• Microbiology and Biotechnology Letters
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• v.37 no.3
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• pp.204-212
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• 2009

A strain producing ${\alpha}$-galactosidase and ${\beta}$-glucosidase was isolated from Kimchi. The isolated strain was identified as Weissella cibaria by 16S rDNA analysis and designated as Weissella cibaria K-M1-4. The enzyme activity of ${\alpha}$-galactosidase and ${\beta}$-glucosidase reached the maximum in the soy medium at $37^{\circ}C$ for 24 hr. The enzymes were purified by ethanol fractionation, DEAE sepharose fast flow, and sephacryl S-100HR column chromatography. ${\alpha}$-Galactosidase specific activity was shown by 576 Units/mg protein and the yield was 3.5% of the total activity of crude extracts. ${\beta}$-glucosidase specific activity was shown by 480 Units/mg protein and the yield was 2.9% of the total activity of crude extracts. The optimum temperature for ${\alpha}$-galactosidase was $60^{\circ}C$ and 43% of its original activity remained when it was treated at $80^{\circ}C$ for 30 min. For ${\alpha}$-galactosidase shows the optimum pH of 8.0 and is fairly stable between pH5.0 and pH9.0. The enzyme activity was increased in the presence of $Fe^{2+}$ and $Cu^{2+}$. The value of Km and Vmax for the enzyme were 0.98 mM and $1.81{\mu}$mole/min, respectively. The ${\beta}$-glucosidase has the optimum temperature of $50^{\circ}C$ and 46% of its original activity remained when it was treated at $80^{\circ}C$ for 30min. Its optimum pH of 7.0 and is fairly stable between pH5.0 and pH9.0. The enzyme activity was increased in the presence of $Fe^{2+},\;Co^{2+}$ and $Cu^{2+}$. The value of Km and Vmax for the enzyme were 1.24 mM and $6.81{\mu}$mole/min, respectively.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.1
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• pp.38-45
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• 1998

To extract insoluble proteins and to improve funtional properties of abolished proteins, an phytase producing Aspergillus sp. SM-15 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. Phytase production reached to maximum when the wheat bran medium containing 1% mannose, 1% yeast extract, 1% (NH4)2HPO4 and 0.2% calcium chloride was cultured for 4 days. Phytase was purified 17.1 fold and specific activity was 244.32unit/mg by a sequencial process of ammonium sulfate fraction, ion exchange chromatography and gel filtrations Pruified enzyme was confirmed as a single band by the polyacrylamide gel electro-phoresis. The molecular weight of phytase was estimated to be 46,000. The optimum pH and temperature for the phytase activity were 5.5 and 5$0^{\circ}C$. The enzyme is stable in pH 4.5~5.5, 6$0^{\circ}C$. The activity of purified enzyme was inhibited by Hg2+ whereas activited by Pb2+ and Fe2+. The activity of phytase was inhibited by the treatment with iodine. The result indicate the possible involvement of histidine at active site. Km and Vmax of the puridied phytase were 37.037mM/L and 159.87umol/min, respectively.

• Journal of Microbiology
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• v.35 no.2
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• pp.134-140
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• 1997

Botrytis cinerea T91-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Mg^{2+}$, and Cu$^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was 5$0^{\circ}C$. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 3$0^{\circ}C$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.19 no.6
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• pp.596-604
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• 1990

Polygalacturonase activity was not detected at turning stage but were 55.01 and 206.70 units/100g -fr. wt. in mature and soft persimmon, respectively. Polygalacturonase have two isoenzymes and its molecular weight was estimated to be 55,000 doltons by the method of gel filtration. Vmax and Km of polygalacturonase 1 were 0.195$\mu$ mole reducing-sugar/$m\ell$/30 min. and 3.50mg/$m\ell$, respectively. The optimum temperature and pH of the enzyme appeared to 4$0^{\circ}C$ and 3.5, respectively. Polygalacturonase I had Vmax of 0.110m mo1e reducing-sugar/$m\ell$/30min. and Km value of 2.50mg/$m\ell$, The optimum temperature and pH of polygalcturonase II appear 4$0^{\circ}C$ and 4.0, respectively. Polygalacturonase I was fairly stable at 6$0^{\circ}C$while polygalacturonase II appeared to be stable up to 4$0^{\circ}C$.

