• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.427-434
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• 1998

An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.5
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• pp.637-642
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• 1996

We isolated a marine bacterium, Pseudomonas sp,, which could produce the enzyme of alginate lyase, and cloned the alginate lyase gene in Escherichia coli. The cloned DNA was overexpressed with approximately $50\%$ amount of total proteins. In addition, the expressed proteins were not secreted into the medium, and most of them existed in the cytoplasm by the soluble form, but not observed any inclusion body by TIM. For the optimum enzyme activity, temperature was $20^{\circ}C$, pH was 7.0, and Km and Vmax values of the enzyme were $0.4\%$ and 625 units/mg, respectively.

• Journal of Life Science
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• v.6 no.3
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• pp.198-203
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• 1996

A thermolabile alkaline phosphatase has been purified through steps of osmotic shock, ammonium sulfate salting-out, and DAEA-cellulose chromatography from the cultured broth of the marine Vibrio sp. M-96 strain. The optimal temperature for the enzyme activity was 35$\circ$C. The optimal pH was pH11.0, and the range of pHstability was pH10.4 to 12.0. Thermal inactivation occured within 6 mintes at 60$\circ$C. The enzyme was considerably inactivated by 0.1mM concentrations of Hg$^{2+}$, Ni$^{2+}$ and Zn$^{2+}$, whereas activated up to 234% by 1mM of Mn$^{2+}$. The activation energy and deactivation energy by the Arrhenius equation were 4.02 Kcal/mol and 9.098 Kcal/mol, respectively. The Km and Vmax values of the enzyme for p-introphenylphosphate were found to be 0.0465mM and 0.001334mM/min, respectively. Active form of the enzyme had a molecular weight of 57,000 dalton determined by the Sephadex G-200 gel filtration method.

• Bulletin of Food Technology
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• v.17 no.4
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• pp.98-106
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• 2004

Two mutant enzymes of $\gamma$-glutamylcysteine synthetase ($\gamma$-GCS) which catalyzed the synthesis of $\gamma$-glutamylcysteine from L-glutamic acid and L-cysteine in the presence of ATP, were prepared bypoint mutation of $\gamma$-GCS gene with site-directed mutagensis in E. coli. Conformational structuresand catalytic reaction kinetics of mutant enzymes were compared with wild type $\gamma$-GCS afterpurification. The S495F mutant enzyme (serine at 495 residue was substituted with phenylalanine),which had no catalytic activity for $\gamma$-glutamylcysteine synthesis, rarely folded even in neutral pH.However, the mutant A494V (alanine of 494 residue was replaced by valnine) which showed 50 %increase of activity, had a high folding structure. The folding structure of A494V also more stable athigh temperature and extreme pH compared to wild type and S495F. Reaction kinetics of wild typeand A494V were also investigated, Km value of A494V was smaller than that of wild type, while itshowed a little difference at Vmax values. This result evolved that alanine at 494 may be involved inbinding site of substrate rather than catalytic site. In addition, change of catalytic activity by onepoint mutation was highly correlated with the folding structure of enzyme.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1216-1220
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• 2001

Effect of hydroquinone (HQ) on rumen urease activity was studied. Hydroquinone at concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm inhibited urease activity of intact rumen microbes in vitro by 25%, 34%, 55% and 64% respectively. In the presence of low concentrations of $\beta$-mercaptoethanol, rumen urease could be solubilized and partially purified. The Km for the enzyme was $2{\times}10^{-3}$ M with Vmax of $319.4{\mu}moles/mg$ min. The kinetics of inhibition with partially purified rumen urease was investigated. The result showed that the inhibitory effect was not eliminated by increasing urea concentrations indicating a noncompetitive effect in nature with an inhibition constant $1.2{\times}10^{-5}$ M. Hydroquinone at the concentration of 10 ppm produced 64% urease inhibition, did not affect ruminal total dehydrogenase and proteolytic enzyme (p>0.05), but increased cellulase activity by 28% (p<0.05) in vitro. These results indicated that hydroquinone was a effective inhibitor of rumen urease and could effectively delay urea hydrolysis without a negative effect. The inhibitor appeared to offer a potential to improve nitrogen utilization by ruminants fed diets containing urea.

• Biomedical Science Letters
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• v.8 no.1
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• pp.47-52
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• 2002

To investigate an effect of cyclohexanone (CHO) treatment on the serum levels of xanthine oxidase (XO) in liver damaged animals, the rats were intraperitoneally pretreated with 50% carbon tetrachloride ($CCl_4$) in olive oil (0.1 mL/ 100 g body weight) 14 times every other day. To the $CCl_4$-pretreated rats, CHO (1.56 g/kg body weight) was injected once and then the animals were sacrificed at 4 hours after CHO treatment. The increasing rate of serum and liver XO activities to the control was higher in CHO-treated animals pretreated with $CCl_4$ than the $CCl_4$-pretreated those. Concomitantly CHO injection to the $CCl_4$-pretreated animals showed somewhat higher Vmax and lower Km value in the kinetics of liver XO enzyme. Furthermore, increasing rate of hepatic malonedialdehyde content to the control was also higher in CHO-treated animals pretreated with $CCl_4$ than $CCl_4$-pretreated those. On the other hand, the injection of CHO to the $CCl_4$-pretreated animals showed the more enhanced liver damage on the basis of liver function finding; liver weight per body weight (%), serum levels of alanine aminotransferase activity and hepatic glucose-6-phosphatase activity. In conclusion, injection of CHO to the $CCl_4$-pretreated rats led to more increased activity of serum XO and it may be caused by acceleration of hepatocyte membrane permeability and induction of enzyme protein.

