• BMB Reports
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• 제32권1호
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• pp.39-44
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• 1999

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1401-1407
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• 2008

The roles of conserved amino acid residues (Va1329-Ala330-Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose. These results propose that the substrate specificity, hydrolysis pattern, and transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.268-273
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• 2019

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with $His^{14}$ and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.

• Journal of Applied Biological Chemistry
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• 제48권2호
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• pp.75-78
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• 2005

Halotolerant protease produced by Halomonas marisflava KCCM 10457 was partially purified through ammonium sulfate precipitation and Sephacryl S-200HR gel permeation chromatography. Optimal pH and temperature of protease were 11.0 and $45^{\circ}C$, respectively. Enzyme activity was inhibited by $Cu^{2+}$, $Hg^{2+}$, $Fe^{2+}$, and $Fe^{3+}$, and selectively inhibited by p-chloromercuribenzoic acid (PCMB), suggesting this enzyme is cysteine protease. The enzyme is halotolerant, because it retained 77% of original activity in presence of 3.33 M NaCl. The protease showed broad substrate specificity to various natural proteins; BSA, casein, egg albumin, gelatin, and hemoglobin.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1997년도 학술발표회
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• pp.37-37
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• 1997

The catalytic efficiency of pyranose oxidase (EC 1.1.3.10.) determined for various sugars showed that D-glucose is the preferred substrate and the enzyme oxidized the various aldonolactones. The specificity constants of pyranose oxidase determined for deoxy- and deoxyfluoro-D-glucoses showed that a hydroxy group at C-4 of D-glucose acts as a hydrogen-bone acceptor, at C-6 as a hydrogen-bond donor, and at C-1 as a hydrogen-bond donor.(omitted)

• 한국미생물·생명공학회지
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• 제51권4호
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• pp.333-352
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• 2023

Transaminase represents the most important biocatalysts used for the synthesis of chiral amines due to their stereoselectivity. They allow asymmetric synthesis with high yields and enantioselectivity from their corresponding ketones. Due to their environmentally friendly access for the preparation of chiral amines, they have attracted growing attention in recent times. Thus, the production of chiral compounds by transaminase catalysed reactions is considered as an important application in synthetic organic chemistry. Therefore, transaminase is considered to be an important enzyme in the pharmaceutical and chemical industries. ω-Transaminase holds great potential because of its wide substrate specificity thus making it a suitable enzyme to be used at an industrial scale. This review highlights the reaction mechanism, classification, substrate specificity, and biochemical properties. The review also showcases the application of ω-transaminase in organic chemistry with a focus on the production of active pharmaceutical ingredients (APIs).

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.581-581
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• 2003

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.174-174
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• 2005

• Bulletin of the Korean Chemical Society
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• 제20권10호
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• pp.1126-1128
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• 1999

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.597-605
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• 2018

Acyl-CoA oxidases (ACOXs) play important roles in lipid metabolism, including peroxisomal fatty acid ${\beta}$-oxidation by the conversion of acyl-CoAs to 2-trans-enoyl-CoAs. The yeast Yarrowia lipolytica can utilize fatty acids as a carbon source and thus has extensive biotechnological applications. The crystal structure of ACOX3 from Y. lipolytica (YlACOX3) was determined at a resolution of $2.5{\AA}$. It contained two molecules per asymmetric unit, and the monomeric structure was folded into four domains; $N{\alpha}$, $N{\beta}$, $C{\alpha}1$, and $C{\alpha}2$ domains. The cofactor flavin adenine dinucleotide was bound in the dimer interface. The substrate-binding pocket was located near the cofactor, and formed at the interface between the $N{\alpha}$, $N{\beta}$, and $C{\alpha}1$ domains. Comparisons with other ACOX structures provided structural insights into how YlACOX has a substrate preference for short-chain acyl-CoA. In addition, the structure of YlACOX3 was compared with those of medium- and long-chain ACOXs, and the structural basis for their differences in substrate specificity was discussed.