• 한국미생물·생명공학회지
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• 제22권5호
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• pp.507-514
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• 1994

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

• 한국미생물·생명공학회지
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• 제15권6호
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• pp.375-380
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• 1987

Alcaligenes eutrophus A52의 무세포 추출액으로부터 유안염석 및 DEAE-cellulose 이온교환 크로마토그래피로서 D-$\alpha$-Amino-$\varepsilon$-Caprolactam(DAC) racemase 와 L-$\alpha$-Amino-$\varepsilon$-Caprolactam(LAC) hydrolase를 분획하였다. A. eutrophus A52에 의한 DAC로부터 L-lysine으로의 전환은 DAC가 racemase에 의해 LAC로 전환된 후 hydrolase에 의해 L-lysine으로 가수분해됨을 확인하였다. DEAE-cellulose 이온교환 크로마토그래피에 의해 분리된 racemase의 분획은 최적온도가 55$^{\circ}C$, 최적 pH는 8.0이었으며 hydrolase의 분획은 최적온도가 $65^{\circ}C$ 최적 pH가 9.0이었다. 이 두 효소를 모두 함유하는 무세포 추출 액에 의한 DAC로부터 L-lysine으로의 전환은 6$0^{\circ}C$와 pH8.5에서 최대를 나타내었고 2% 이상의 DAC와 L-lysine에 의해 상당한 저해를 받았으나 0.5% DAC를 3.1mg의 단백질에 상당하는 무세포 추출액으로서 전환시킨 결과 55$^{\circ}C$에서 10시간 동안에 약 98%가 L-lysine으로 전환되었다.

• 한국식품과학회지
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• 제25권5호
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• pp.576-581
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• 1993

김치 조직의 연화에 관여하는 효소인 polygalacturonase(PG)를 배추에서 추출하여 황산암모늄 분획, 이온교환크로마토그래피 및 FPLC를 이용하여 D-PG, C-1, C-2 PG 세 분획으로 분리 정제하여 특성을 조사하였다. 분리된 세 분획의 활성 최적 온도는 $65^{\circ}C$, 최적 pH가 5.2였으며 pH$4.5{\sim}8.0$ 범위에서 안정하였다. NaCl에 의한 영향은 0.3M NaCl에서 최대의 활성을 보였으나 0.6M 이상에서는 저해를 받았으며 $CaCl_2$의 경우 $0{\sim}0.5mM$ 농도에서는 활성이 크게 영향을 받지 않았으나 0.8mM에서는 저해를 받았다. 열불활성화 특성은 isozyme간에 큰 차이가 없었으며 1차 반응을 따랐다. 이 효소 isozyme의 z값은 $8.4{\sim}9.3^{\circ}C,\;80^{\circ}C$에세 D값은 $102{\sim}126$초였다. 이 호소의 불활성화 값을 이용하여 retort pouch 김치 살균공정에 적용하고 역가의 잔존 가능성에 대하여 고찰하였다.

• 한국축산식품학회지
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• 제29권2호
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• pp.157-163
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• 2009

This study was conducted for the isolation, purification, and characterization of a protease from Korean pear, to see its proteolytic activity on chicken actomyosin and to find the optimum pH and temperature of activity on chicken actomyosin. The protease was isolated from crude extract of Korean pear by ammonium sulfate precipitation. Further purification was done by DEAE-Sepharose ion-exchange chromatography, Mono-Q and Mini-Q column chromatography. The purified enzyme gave a single protein band on SDS polyacrylamide gel electrophoresis and the molecular weight was found to be 38 kDa. The specific activity of purified enzyme was 34,907 unit/mg with 25 fold purification and the yield was 2%. The purified enzyme incubated with chicken actomyosin showed high activity. The optimum pH and temperature for enzyme activity on chicken actomyosin were 6.5 and $70^{\circ}C$, respectively. A protease was purified from Korean pear for the first time and characterized. It was found to be promising for meat tenderization.

• Applied Biological Chemistry
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• 제46권3호
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• pp.176-182
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• 2003

한국재래간장으로부터 혈전용해효소를 강하게 생산하는 균주를 선발하고 이를 Bacillus subtilis K7로 동정하였다. B. subtilis K7이 생산하는 혈전용해성 protease를 정제하여 분자량을 확인한 결과 21,500 Da이었다. 정제된 효소의 최적반응조건은 $40^{\circ}C$와 pH 9.0이었으며 pH 5.0라서 12.0까지 안정하고 $50^{\circ}C$에서 20분간 열처리한 후에도 50%이상의 효소활성을 가지며 EDTA, CDTA 및 iodoacetat에 실활하는 효소이었다. 이 효소의 fibrin에 대한 Km 값은 $1.8{\times}10^{-2}$ M이었다.

