• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.969-975
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• 2003

Two conserved amino acid residues in the extra sugar-binding space near the catalytic site of Thermus maltogenic amylase (ThMA) were analyzed for their role in the hydrolysis and transglycosylation activity of the enzyme. Site-directed mutagenesis was carried out by replacing N33l with a lysine (N331K), E332 with a histidine (E332H), or by replacing both residues at the same time (N331K/E332H). The measured $K_m$ values indicated that affinities toward all substrates tested, including starch, pullulan, ${\beta}-cyclomaltodextrin$, and acarbose, were lower in all the mutants compared to that of wild-type ThMA, leading to reduced hydrolysis activity. In addition, the lower ratio of transglycosylation to hydrolysis in the mutants compared to that in the wild-type ThMA indicated that these mutants preferred hydrolysis to the transglycosylation reaction. These results demonstrated that the conserved dipeptide at 331 and 332 of ThMA is directly involved in the formation and accumulation of transfer products by accommodating acceptor sugar molecules.

• Bulletin of the Korean Chemical Society
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• v.30 no.7
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• pp.1547-1552
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• 2009

Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

• Bulletin of Food Technology
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• v.8 no.4
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• pp.179-191
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• 1995

Thermoanaerobacter ethanolicus유래 thermophilic amylopullulanase의 thermophilicity와 thermostability의 기작을 규명하기 위하여 N-말단과 C-말단으로부터 nested deletion mutatnt와 sitedirected mutagenesis등에 의한 변이효소를 제조, 분석하였다. 이러한 까다로운 변이효소를 제조하여 amylopullulanase의 특정부위가 효소의 thermophilicity와 thermostability에 관여하고 있는 것을 확인했다. N-말단의 start amino acid에서 194와 324 아미노산잔기에 이르는 부위 (TPR)가 이 효소의 높은 최적반응온도의 유지에 관련되어 있고 1102와 1224잔기에 이르는 부위 (TSR)는 thermodenaturation이 잘 일어나지 않도록 하고 있었다. 야생형 amylopullulanase (Apu), 변이효소중 ApuN342와 ApuN106/C379는 비슷한 효소비활성과 Km값을 가지고 있었다. TPR부위의 site-directed mutagenesis에 의한 변이효소중 P240A (proline$\rightarrow$alanine), P244A, P240A-P244A는 야생형의 최적반응온도 $80^{\circ}C$와 똑같았지만 효소의 열안전성(반감기)는 $85^{\circ}C$에서 21, 105, 128분을 보여 주었다. TSR에서의 변이효소중 P1159A, P1202A는 열안전성은 비슷하였지만 반응최적온도는 $85^{\circ}C$와 $90^{\circ}C$로 야생형 Apu보다 오히려 높아졌다. 따라서 proline은 분자내에서 thermophilicity와 thermostability를 항상 증가시키는 쪽으로 영향을 주지 않는 것으로 보인다. Proline은 그 위치와 주위의 다른 아미노산잔기와 같이 종합적으로 분자의 구조에 영향을 미치고 있는 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.48-54
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• 2008

A putative ${\omega}$-aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the ${\omega}$-aminotransferase was investigated. AptA showed high activity for short-chain ${\beta}$-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the ${\omega}$-aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase (PDB ID: 1DGE) as a template. Then, the ${\omega}$-aminotransferase was rationally redesigned to increase the activity for 3-amino-3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1433-1441
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• 2015

Pollution resulting from the discharge of textile dyes into water systems has become a major global concern. Because peroxidases are known for their ability to decolorize and detoxify textile dyes, the peroxidase activity of Vitreoscilla hemoglobin (VHb) has recently been studied. It is found that VHb and variants of this enzyme show great promise for enzymatic decolorization of dyes and may play a role in achieving their successful removal from industrial wastewater. The level of VHb peroxidase activity correlates with two amino acid residues present within the conserved distal pocket, at positions 53 and 54. In this work, sitedirected mutagenesis of these residues was performed and resulted in improved VHb peroxidase activity. The double mutant, Q53H/P54C, shows the highest dye decolorization and removal efficiency, with 70% removal efficiency within 5 min. UV spectral studies of Q53H/P54C reveals a more compact structure and an altered porphyrin environment (λ_Soret = 413 nm) relative to that of wild-type VHb (λ_Soret = 406), and differential scanning calorimetry data indicate that the VHb variant protein structure is more stable. In addition, circular dichroism spectroscopic studies indicate that this variant's increased protein structural stability is due to an increase in helical structure, as deduced from the melting temperature, which is higher than 90℃. Therefore, the VHb variant Q53H/P54C shows promise as an excellent peroxidase, with excellent dye decolorization activity and a more stable structure than wild-type VHb under high-temperature conditions.

• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.