• 한국미생물·생명공학회지
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• 제27권4호
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• pp.298-303
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• 1999

An $\alpha$-D-galactosidase ($\alpha$-D-galactoside galactohydrolase, EC 3. 2. 1. 22) from earthworm was purified by affinity chromatography using N-$\varepsilon$-aminocaproyl-$\alpha$-D-galactopyranosylamine coupled to sepharose and its properties were examined. The specific activity of the purified enzyme, tested with p-nitrophenyl-$\alpha$-D-galactopyranoside as substrate, was 314 units/mg protein, representing an 122-fold purification of the original crude extract. The final preparation obtained from by Sephadex G-25 chromatography showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 48,000 by SDS-polyacrylamide gel electrophoresis. The purified galactosidase was showed maximum activity at pH 4.5 and 4$0^{\circ}C$, and was stable in the pH and temperature ranges from 4.0 to 5.5 and 30 to 5$0^{\circ}C$, respectively. The enzyme activity was inhibited by Zn2+, Hg2+ and Co2+. When the purified $\alpha$-galactosidase treated to guar gum for 6 hour, gel-promoting property was increased. It was clear that enzymatic elimination of galactose from guar gum by purified $\alpha$-galactosidase would lead to a significant increase in gelation ability.

• Applied Biological Chemistry
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• 제48권2호
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• pp.109-114
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• 2005

토양 시료로부터 n-hexadecane 대사능을 가지며 배양액의 표면장력을 감소시키고, 탄화수소를 가장 잘 유화시키는 세균을 oil-film collapsing 방법을 통해 선별하였다. 세균은 Bacillus sp.로 부분동정되었으며, BJS-51로 명명하였다. 생물계면활성제의 최적 생산에는 n-hexadecane이 가장 효과적인 탄소원이었으며, 3%의 농도일 때 표면장력이 76 dyne/cm에서 31 dyne/cm로 감소하였다. 이 때, CMD(critical micelle dilution)가 5.7로 가장 높았다. 질소원으로는 $(NH_4)_2HPO_4$가 가장 효과적이었으며, C/N ratio가 60인 경우 표면장력이 29 dyne/cm, CMD가 9.2로 가장 활성이 좋았다. 생산 최적 pH는 7.2였으며, 최적 온도는 $30^{\circ}C$였다. Bacillus sp. BJS-51에 의해 생산된 생물계면활성제를 유기용매추출법과 preparative HPLC systems을 통해 추출, 정제하였다. 각종 발색 시약으로 정제된 생물계면활성제의 생화학적 성질을 조사한 결과, 지질다당임을 확인 할 수 있었다. 생산된 생물계면활성제의 표면장력은 27 dyne/cm까지 감소하였으며, CMC(critical micelle concentration)는 0.08 g/l였다. 상기의 최적조건에서 생물계면활성제의 생산량은 CMD값이 9.2이었으므로 약 0.74 g/l이었다. 생산된 생물계면활성제는 $pH\;2{\sim}12$ 사이에서 표면장력 $29{\sim}31\;dyne/cm$로 안정하였으며, $100^{\circ}C$에서 60분간 열을 가한 후에도 표면장력 $29{\sim}30\;dyne/cm$로 안정하였다.

• 한국균학회지
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• 제36권2호
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• pp.183-188
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• 2008

3단계를 거처 식용 가능한 다발구멍장이버섯으로부터 분리한 혈전용해효소의 비활성은 30.49 U/mg이었으며, MALDI-TOF/MS로부터 분자량이 30086.41 Da이었다. 일차 아미노산 구조는 Glu-Thr-Val-Thr-Glu-Thr-Thr-Ala-Pro-Trp-Gly-Leu-Ser-Arg-Ile으로 밝혀졌으며, pH 8.0에서 pH 10.0의 넓은 범위에서 큰 활성을 나타내는 alkaline protease로, 최적온도는 $50^{\circ}C$이며, $30^{\circ}C$까지는 대체로 열에 안정한 효소였다. 이 효소는 합성 기질 N-Suc-Ala-Ala-Pro-Phe pNA을 강하게 분해하였으며, $Hg^{2+}$ 금속이온의 첨가로 효소 활성이 완전히 사라졌다. 효소저해제인 PMSF의 첨가로 혈전용해활성이 사라지는 결과로부터 serine 분해효소임을 알 수 있었다.

