• Microbiology and Biotechnology Letters
• /
• v.26 no.5
• /
• pp.413-419
• /
• 1998

Aryl acylamidase [EC 3.5.1.13] present in an acetaminophen-assimilating Pseudomonas sp. has been purified to a homogeneity using series of ammonium sulfate fractionation, DEAE-Sephacel anion exchange, Phenyl-Sepharose CL-4B hydrophobic, and Sephadex G-100 gel-permeation chromatography. The molecular weight, which was estimated by gel-permeation filtration and sodium dodecyl sulfate polyacylamide gel electrophoresis, was about 57 kDa and 56 kDa, respectively, indicating that this enzyme is a monomeric protein. The optimum pH was 10.5 and the optimum temperature was 40$^{\circ}C$. After incubation of the enzyme at 50$^{\circ}C$ for 30 min, residual activity of the enzyme was 34% compared to its original activity. The Km values for acetaminophen and 4'-nitroactanilide were 0.10 mM and 0.11 mM, respectively.

• Microbiology and Biotechnology Letters
• /
• v.19 no.6
• /
• pp.592-597
• /
• 1991

An extracellular xylanase of Bacillus stearothemophilus was purified to a single protein through a sequency of operations including ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography, Sephadex G-100 gel filtration and heat treatment. The purified enzyme had a moleular weight of 170, 000. the pH and temperature optima for the enzyme activity were pH 9.0 and $55^{\circ}C$, respectively. The activity was enhanced by $co^{2+} \; and\; Mn^{2+}$, and inhibited by $Hg^{2+}$. Pattern of hydrolysis demonstrated that the xylanase was an endo-splitting enzyme able to break down larchwood xylan at random giving xylobiose and xylotriose as the main end products.

• Korean Journal of Microbiology
• /
• v.37 no.1
• /
• pp.21-27
• /
• 2001

Recently many investigators have devoted considerable attention to the production and purification of PA for antigens, and the preparation of new synthetic medium (RM medium) have solved to increase the yields of the PA but, the low sensitivity of the PA to detect B. anthracis infections has remained as a problem to be solved. This study was undertaken to evaluate the yields of the PA from culture filtrates of B. anthracis Sterne $34F_2$ strain in modified RM medium in which 10 g/l of $NaHCO_3$ and 10g/l of glucose were replaced by 8 g/l and 5 g/l, and the first purification step of PA from culture broth was used hydroxyapatite. The PA was purified by hydroxyapatite column chromatography, DEAE-Sepharose CL-4B column chromatography and Toyo-pearl gel filtration chromatography. The yield of PA from the modified RM medium, 8.6 mg/l.

• Korean Journal of Microbiology
• /
• v.26 no.4
• /
• pp.324-331
• /
• 1988

Ascorbate oxidizing enzyme from the crude extract of Pleurotus ostreatus was purified by ammonium sulfate precipitation, preparative polyacrylamide gel electrophoresis, DEAE Sepharose CL-6B ion exchange chromatography and Sephadex G-150 gel filtration chromatography. The molecular weight of the enzyme estimated by Sephadex G-150 gel filtration chromatography was 140,000 and that of its subunit by SDS-polyacrylamide gel electrophoresis 66,000. The optimum pH for the maximum activity of the enzyme was 5.2 and the isoelectric point of the enzyme was 6.0 Km values for L-ascorbic acid and D-isoascorbic acid were both 2.2.$\mu$M, which indicates that the enzyme has the asme affinity towards both substrates.

• Korean Journal of Microbiology
• /
• v.30 no.4
• /
• pp.291-298
• /
• 1992

Srrepromycec. corlirolar produces at least 4 catalase activity bands with different electrophoretic mobilities on polyacrylamide gel which vary during development. Spores and mycelia at stationary phase produced all the activity bands(Cat1. 760 kr); Cat3-I, 170 kD: Cat3-2, 140 kD: Cat3-3. 130 kD; Cat4, 70 kD) except for Cat2 (300 kD). Mycelia at mid-logarithmic phase produced only Cat2 and Cat3-2 bands, and mycelia at late-logarithmic phase produced bands except Catl and Cat\ulcorner. Catalase-deficient mutants were screened in S. coelicalur by H201 bubbling test following NTG mutagenesis. Wc tested sevcral non-bubbling or slow-bubbling mutants for their catalase activities. The overall activities in cell extracts decreased more than 5 fold. Activity bands in native gel selectively decreased in intensity or disappeared. In all the non-bubbling mutants testcd, Cat3-2 band decreased significantly or disappeared. suggesting that Cat3-2 is the major catalase. The selective disappearance of bands in mutants suggest that each band is governed by different genes. We purified catalase activity from -:ell extracts obtained at late-logarithmic phase. Following chromatographies on Sepharose CL-4B. DEAE Sepharose CL-6B. Phcnyl Sepharose CL-4B. and hydroxylapatite columns. only the Cat3-2 activity was obtained. The native form of Cat3-2 has molecular weight of approximately 140 kD, judged by gel electrophoresis. Thc electrophoretic mobility on SDS-polyactylamide gel suggests that this enzyme contains 2 identical subunits of 67 kD.

