• Archives of Pharmacal Research
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• v.19 no.6
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• pp.497-501
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• 1996

A gene coding for triosephosphate isomerase (TPI) from a rat skeletal muscle cDNA library was cloned and its nucleotide sequence was determined. The 1, 348-bp cDNA clone contains 24 bp $5^I$ noncoding region, the entire 750 bp coding region corresponding to a protein of 249 amino acids, $547bp 3^I$ noncoding region and part of a poly(A) tail. It also contains a polyadenylation signal, AATAAA, starting from 17 bp upstream of the poly(A) tail. The calculated molecular weight of rat TPI is 27.8 kDa and the net charge is +4. The deduced amino acid sequence from rat TPI CDNA sequence has 93% and 94% homology with that of mouse and human clones, respectively. The amino acids at the residue of Asn12, Lys14, His96, Glu 166, His96, His101, Ala177, Tyr165, Glu13O, Tyr2O9, and Ser212 in catalytic site are completely identical, confirming that the functional residues in TPI proteins are highly conserved throughout evolution. The most profound characteristic of rat TPI enzyme, compared with other TPIs, is that there are five cysteine substitutions at the residue of 21, 27, 159, 195 and 204. A Glu123 instead of Gly was found in rabbit, rhesus, mouse and human sequences. Through the method of RT-PCR, the mRNA transcription level of TPI gene was found to be different among various tissues and was highest in muscle.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.838-844
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• 2001

The enzyme Fuc aldolase from Methanococcus jannaschii that catalyzes the aldol condensation of DHAP and L-lactaldehyde to give fuculose-1-phosphate was inactivated by DEP. The inactivation was pseudo first-order in the enzyme and DEP, which was biphasic. A pseudo second-order rate constant of 120$M^{-1}min^{-1}$ was obtained at pH 6.0 and $25{\circ}C$. Quantifying the increase in absorbance at 240nm showed that four histidine residues per subunit were modified during the nearly complete inactivation. The statistical analysis and the time course of the modification suggested that two or three histidine residues were essential for activity. The rate of inactivation was dependent on the pH, and the pH inactivation data implied the involvement of the amino acid residue with a $pK_a$ value of 5.7. Fuc aldolase was protected against DEP inactivation by DHAP, indicating that the histidine residues were located at the active site of Fuc aldolase. DL-Glyceraldehyde, as an alternative substrate to L-lactaldehyde, showed no specific protection for the Fuc aldolase.

• Environmental Engineering Research
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• v.10 no.3
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• pp.131-137
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• 2005

The catalytic activity of precious metals(Rh, Pd, Pt) and nickel catalysts supported on ${\gamma}-Al_2O_3\;and\;CeO_2$ in the partial combustion of methane(PCM) to syngas was investigated based on the product distribution in a fixed bed now reactor under atmospheric condition and also on analysis results by SEM, XPS, TPD, BET, and XRD. The activity of the catalysts based on the syngas yield increased in the sequence $Rh(5)/CeO_2{\geq}Ni(5)/CeO_2>>Rh(5)/Al_2O_3>Pd(5)/Al_2O_3>Ni(5)/Al_2O_3$. Compared to the precious catalysts, the syngas yield and stability of the $Ni(5)/CeO_2$ catalyst were almost similar to $(5)/CeO_2$ catalyst, and superior to these of any other catalysts. The syngas yield of $Ni(5)/CeO_2$ catalyst was 90.66% at 1023 K. It could be suggested to be the redox cycle of the successive reaction and formation of active site, $Ni^{2-}$ and the lattice oxygen, $O^{2-}$ produced due to reduction of $Ce^{4-}$ to $Ce^{3-}$.

• Proceedings of the KIEE Conference
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• 2005.07b
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• pp.1681-1683
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• 2005

The molten carbonate fuel tell(MCFC) is endowed with the high potential especially in future electric power generation industry by its own outstanding characteristics. KEPCO(KEPRI) started a 100 kW MCFC system development program in 1993 and has been executed 100kW system develpilot plant successfully completed first phaseopment by 2005 on the basis of successful results of 25kW system development. In this program, the components and mechanical structure for 100 kW stack and system construction were completed on last year and now system pre-commissioning was being executed. A 100 kW MCFC power plant was constructed at the site of Boryeong Thermal Power Plant. A 100 kW MCFC system has characterized as a high pressure operation mode, $CO_2$ recycle, and externally reforming power generation system. The 100 kW MCFC system consisted with stacks which was made by two 50 kW sub-stacks, 90 cells with 6,000 cm2 active area and BOP including a reformer, a recycle blower, a catalytic burner, an inverter, and etc. The system will be operated under 3 atm pressure condition and expected to last over 5,000 hours by the end of this year.

• 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.

• Bulletin of the Korean Chemical Society
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• v.13 no.5
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• pp.511-516
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• 1992

The catalytic oxidation of CO on perovskite $Eu_{1-x}Sr_xCoO_{3-y}$, has been investigated at reaction temperatures from 100 to $250^{\circ}C$ under stoichiometric CO and $O_2$ partial pressures. The microstructure and Sr-substitution site of the catalyst were studied by means of infrared spectroscopy. The reaction rates were found to be correlated with 1.5-and 1.0-order kinetics with and without a $CO_2$ trap, respectively; first-and 0.5-order with respect to CO and 0.5-order to $O_2$ with the activation energy of 0.37 eV $mol^{-1}$. It was found from IR, ${\sigma}$ and kinetic data that $O_2$ adsorbs as an ionic species on the oxygen vacancies, while CO adsorbs on the lattice oxygens. The oxidation reaction mechanism is suggested from the agreement between IR, ${\sigma}$ and kinetic data.

• Journal of Microbiology and Biotechnology
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• v.34 no.6
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• pp.1322-1327
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• 2024

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1075-1080
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• 2004

Dextransucrase (DSRB742) from Leuconostoc mesenteroides NRRL B-742CB is a glucosyltransferase that catalyzes the synthesis of dextran using sucrose, or the synthesis of oligosaccharides when acceptor molecules, like maltose, are present. The DSRB742 gene (dsrB742) was cloned and the properties were characterized. In order to identify critical amino acid residues, the DSRB742 amino acid sequence was aligned with glucosyltransferase sequences, and three amino acid residues reported as sucrose binding amino acids in Streptococcus glucosyltransferases were selected for site-directed mutagenesis experiments. Asp-533, Asp-536, and His-643 were independently replaced with Ala or Asn. D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether. Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities. However, modifications at Asp-533 or at His-643 retained acceptor reaction activities in the range of 8.4% to 21.3% of DSRB742 acceptor reaction activity. Thus at least two carboxyl groups of Asp-533 and Asp-536, and His-643 as a proton donor, are essential for the catalysis process.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.79-86
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• 2012

ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.