• BMB Reports
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• 제35권2호
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• pp.165-171
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• 2002

A serine collagenolytic protease was purified from the internal organs of filefish Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and $55^{\circ}C$. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

• 한국식품영양학회지
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• 제15권2호
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• pp.104-111
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• 2002

호열균 B. stearotheymophilus으로부터 단백질 고차구조 형성을 촉진하는 내열성 PPIase를 정제하기 위해, 이 균체를 대량으로 배양 집균, 파쇄하여 효소활성을 측정하였다. 효소의 활성측정은 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pAN)를 기질로 사용하였다. chymotrypsin은 기질 이성체(cis-trans 형)의 한쪽(trans)만을 특이적으로 분해하는 반응을 이용하여 PPIase 활성을 측정하였다. 호열균 추출시료로 부터 효소활성을 확인한 후, DEAE-sepharose CL-6B, Sephadex G-75로 정제 후, 최종적으로 Superose TM-12 (FPLC) gel-필트레이션으로 분자량 18kDa의 본 효소를 정제하였다. 정제한 효소의 화학적 특징을 조사한 결과 pH 7.5~8.0사이에 안정하였으며, 최적 pH는 8.0으로 나타났다. 그리고 $65^{\circ}C$에 30분간 열처리 후, 효소활성을 측정한 결과 50%이상의 잔존 활성을 갖는 내열성 효소임을 확인하였다. 정제 단백질의 N-말단 아미노산 분석은 Edman 분해법으로 39 아미노산 잔기를 결정하였다. 그리고 PPIase의 재구성(refolding) 반응은, 요소로 변성시킨 기질 RNase 1을 이용하여 이 단백질의 재구성 (refolding) 실험을 조사한 결과, PPIase는 변성 기질 RNase 1의 재구성(refolding)을 촉진하는데 높은 효과를 가지고 있었다. 호열균 유전자 라이브러리로부터 PPIase 유전자 약 3kb을 클로닝하였다. 재조합 플라스미드 cPI-40에서 프라이머(A-1, B-2)를 이용하여 PPIase N-말단을 코드하는 유전자를 PCR법으로 증폭하여, 염기배열을 결정한 결과 증폭된 단편은 165염기로 형성된 55 아미노산 잔기를 코드하는 open reading frame(ORF)가 연속되고 있었다. 그리고 Edman법으로 결정한 PPIase의 39아미노산 잔기가 이 배열내에 완전히 보존되어 있었다. 이 결과로부터 이 ORF는PPIase구조 유전자의 1/3에 해당하는 단편임을 확인하였다.

• Journal of the Korean Wood Science and Technology
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• 제35권2호
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• pp.85-95
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• 2007

Manganese dependent peroxidase (MnP) is the most ubiquitous enzyme produced by white-rot fungi, MnP is known to be involved in lignin degradation, biobleaching and oxidation of hazardous organopollutants. Bjerkandera fumosa is a nitrogen-unregulated white-rot fungus, which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, and incubation temperature. The growth of fungus was optimal in pH 4.5 at $30^{\circ}C$, $N_2$-unregulated white-rot fungus produces high amounts of MnP in the excess N-nutrients. The fungus produced the highest level of MnP (up to $1000U/{\ell}$) with $0.25g/{\ell}$ asparagine and $1g/{\ell}$ $NH_4Cl$ as N source at 1.5 mM $MnCl_2$ concentration, pH value of 4.5 at $30^{\circ}C$. Purification of MnP revealed the existence of two isoforms: MnPl and MnP2. The molecular masses of the purified MnPl and MnP2 were in the same range of 42~45 kDa. These isoforms of B. fumosa strictly require Mn to oxidize phenolic substrates. Concerned to kinetic constants of B. fumosa MnPs, B. fumosa has similar Km value and Vmax compared to the other white-rot fungi.