• The Korean Journal of Mycology
• /
• v.40 no.2
• /
• pp.109-113
• /
• 2012

We have isolated an anti-complementary polysaccharide from the hot water extracts of Phellinus linteus mycelia. Anti-complementary polysaccharide, PL-5-IIIa, was purified by ultrafiltration, gel permeation chromatography using Sepharose CL-4B. GPC (Sepharose CL-4B) and its homogenicity was demonstrated by HPLC. Using gel permeation chromatography with standard dextrans, its molecular weight was determined as about 800,000 dalton. The purified PL-5-IIIa was identified as a protein bound polysaccharide comprising of 29.6% protein and 64.2% carbohydrate which was composed of fucose(15.8%), galactose(43.1%) and mannose(40.6%).

• Korean Journal of Environmental Biology
• /
• v.29 no.4
• /
• pp.305-311
• /
• 2011

The distribution of flocculating activity of culture broth was examined and the major constituent with flocculating activity was identified. Most of flocculating activity was found in culture broth without cells. As the activity was maintained by the digestion with pronase, it suggests that the activity is due to the polysaccharide. The bioflocculant obtained from $Lactobacillus$ $jensenii$ YW-33 was precipitated by 60~80% EtOH fractionation (LJ-80). LJ-80 was separated by ion-exchange chromatography using DEAE-Toyopearl 650C and LJ-80-II showed more potent flocculating activity than those of other fractions. The major activity fraction LJ-80-II was further purified on the gel permeation using Sepharose CL-6B to LJ-80-II-1. GPC (Sepharose CL-6B) and HPLC were used to determine whether LJ-80-II-1 has a homogenecity. The molecular weight of purified LJ-80-II-1 was estimated over 800,000 dalton by gel permeation chromatography. Purified LJ-80-II-1 contained 98.4% total sugar, 0.6% protein. Main sugar of purified LJ-80-II-1 was composed of mannose : galactose : glucose with a molar ratio of 1.61 : 0.25 : 1.00.

• BMB Reports
• /
• v.28 no.2
• /
• pp.162-169
• /
• 1995

Rat brain succinic semialdehyde dehydrogenase (EC 1.2.1.24 SSADH) activity was detected in mitochondrial, cytosolic and microsomal fractions. Brain mitochondrial soluble SSADH was purified by ammonium sulfate precipitation, DEAE Sephacel, and 5'-AMP Sepharose 4B affinity chromatography. The purified enzyme was shown to consist of four identical subunits, and the molecular weight of a subunit was 55 kD. The $K_m$ for short chain aliphatic aldehydes and aromatic aldehydes were at the $10^{-3}M$ level but that for succinic semialdehyde was 2.2 ${\mu}M$. Either $NAD^+$ or $NADP^+$ can be used as a cofactor but the affinity for $NAD^+$ was 10 times higher than that for $NADP^+$. The brain cytosolic SSADH was also purified by ammonium sulfate precipitation, DEAE Sephacel, Blue Sepharose CL-6B and 5'-AMP Sepharose 4B affinity chromatography and its Km for short chain aliphatic aldehydes was at the $10^{-3}$ level but that for succinic semialdehyde was 3.3 ${\mu}M$. $NAD^+$ can be used as a cofactor for this enzyme. We suppose that both enzyme might participate in the oxidation of succinic semialdehyde, which is produced during GABA metabolism. The activity of both cytosolic and mitochondrial SSADH was markedly inhibited when the concentration of succinic semialdehyde was high. The reciprocal plot pattern of product inhibition and initial velocity indicated a sequential ordered mechanism for mitochondrial matrix SSADH. Chemical modification data suggested that amino acid residues such as cysteine, serine and lysine might participate in the SSADH reaction.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.108-108
• /
• 2002

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.

• KSBB Journal
• /
• v.15 no.3
• /
• pp.318-322
• /
• 2000

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the Enterococcus faecalis RKY1 grown anaerobically on a defined medium containing glycerol and fumarate. A major portion of the purification was performed with employing Triton X-100 and reducing agents by Phenyl-sepharose CL-4B DEAE-sepharose and Dephadex G-150 The final activity was 0.42 unit/mg. The deduced molecular mass of active band was 66 kDa. The optimal pH and temperature for the activity were 7.0 and 38$^{\circ}C$ respectively. The enzyme activity was not affected by 1mM metal ions such as bacl2 $.$2H2O HgCl2 MnCl2$.$4H2O ZnCl2 CuCl2$.$2H2O Mgcl2$.$6H2O FeSo4$.$7H2O and by EDTA. Partially purified enzyme ws yellow in color ; spectroscopic study indicated the presence of flavins as a cofactor.