• BMB Reports
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• 제40권3호
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• pp.315-324
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• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• BMB Reports
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• 제30권2호
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• pp.113-118
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• 1997

Aspartase from Hafnia alvei was inactivated by N-ethylmaleimide (NEM) and 5,5' -Dithiobis-(2-znitrobenzoic acid) (DTNB) following pseudo-first order kinetics. Their apparent reaction orders were 0.83 and 0.50 for NEM and DTNB modifications, respectively, indicating that inactivation was due to a sulfhydryl group in the active site of aspartase and participation of the sulfhydryl group in an essential step in the catalytic reaction. When aspartase was modified by DTNB, the enzyme activity was restored by dithiothreitol treatment, indicating that cysteine residuetsl islarel possibly at or near the active site. The pH-dependence of the inactivation rate by NEM suggested that an amino acid residue having pK value of 8.3 was involved in the inactivation. When aspartase was incubated with NEM and L-aspartate together, L-aspartate markedly protected the enzyme from inactivation by NEM, but the other reagents used did not.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2004년도 추계학술대회 논문집
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• pp.269-272
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• 2004

The target of this paper is the development of Web-based monitoring & control system which is for effective and economic management of mold manufacturing process. This system has three module; G-code Control, Monitoring Module and Result analysis module. Also, as the environment of development is based on internet, this system which is possible to the remote site management of manufacturing process works on Web. To be possible to control the manufacturing monitoring by client, each module is made ActiveX control and is based on socket communication. This system makes the foundation which is possible to manage the mold manufacturing process efficiently from remote site by matching real-time monitoring with manufacturing process in factory using G-code control and displaying the result of manufacturing using Ch-CGI.

• 통합자연과학논문집
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• 제8권4호
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• pp.267-272
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• 2015

Galactosemia is a potentially lethal disorder caused by the deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT) within the Leloir pathway. Galactokinase (GALK) is the enzyme in Leloir pathway which converts ${\alpha}$-D galactose to galactose 1-phosphate. The elevated level of galactose-1-phosphate, the product of GALK plays a major role in Galactosemia. Therefore the inhibition of GALK is a novel therapy for this disorder. Hence in the present study, we performed molecular docking of twenty inhibitors with different activity against galactokinase into the active site of galactokinase enzyme. The binding mode of these inhibitors was obtained using Surflex dock program interfaced in Sybyl-X2.0. The residues such as SER141, TYR109, ARG105, ARG228, TYR106, GLY346, GLY136, ASP86, ASP186 and SER142 found to interact with inhibitors.

• BMB Reports
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• 제30권4호
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• pp.280-284
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• 1997

Phenylglyoxal diethyl pyrocarbonate (DEPC), and 1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimide metho-p-toluenesulfonate (CMC) are modifying reagents specific for arginine, histidine, and aspartate or glutamate, respectively. They were found to inactivate S. cerevisiae farnesyl protein transferase (FPTase). The peptide substrate protected the enzyme against inactivation by CMC and the other substrate farnesyl pyrophosphate showed protection against inactivation by phenylglyoxal. while neither of the two substrates protected the enzyme against DEPC inactivation. These results suggest the presence of aspartate/glutamate, arginine and histidine residues at the active site of this enzyme.

• BMB Reports
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• 제31권2호
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• pp.139-143
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• 1998

The catabolic ${\alpha}-acetolactate$ synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the tryptophane-specific reagent, N -bromosuccinimide, and the arginine-specific reagent, phenylglyoxal. The enzyme was inactivated slowly by the cysteine-specific reagent N-ethylmaleimide. The second-order rate constants for the inactivation by N-bromosuccinimide, phenylglyoxal. and N -ethylmaleimide were $114,749M^{-1}min^{-1}$, $304.3M^{-1}min^{-1}$, and $5.1M^{-1}min^{-1}$, respectively. The reaction order with respect to N-bromosuccinimide, phenylglyoxal, and N-ethylmaleimide were 1.5,0.71, and 0.86, respectively. The inactivation of the catabolic aacetolactate synthase by these modifying reagents was protected by pyruvate. These results suggest that essential tryptophane, arginine, and cysteine residues are located at or near the active site of the catabolic ${\alpha}-acetolactate$ synthase.

• The Korean Journal of Physiology and Pharmacology
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• 제7권1호
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• pp.33-37
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• 2003

Oxidative damage to mitochondria is a critical mechanism in necrotic or apoptotic cell death induced by many kinds of toxic chemicals. Thioredoxin (Trx) family proteins are known to play protective roles in organisms under oxidative stress through redox reaction by using reducing equivalents of cysteines at a conserved active site, Cys-X-X-Cys. Whereas biological and physiological properties of Trx1 are well characterized, significance of mitochondrial thioredoxin (Trx2) is not well known. Therefore, we addressed physiological role of Trx2 in PC12 cells under oxidative stress. In PC12 cells, transiently overexpressed Trx2 significantly reduced cell death induced by hydrogen peroxide, whereas mutant Trx2, having serine residues instead of two cysteine residues at the active site did not. In addition, stably expressed Trx2 protected PC12 cells from serum deprivation. These results suggest that Trx2 may play defensive roles in PC12 cells by reducing oxidative stress to mitochondria.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.35-35
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• 2001

The conformational study on cyclic Ac-Cys-Pro-Xaa-Cys-NHMe (Ac-CPXC-NHMe; X = Ala, Val, Leu, Aib, Gly, His, Phe, Tyr, Asn, and Ser) peptides has been carried out using the ECEPP/3 force field and the hydration shell model in the unhydrated and hydrated states. This work has been undertaken to investigate structural implications of the CPXC sequence as the chain reversal for the initiation of protein folding and as the motif for active site of disulfide oxidoreductases.(omitted)

• 미생물학회지
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• 제29권4호
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• pp.238-242
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• 1991

Extracellular laccase secreted from Pleurotus ostreatus was activated by $Cu^{2+}$ and $Cu^{+}$ . The enzyme was strongly inactivated by 8-hydroxyquinoline, potassium cyanide, sodium azide, sodium bisulfite and 2-mercaptoethanol. The two ionogenic groups, which have pKa values of 5.60-5.70 and 6.70-6.85 respectively, were found to relate with the active site of this enzyme. The oxidation reactions were brought about by initial single electron transfer process on the active site. The enzyme was found to be a metalloprotein which had about 3.9 cupric ions per molecule of protein as a prosthetic group. The enzyme showed a strong peak at 605 nm and a weak shoulder at 330 nm in UV-Visible absorption spectrum. Both signals disappeated upon treatment of the enzyme with 4 electron equivalent ascorbate. These results indicate that type I Cu peak and type III Cu shoulder are present in laccase.