• Archives of Pharmacal Research
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• 제29권5호
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Journal of Microbiology
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• 제39권4호
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• pp.279-285
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• 2001

Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1:K1 was isolated from the environment and identified . A polymerase chain reaction assay revealed that this strain harbored both the tdh and the genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6h of culti-vation at 37$\^{C}$ under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85kDa, 59kDa, 41kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255kDa. The purified urease was stable at pH 7.5 and the opeimal pH in HEPES buffer was 8.0 The enzyme was stable at 60$\^{C}$ for 2 h with a residual activity of 32% . The addition of 10$\mu$M if NiCl$_2$maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD$\_$50/(median toxic dose) of the purified urease was 2.5$\mu\textrm{g}$/ml on human leukemia cells.

• 공업화학
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• 제9권4호
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• pp.569-573
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• 1998

1-부틸아민과 에피클로로히드린의 축합체 (PBE)인 새로운 수용성고분자를 합성하고, 적외선 스펙트럼과 레이저 광산란에 의한 산란광의 강도 측정에 의한 분자량 결정, 그리고 제타전위를 측정하여 PBE의 특성을 결정하였으며, PBE와 콜로이드상 벤토나이트입자들의 상호작용에 관해서도 연구 하였다. PBE는 그 골격에 3차 암모늄기를 가지고 있으며 분자량은 1600정도로 비교적 작은 편이었으며, PBE가 콜로이드성 벤토나이트입자에 흡착되는 현상은 Langmuir흡착등온식으로 잘 묘사되었다. 음전기를 띤 콜로이드성 벤토나이트입자 표면에 PBE의 흡착이 진행되면서 $\zeta$ 전위가 음의 값에서 양의 값으로 변하는 결과로부터 PBE가 양이온성고분자전해질인 것과 pH변화 및 $Ca^{2+}$ 이온의 농도변화로부터 PBE가 벤토나이트 표면에 흡착되는 현상은 양이온교환형식으로 이루어지는 것을 확인하였다. 또한 PBE는 무기계 콜로이드인 벤토나이트 현탁액과 소화슬러지에 대해서 응집효과를 가지고 있음이 밝혀졌다.

• 농업과학연구
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• 제39권2호
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• pp.255-261
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• 2012

A bacterium AB-55, isolated from waste mushroom bed of Agaricus bisporus in Sukseong-myeon, Buyeo-gun, Chungcheongnam-do, Korea, was screened onto xylan agar congo-red plate by the xylanolysis method and was used to produce an xylanase in shaker buffle flask cultures containing oat spelt xylans. The phylogenetic analysis using 16S rRNA gene sequence data showed that the strain AB-55 had the highest homology (99.0%) with Bacillus subtilis and it was named as Bacillus subtilis AB-55. A xylanase was purified by ammonium sulfate precipitation (50~80%), gel filtration on sephacryl S-300, and ion exchange chromatography on DEAE sepharose FF. The molecular weight of the xylanase was estimated as 44 kDa by SDS-PAGE. Optimal pH and temperature for the xylanase activity was pH 7 and $50^{\circ}C$, respectively. N-terminal amino acid sequence of the enzyme was identified as Ser-Ala-Val-Lys-His-Gly-Ala-Ile-Val-Phe. The substrate specificity of the enzyme exhibited that it hydrolyzed efficiently oat spelt xylan as well as beechwood xylan, but showed no activity against Avicel and carboxymethyl clellulose (CMC). The enzyme activity was enhanced by $Fe^{2+}$ and $Mn^{2+}$ whereas was entirely inhibited by $Hg^+$.

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.201-208
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• 1998

A protease, which had no tryptic and chymotryptic activity, was purified from the hepatopancreas of shrimp, P. japonicus, through ammonium sulfate fractionation, Q­Sepharose ionic exchange, benzamidine Sepharose 6B affinity, and Sephacryl S-100 gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 24 kDa by gel filtration and showed a single peptide band by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protease had a low ratio of acidic to basic amino acids, which is different with pro teases from marine animals. The enzyme was partially inhibited by benzamidine, tosyl-L-lysine chioromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), and pepstatin. The enzyme did not have any activity against benzoyl-D,L-arginine p-nitroanilide (BAPNA) or benzoyl-L-tyrosine ethyl ester (BTEE) which is a specific substrate of trypsin and chymotrypsin, respectively. However, the enzyme showed activity forward N-CBZ-L-tyrosine p-nitrophenyl ester (CBZ-Tyr-pNE), N­CBZ-L-tryptophan p-nitrophenyl ester (CBZ-Trp-pNE), and N-CBZ-L-proline p-nitrophenyl ester (CBZ-Pro-pNE). The protease did not showed tryptic and chymotryptic activity, which was not reported in shrimp hepatopancreas.