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1.
A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54 900 as estimated from SDS gel electrophoresis and 21 500 from gel filtration. The optimum pH and temperature were 5.0 and 35°C, respectively. The enzyme was stable at temperatures up to 45°C and in a pH range between 4.0 and 6.0. The Km was 2.5 mg ml-1 for colloidal chitin, Hg2+ and Ag+ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme. 相似文献
2.
Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The Km and Vmax values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)-1 h-1 , respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca2+ and Zn2+ , whereas it was strongly inhibited by Cu2+ , Ag2+ , Hg2+ , p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls. 相似文献
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K
3.
Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu2+ , Cr2+ and Hg2+ were most inhibitory, decreasing Ala2 breakdown to 15, 15 and 5% of control activity, whereas Co2+ , Mn2+ and Zn2+ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l−1 and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo. 相似文献
4.
M. Esther Fárez-Vidal Antonia Fernández-Vivas F. González J.M. Arias 《Journal of applied microbiology》1995,78(1):14-19
M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg2+ , Zn2+ , Cu2+ , Ag+ , Pb2+ , Fe2+ and Fe3+ , EDTA and glutardialdehyde, but was less affected by Ni2+ and Cd2+ . Li+ , Mg2+ , Ba2+ , Ca2+ , N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K m (45°C, pH 8) for starch hydrolysis was 2.0 times 10-3 gl-1 . Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase. 相似文献
5.
Deoxyribonuclease activities in Myxococcus coralloides D 总被引:2,自引:1,他引:1
M. Magdalena Martínez-Cañamero J. Muñoz A.L. Extremera J.M. Arias 《Journal of applied microbiology》1991,71(2):170-175
Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+ ); however, other metal ions (Fe2+ , Ni2+ , Zn2+ ) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P). 相似文献
6.
Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with Km values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg2+ concentration. pH had no effect on the Km value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg2+ , although it was essential for maximum activity. Mg2+ could not be replaced by Cu2+ , Ca2+ , Ba2+ , Co2+ or Ni2+ . Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively. 相似文献
7.
A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the Km for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (Km = 2.4 m M ), NADP+ (Km = 4.0 m M ), 5'-AMP (Km = 4.5 m M ), 5'-ADP (Km = 6.1 m M ) and ribose-5P (Km = 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn2+ , K+ , Hg2+ and Mo6+ were strong inhibitors, whereas F− and Ca2+ inhibited weakly; Co2+ and Ni2+ were activators. 相似文献
8.
J. Rodriguez J.L. Copa-Patino F. Reyes M.I. Pérez-Leblic 《Letters in applied microbiology》1994,19(4):217-220
High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K m of 0.80 mmol 1-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1-1 . Hg2+ , Ag+ and Fe3+ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine. 相似文献
9.
Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation 总被引:2,自引:0,他引:2
Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The Km and Vmax values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)−1 h−1 , respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba2+ , Cu2+ , Mn2+ and Hg2+ . The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures. 相似文献
10.
NADP+ -malic enzyme ( l -malate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn2+ or Mg2+ , for its activity. Km values at pH 7.8 for malate, NADP+ and Mn2+ were 4.0, 0.031 and 0.71 m M , respectively. Mn2+ -dependent activity was inhibited by heavy metal ions such as Cd2+ , Zn2+ , Hg2+ , and to a lesser extent by Pb2+ and Al3+ . Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP+ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP+ , pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP+ -malic enzyme is controlled by intracellular concentrations of substrates and products. 相似文献
11.
Purification and properties of a neutral invertase from the roots of Cichorium intybus 总被引:1,自引:0,他引:1
Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)−1 min−1 . The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a Km between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl2 and AgNO3 and partially inhibited by CoCl2 , and ZnSO4 (1 mM). Pyridoxal phosphate (Ki ≅ 500 μ M ), Tris (Ki ≅ 1.2 m M ), glucose and fructose (Ki ≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed. 相似文献
12.
Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and Km values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca2+ , Mg2+ and Mn2+ activate the enzyme, but Zn2+ and Cu2+ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing Ki values of 0.38 m M , 0.27 m M and 0.16 m M , respectively. 相似文献
13.
Abstract— A novel phospholipase A1 , present in human cerebral cortex and active at pH 9.25 upon ultrasonicated phosphatidylethanolamine is described. It has been purified 39-fold from acetone-dried powders of cortex by ammonium sulphate fractionation at 25% saturation, followed by Sephadex G150 gel filtration. A molecular weight of approx 500,000 has been found by Sepharose 6B gel filtration. The enzyme was slightly stimulated by 2 mM-Ca2+ and was inhibited by 1 mM-Hg2+ and by all detergents tested. The enzyme hydrolysed phosphatidylcholine at 18% of the rate for phosphatidylethanolamine, lysophospholipids to a lesser degree and neutral lipids not at all. There appeared to be a preference for fluid phosphatidylethanolamine substrates. The similarities of the enzyme to other phospholipases A1 are discussed. 相似文献
14.
Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K m and V max values have been found to be 1.1 mM and 25.6 μmol min−1 (mg protein)−1 , respectively. Enzyme activity was inhibited by Hg2+ , Ag2+ , and Cu2+ at a concentration of 1 × 10−3 M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product. 相似文献
15.
An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca2+ concentration required for PLI and PLII activity was determined to be 0·5 mmol l−1 . The Ca2+ requirement could not be replaced by other metal cations such as Mg2+ , Cu2+ , Zn2+ , Fe3+ and Co2+ . Optimal pH for activity was determined to be between 8·5 and 9·0. The K m values for sodium polygalacturonate were 1·28 and 1·11 mg ml−1 for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl2 or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII. 相似文献
16.
Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata 总被引:1,自引:1,他引:0
The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg−1 when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K m and V max values were 7.33 mg ml−1 and 833 μmol min−1 , respectively. EG activity was inhibited by Fe2 + , Hg2 + , Ag+ and Pb2 + , and enhanced by dithiothreitol and Zn2 + . The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found. 相似文献
17.
Abstract— 45 Ca2+ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K+ at all times investigated. The accumulated 45 Ca2+ was bound within the synaptosome. 45 Ca2+ -ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal 45 Ca2+ then returned to control values. A23187 also increased intra-synaptosomal Na+ and Cl− contents. Botulinum toxin inhibits the K.+ -stimulated release of [14 C]ACh from synaptosomes but the ionophore released [14 C]ACh from both normal and botulinum-treated preparations in a Ca2+ -dependent manner. However, it also elicited Ca2+ -dependent release of [choline. Increased extracellular Ca2+ (10 mM and 20 mM) released [14 C]ACh (but not [14 C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca2+ essential for the mechanism of ACh release. 相似文献
18.
An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg2+ . Ca2+ , Hg2+ , Fe3+ , arsenate, K+ and Zn2+ were inhibitory. Mn2+ , dithiothreitol and EDTA had no effect, and polyamines were activators. 相似文献
19.
Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1 总被引:2,自引:0,他引:2
Masaru Sakurada Diego P. Morgavi Kenji Komatani Yoshifumi Tomita ryoji Onodera 《FEMS microbiology letters》1996,137(1):75-78
Abstract A chitinase was purified from the cytosolic fraction of the anaerobic rumen fungus Piromyces communis OTS1 by affinity chromatography using regenerated chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 6.2 and at 60 °C in a 20-min assay. The molecular mass of the purified protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme activity, which was of the 'endo' type, was inhibited by A+ , Hg2+ and allosamidin. N -Acetyl- β -glucosaminidase and 'exo' type chitinase activity were absent from the purified preparation. 相似文献
20.
Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species 总被引:1,自引:1,他引:0
Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met5 ]-enkephalin-Arg6 -Gly7 -Leu8 . The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu5 ]- enkephalin at the C-terminus. 相似文献