• Biomolecules & Therapeutics
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• 제3권2호
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• pp.165-170
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• 1995

The biochemical properties of the fibrinolytic protease of 50,800 Da isolated from the venom of Kgdistrodon blomhoffi brevicaudus were characterized. The enzyme hydrolyzed the carboxyl side of arginine in the synthetic chromogenic peptides, N-Benzoyl-Phe-Val-Arg-pNA and N-p-Tosyl-Gly-Pro-Arg-pNA, and the enzyme activity was inhibited by phenylmethylsulfonylfluoride indicating that the enzyme belongs to the serine protease family. The pretense showed maximum activity at pH 7.5 and inhibited by ZnCl$_2$, CuSO$_4$, but not by soybean trypsin inhibitor, pepstatin A, 2-mercaptoethanol and EDTA. The fm value determined with N-p-Tosyl-Gly-Pro-Arg-pNA was 0.2 mM. The thrombolytic activity of the purified enzyme was evaluated by platelet aggregation test in rabbits. While the platelet count ratio in blood of the rabbits injected with thrombin alone declined from 1.0 to 0.6 within 7 min and maintained around 0.6 for 24 hours thereafter, the ratio rapidly recovered from around 0.6 to 0.8 in 1 hr, to 1.0 in 24 hrs when the rabbits were sequentially treated with thrombin and the purified enzyme. The result showed that the serine protease from A. blomhoffi brevicoudus of 50,800 Da had a thrombolytic activity in vivo and the enzyme might be developed as a therapuetic agent for the treatment of thrombic disease.

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.238-245
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• 2009

Pure culture experiments were conducted to assess the bacteriostatic and bactericidal effects of phlorotannins (PT) isolated from Ascophyllum nodosum (brown seaweed) on Escherichia coli O157:H7. In Exp. 1, one non-O157:H7 strain (25922) and three strains of E. coli O157:H7 (3081, EDL933 and E318N) were cultured in M9 medium with PT included at 0 (control), 25, 50 or $100{\mu}g/ml$ (n = 3). Bacterial growth was monitored by $OD_{600}$ at 0, 4, 6, 12 and 24 h, and by dilution plating at 0, 4, 6 and 24 h. All strains were inhibited (p<0.001) by PT to varying degrees. At 50 or $100{\mu}g/ml$, PT prevented growth of all four strains. At $25{\mu}g\;PT/ml$, growth of 25922, 3081, E318N and EDL933 was inhibited for 6, 12 and 24 h, respectively, but 25922 and 3081 resumed growth by 12 and 24 h. Direct plating confirmed bactericidal effects of PT on all four strains at $100{\mu}g/ml$, and on EDL933 and E318N at $50{\mu}g/ml$. In Exp. 2, strains 25922 and 3081 were incubated with no tannins or with $50{\mu}g/ml$ of PT, purified condensed tannins (CT) from Quebracho (Schinopsis balansaei), or purified tannic acid from Rhus semialata (Anacardiaceae) as hydrolysable tannins (HT). Strain 3081 was unaffected by HT or CT, but was completely inhibited (p<0.001) by PT at 4, 6 and 24 h. Strain 25922 was unaffected by HT, slightly inhibited by CT, and almost eradicated by PT at 4 and 6 h. Transmission electron microscopy revealed tannin-mediated alterations to bacterial cell walls. Phlorotannins from A. nodosum exhibit growth-inhibiting and bactericidal effects in vitro against the strains of E. coli O157:H7 investigated. Anti-E. coli efficacy of A. nodosum PT is superior to that of terrestrial tannins purified from Quebracho and from Rhus semialata.

• BMB Reports
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• 제39권6호
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• pp.671-676
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• 2006

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748-fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.