• Archives of Pharmacal Research
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• v.20 no.6
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• pp.519-524
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• 1997

Steroid $9{\alpha}$-hydroxylase is an enzyme found in nocardioform microorganisms which can utilize steroids as a sole carbon source. After fractional centrifugation of the cell homogenates, the enzyme activity in Nocardia and Rhodococcus was found in cytoplasmic membrane fraction. On the contrary, Mycobacterium had its 9.alpha.-hydroxylation activity in cytosolic fraction. To characterize the enzyme in these microorganisms, several potential inhibitors of 9.alpha.-hydroxylase were tested and the cofactor requirement for the same enzyme was also examined. The inhibitory effect of ferrous ion chelators indicated involvement of iron containing proteins in the 9.alpha.-hydroxylase system. On the other hand, metyrapone, an inhibitor known to be specific for cytochrome P450 interfered with the enzyme in Mycobacterium, but didn't inhibit the enzyme activity in Nocardia and Rhodococcus. While the $9{\alpha}$-hydroxylase system in Nocardia and Rhodococcus required NADPH, NADH was required as an election donor in Mycobacterium.

• BMB Reports
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• v.28 no.1
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• pp.12-16
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• 1995

The branched-chain ${\alpha}$-keto acid dehydrogenase (BCKAD) complex is a rate limiting enzyme which catalyzes the oxidative decarboxylation of branched-chain ${\alpha}$-keto acids. Numerous studies have suggested that BCKAD is subject to covalent modification in vitro via phosphorylation and dephosphorylation, which are catalyzed by a specific kinase and phosphatase, respectively. The biggest difficulty in the assay of BCKAD activity is to arrest the interconversion between the active and inactive forms. BCKAD activity was determined from fresh rat heart and liver tissues using homogenizing and assay buffers containing inhibitors of phosphatase and kinase. The results suggest that a radiochemical assay using ${\alpha}$-keto[1-$^{14}C$]-isovalerate as a substrate for the enzyme can be applied as a reliable method to determine in vitro enzyme activity with arrested interconversion between the active and inactive forms of the BCKAD complex.

• BMB Reports
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• v.31 no.3
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• pp.254-257
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• 1998

The gshI gene from the Escherichia coli K-12 strain codes for ${\gamma}-glutamylcysteine$ synthetase which mediates the rate-limiting step of glutathione biosynthesis. The isolated gshI gene from E. coli K-12 has an unusual translation initiation codon, UUG. The 494th amino acid is Ala rather than Gly which was found in a mutant strain E. coli B. In order to improve the translational rate of the gshI gene of E. coli K-12, the initiation codon, UUG, was changed to the usual AUG codon by the site-specific mutagenesis. This change has resulted in a 53% increase of ${\gamma}-glutamylcysteine$ synthetase activity. The enzyme activity was also improved by replacing $Ala^{494}$ with Val (A494V) or Leu (A494L). The replacement of $Ser^{495}$ with Thr (S495T) also resulted in a 62% increase of the enzyme activity. Therefore, the specific activity of ${\gamma}-glutamylcysteine$ synthetase was increased with the increasing chain length of the aliphathic amino acid at the site of the 494th amino acid (Ala<$Val{\leq}Leu$).

• YAKHAK HOEJI
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• v.38 no.1
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• pp.12-19
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• 1994

Capsaicin(8-methyl-N-vanillyl-6-nonenamide) is the principal pungent component of Capsicum fruits. This work is directed to the capsaicin-hydrolyzing enzyme playing a key role in the rate limiting and critical step of capsaicin metabolism. In order to get precise information on the enzyme's subcellular location, rat liver homogenate was divided into six subcellular fractions by differential centrifugation technique: crude nuclear pellet, PNS(post nuclear supernatant) fraction, lysosomal pellet, cytosol, Tris wash fraction, micrisomes. Capsaicin-hydrolysing enzyme activity was analysed by high performance liquid chromatography(HPLC). This enzyme was found at the highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum, NADPH-cytochrome c reductase and nucleoside diphosphatase. This is compatible with the result of ninhydrin color reaction of vanillylamine, primary metabolite of capsaicin hydrolysis, on thin layer chromatography(TLC). This enzyme is most active at pH $8.0{\sim}9.0$. Definite subcellular location of this enzyme will make it easy to proceed with further study.

• Journal of Pharmaceutical Investigation
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• v.19 no.2
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• pp.81-86
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• 1989

The proteolytic enzyme extracted from Neungee [Sarcodon aspratus (Berk.) S. Ito] was purified by using Tris-acryl CM-cellulose column chromatography and chromatofocusing. The specific activity of the purified enzyme increased 15.8 times as compared with that of the crude enzyme. The enzyme was homogeneous on polyacrylamide gel electrophoresis and stable at pH values ranging from 4.0 to 10.8. The enzyme activity remained unchanged when the mushroom and the purified enzyme were stored for 3 years and 6 months at 4°C, respectively. The enzyme was found to be an endogeneous protease.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.33-38
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• 2002

Purification and characterization of enzyme property of a cell-wall lytic enzyme against Lactobacillus plantarum were carried out. Final specific activity of purified enzyme was 5.8 units/mg and purity of the enzyme was increased 8.3 fold compared with the enzyme activity in culture broth. The molecular weight of purified enzyme was estimated to be 60,000 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. Optimal pH and temperature for the activity of this enzyme were 3.0 and 4$0^{\circ}C$, respectively. The cell-wall lytic enzyme activity was maintained at 3$0^{\circ}C$ when treating the enzyme for 30 mins, whereas the activity was decreased to 80% of the maximum level at 4$0^{\circ}C$ The enzyme activity exhibited good stability at the range of pH 4~7.

