivation of the enzyme. About 1 isoindole group per mol of the enzyme was formed following complete loss of enzymatic activity. These results suggest that the amino acid residues of the enzyme participating in a reaction with o-phthalaldehyde are cysteinyl and lysyl residues at or near the NADPH binding site.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.713-717
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• 2009

The dependence of the catalytic properties of horseradish peroxidase on the structural changes of ionic liquids was investigated with two water-miscible ionic liquids, N-butyl-3methypyridinium tetraftuoroborate ([$BMP_y$][$BF_4$]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIM][$MeSO_4$]), each of which shares an anion ($BF_4^-$) or a cation ($BMIM^+$) with 1-butyl-3-methylimidazolium tetraftuoroborate ([BMIM][$BF_4$]), respectively. The oxidation of guaiacol (2-methoxyphenol) with $H_2O_2$was used as a model reaction. In order to minimize the effect of solution viscosity on the kinetic constants of the enzymatic catalysis, the enzymatic reactions for the kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. Similarly to the previously reported results for [BMIM][$BF_4$], as the concentration of [$BMP_y$][$BF_4$] increased, the $K_m$value increased with a decrease in the $k_{cat}$value: the $K_m$value increased markedly from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to the enzyme. On the contrary, [BMIM][$MeSO_4$] decreased the Km value to 1.4 mM in 25% (v/v) ionic liquid. [BMIM][$MeSO_4$] also decreased $k_{cat}$more than 3-folds [from 13.8 $s^{-1}$in 100% water to 4.1 $s^{-1}$in 25% (v/v) ionic liquid]. These results indicate that the ionic liquids interact with the enzyme at the molecular level as well as at a macroscopic thermodynamic scale. Specifically, the anionic component of the ionic liquids influenced the catalysis of horseradish peroxidase in different ways.

• 약학회지
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• 제38권2호
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• pp.202-210
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• 1994

To investigate the feasibility of mucosal delivery of $[D-Ala^6]$ LHRH, a potent analogue of LHRH, enzymatic proteolysis of $[D-Ala^6]$ LHRH and inhibitory effect of medium chain fatty acid salts(MFA) were studied using rabbit mucosal homogenate. $[D-Ala^6]$ LHRH incubated in homogenates of rectal(RE), nasal(NA) and vaginal(VA) mucosa were assayed by HPLC. The degradation of $[D-Ala^6]$ LHRH followed the first order kinetics. The degradation products were found as $[D-Ala^6]$ $LHRH^{1-7}$(m-i), to a lesser extent, $[D-Ala^6]$ $LHRH^{1-9}$(m-ii) and $[D-Ala^6]$ $LHRH^{1-3}$(m-iii) by the method of amino acid analysis(PITC method). The formation of$[D-Ala^6]$ $LHRH^{1-7}$ was not inhibited by the addition of disodium ethylenediaminetetraacetic acid but inhibited by sodium tauro-24,25-dihydrofusidate, suggesting that endopeptidase 24.11(EP 24.11) cleaves the $Leu^7-Arg^8$ bond of $[D-Ala^6]$ LHRH and is the primary $[D-Ala^6]$ LHRH degrading enzyme. The patterns of $[D-Ala^6]$ LHRH degradation indicated that EP 24.11 exists in each mucosal homogenate with the order of RE>NA>VA. MFA significantly inhibited the proteolysis of $[D-Ala^6]$ LHRH. The addition of sodium caprate(1.0%) or sodium laurate(0.5%) to the each mucosal homogenate completely protected $[D-Ala^6]$ LHRH from the degradation.

• Toxicological Research
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• 제12권2호
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.