• Journal of Environmental Health Sciences
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• v.29 no.2
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• pp.80-86
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• 2003

To evaluate the effect of cyclohexane(CH) treatment on the serum levels of glutathion S-transferase(GST) activity in liver damaged animals, damaged liver was induced with pretreatment of 50% $CCl_4$ dissolved in olive oil (0.1 m1/100g body weight) intraperitoneally 17 times every other day. To $CCl_4$-treated rats, CH (1.56 g/kg body weight, i.p) was injected once and then the animals were sacrificed at 4 hours after injection of CH. The $CCl_4$-treated animals were identified as severe liver damage on the basis of liver functional findings, 1,e, increased serum levels of alanine aminotransferase(ALT), alkaline phosphate(ALP) and xanthine oxidase(XO) activities. On the other hand, $CCl_4$-treated animals injected with CH once($CCl_4$-pretreated animals) showed more decreased serum levels of ALT and XO, and more increased those of ALP rather than $CCl_4$-treated animals. In case of comparing the GST with ALT activity in liver, both $CCl_4$-treated and pretreated animals showed similar changing pattern of enzyme actvity. Especially $CCl_4$-pretreated animals showed significantly increased serum level of GST actvity compared with the $CCl_4$-treated those, whereas those of ALT showed reversed tendency. In aspects of GST enzyme kinetics, $CCl_4$-pretreated animals showed higher Vmax of liver GST enzyme than $CCl_4$-treated animals. In conclusion, injection of CH to the liver damaged rats led to enhanced liver damage and more increased activity of serum GST which may be chiefly caused by the enzyme induction.

• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.208-214
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• 1999

The condition for immobilization of the partially purified agarase from Bacillus cereus ASK202 and the properties of the immobilized enzyme have been investigated. Agarase was immobilized on various supports by entrapment method. The enzyme immobilized on Na-alginate bead showed the highest activity among those studied. The optimal reaction conditions of the immobilized agarase were obtained in 3%(w/v) Na-alginate for the matrix, bead diameter of 2.5mm, 1 unit of agarase solution and 1.0%(w/v) calcium chloride solution. The optimum pH and temperature of the immobilized agarase were pH and temperature of the immobilized agarase were pH 7.0 and 4$0^{\circ}C$, respectively. Km and Vmax values were 0.5mg/ml.min, respectively. The immobilized agarase conerted agar to agarobiose, and their total conversion ratio under the optimal condition was 89%.

• Applied Biological Chemistry
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• v.41 no.7
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• pp.489-495
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• 1998

The optimum culture condition of Scopulariopsis brevicaulis for the production of ${\alpha}$-galactosidase was as follows: Tryptone 1.5%, $NH_4NO_3$ 0.2%, Raffinose 2.5%, $KH_2PO_4$ 0.5%, yeast extract 0.5%, pH 7.0, $27^{\circ}C$. The optimum pH and temperature for the enzyme activity of ${\alpha}$-galactosidase producing Scopulariopsis brevicaulis were pH 7.0 and $27^{\circ}C$, respectively. The enzyme was relatively stable at $pH\;6.0{\sim}8.0$ and at temperature below $40^{\circ}C$. The activity of the enzyme was inhibited by $Ag^{2+},\;Hg^{2+},\;Cu^{2+}$, p-chloromercuribenzoic acid and Iodine. These results would indicate the presence of -SH groups in the catalytic site of the enzyme. Km value was 1.9 mM for $p-nitrophenyl-{\alpha}-D-galactopyranoside$ and Vmax value was $9.66{\times}10^2\;{\mu}M/min$. Sugar constituents of culture broth were identified by HPLC that the enzyme liberated sucrose, glucose and fructose from raffinose and raffinose was significantly decreased.

• Clinical and Experimental Reproductive Medicine
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• v.10 no.2
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• pp.1-11
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• 1983

${\beta}$-Glucuronidase from bull seminal plasma was partially purified by $(NH_4)_2SO_4$fractionation, two successive DEAE-cellulose columns, isoelectric focusing (pH 4 to 6) and Gel filtration on Sephadex G-200. Only one form of ${\beta}$-glucuronidase was obtained by isoelectric focusing at pH 5.13. Highly purified ${\beta}$-glucuronidase had specific activity of 34 units/mg protein and showed one major and some minor contaminants by disc gelk electrophoresis. The enzyme showed maximum activity at pH 5.2 and at $48^{\circ}C$. The enzyme was completely inhibited by 1,4 saccharo-${\alpha}$-lactone (5 mM). Albumin and 0.15 M NaCl increased the ${\beta}$-glucuronidase activity. Km of ${\beta}$-glucuronidase using phenolphthalein mono-${\beta}$-glucuronic acid as substrate was 2.9 mM and Vmax was $0.8{\mu}$mole/min. The enzyme appeared to be a glycoprotein by its binding to concanvalin·A. Rabbit and human sperm-acrosomal extracts and seminal plasma showed high ${\beta}$-glucuronidase activity.