• Applied Biological Chemistry
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• v.33 no.1
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• pp.79-86
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• 1990

Cyclodextrin hydrolase from Bacillus stearothermophilus KFCC 21203 was purified and the properties of the purified enzyme were investigated. The enzyme was purified 15 folds with 77 % recovery by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Ultro AcA 34 gel filtration. The specific activity and the molecular weight of the enzyme were 1.30 units/mg protein and about 29,500, respectively, The maximum activity of the enzyme was shown at $55^{\circ}C$ and pH 5.5. However, stable temperature and pH were $40^{\circ}C$ and $5.0{\sim}8.0$, respectively. The Km value for ${\gamma}-cyclodextrin$ was $3.78{\times}10^{-3}$ M. The degradation activity of the enzyme was selectively high for ${\gamma}-cyclodextrin$, and very low for ${\beta}-cyclodextrin$, but not for ${\alpha}-cyclodextrin$. The decomposed products of ${\gamma}-cyclodextrin$ were mainly glucose and maltose, and a little mlatotriose. The activity of the enzyme was very high for amylose, potato starch, corn starch, amylopectin and maltooligomer, and relatively high for glycogen and dextrin. The decomposed products of them were mainly glucose and maltose.

• Korean Membrane Journal
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• v.8 no.1
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• pp.58-66
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• 2006

The ${\beta}-glucosidase$ from olive fruit is of particular interest compared to the ones from other sources because it has shown to have high specifity to convert the oleuropein into dialdehydes, which have antibacterial activity and are of high interest for their application in the food and pharmaceutical fields. The enzyme is not yet commercially available and advanced clean and safe technologies for its purification able to maintain the functional stability are foreseen. The purification of this protein from fruit extracts has been already tempted by electrophoresis but either enzyme deactivation or high background with unclear profiles occurred. In this work, fruit extracts obtained from the ripening stage that showed the highest enzyme activity have been processed by diafiltration and ultrafiltration. Asymmetric membranes made of polyamide or polysulphone having 50 and 30 kDa molecular weight cut-off, respectively, were tested for the diafiltration process. Ultrafiltration membranes made of polyethersulfone with 4 kDa molecular weight cut-off were used to concentrate the dia-filtered permeate solutions. The efficiency of the separation processes was evaluated byenzyme activity tests using the hydrolysis of p-D-nitrophenyl-${\beta}$-D-glucopyranoside (pNPGlc) as reaction model. Qualitative and quantitative electrophoresis were applied to analyze the composition of protein solution before and after the membrane separation; in addition dot blot and western blot analyses were applied to verify the presence of ${\beta}-glucosidase$ in the processed fractions. The overall results showed that the ${\beta}-glucosidase$ functional stability was preserved during the membrane operations and the removal of 20 kDa proteins allowed to increase the specific activity of the enzyme of about 52% compared to the one present in the initial fruit extract.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.462-469
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• 2000

Sixty-one strains representing the main genera of wood-decaying xylariaceous fungi (mainly in Daldinia, Hypoxylon, Kretzschmaria, Rosellinia, Penzigia, and Xylaria) were tested for their ability to produce ligninolytic enzymes. The phenol oxidase activity and fungal growth of the xylariaceous fungi on gallic aicid and tannic acid media showed a variation in their ability to degrade lignocellulose. A number of species showed equal 개 betterligninolytic enzyme activities than Coriolus versicolor, a known basidiomycete wood-degrader. A large variation of the enzyme activity was observed by individual strains as well as a substantial variation between the isolates of the same species. The most frequent ligninolytic enzymes were peroxidase and general oxidase. With 19% of the strains tested, peroxidase showed the strongest ligninolytic enzyme activity, while tyrosinase activity was detected only in 7% of the strains. All strains of Kretzschmaria and Rosellinia tested was positive for laccase. Xylariaceous fungi were able to degrade the macromolecule, lignin, using each specific ligninolytic enzyme in the specfic lignin degradation pathway.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.132-138
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• 1994

L-Ascorbic acid-producing enzyme in Neurospora crassa was found to exist in mitochondria and the activity of this enzyme was increased by the addition of D-fluconno-${\gamma}$-lactone or L-gulono-${\gamma}$-lactone in the media. L-Ascorbic acid-producin enzyme in N. crassa has been purified with ammonium sulfate precipitation. DEAE Sepharose CL-6B ion exchange chromatography. Sephacryl S-200 gel filtration chromatography and Reactive yellow 3-agarose dye affinity column chromatography. The specific activity of this enzyme was increased to 239.6 fold and the yield was 2.1%. The molecular weight of the native enzyme was 150.000 dalton when it was estimated with Sephacryl S-200 gel filtration chromatography. Its molecular weight appeared as 75.000 dalton by SDS-polyacrylamide gel electrophoresis. which suggested that this enzyme was consisted with two identical subunits. The optimal pH for this enzyme was 9.0 and the $K_m$ value for D-galactono-${\gamma}$-lactone was 0.073 mM.