• KSBB Journal
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• v.15 no.1
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• pp.9-13
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• 2000

In order to determine the characteristics of $\beta$-glucosidase associated with cellulose degradation, the enzyme produced extracellularly by the mycelia of Tricholoma matsutake DGUM 26001 in culture broth was partially purified. The enzyme activity was maintained in the range of temperatures trom 55 to $70^{\circ}C$ and its optimum temperature was $65^{\circ}C$. The $\beta$-glucosidase enzyme showed relatively high activity in the range of pH 3.0-5.0 and its optimum pH was 4.0. Under the optimal conditions, the specific activity of $\beta$-glucosidase for salicin as a substrate was 18.7 unit/mg protein. After thermal treatment of the enzyme at $55^{\circ}C$ for 60 min, more than 90% of the enzyme activity was still sustained. Iron($Fe^{++}$) stimulated enzyme activity, whereas mercury($Hg^{++}$) and copper($Cu^{++}$) inhibited. Compared to salicin as a substrate, the relative activity for cellobiose was observed to be 48.6%. The apparent $K_m$ and $V_{max}$ of the enzyme with cellobiose were 0.12 mM and 0.02 umol/min, respectively.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.92-97
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• 1988

Cysteinylglycinase was partially purified from Proteus mirabilis by consecutive procedure. The specific activity was increased about 16-fold to that of cell-free extract. The enzyme was found rather unstable on ammonium sulfate precipitation ann the precipitated enzyme protein became partially insoluble during dialysis. The precipitated enzyme was found to be solubilized by treatment of 4% Triton X-100 effectiviely, The optimum temperature and pH of the enzyme activity were 35$^{\circ}C$ and 7.3, respectively. After heat treatment of the enzyme at 5$0^{\circ}C$ for 30 min, it lost the activity to 70%. The enzyme was stable at pH 7.0-8.0. The molecular weight of the cysteinylglycinase was found to be about 190,000 by Sephadex G-150 gel filtration. The enzyme was activated by the addition of Mn$^{2+}$ and $Mg^{2+}$ ions. The maximal activation was obtained in preincubation with $Mg^{2+}$ ion for 30 min. The enzyme catalyzed the hydrolysis of various dipeptides and tripeptides. The Km and Vmax values for cysteinylglycine were 1.60 mM and 0.24 m unit/ mg, respectively.

• Korean Journal of Microbiology
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• v.20 no.4
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• pp.189-194
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• 1982

Acid protease produced from Aspergillus tubingensis was pruified by ethanol fractionation, dialysis, and DEAE cellulose column chromatography. As a result of purification its specific activity increased to 5.4 times, and percent recovery was 39. The kinetic constants of the enzyme were studied. Km and Vmax was $1.5{\times}10^{-7}M\;and\;0.11{\Delta}O.D/min$ , respectively, when casein was used as substrate. The order of Km value of several proteins is : casein

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.5
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• pp.1010-1016
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• 1999

Protease related mold was isolated and selected as a starter culture for commercial production of meju. Isolated microorganism was identified as Syncephalastrum racemosum PDA 132 2. To obtain basic data about protease for production of soybean peptides and application of the strain in meju fermentation, we extrated and purified protease and charateristics of the enzyme were investigated. The optimum condition for the production of enzyme was pH 4.0, 30oC, 5 days. The protease was purified 19.7 folds by gel filtration and ion exchange chromatography and specific activity was 12.4unit/mg. The purified enzyme was 34kDa in size, thiol protease(100% inhibited by PCMB), and was acidic protease(stable between pH 2.0~5.0). Vmax of the enzyme was 2.14 g/min which was lower(1/50) than that of by Asp. wentti and B. subtilis.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.