Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates

A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).     

Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.     

Purification and characterization of alpha-galactosidase from watermelon

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.     

Purification of acetyl coenzyme A: deacetylacephalosporin C O-acetyltransferase from Acremonium chrysogenum.

Acetyl CoA: deacetylcephalosporin C O-acetyltransferase, which catalyzes the final step of the biosynthetic pathway to cephalosporin C, was stabilized by a buffer solution containing 7-aminocephalosporanic acid and purified over 1300-fold from Acremonium chrysogenum. The purified enzyme has a molecular weight of 55,000 as measured by gel filtration. SDS-polyacrylamide gel electrophoresis showed two subunit bands corresponding to molecular weights of 27,000 and 14,000. The enzyme has an isoelectoric point at pH 4.0 and optimum activity at pH 7.5.     

Pisiferdiol,a Novel Phenolic Diterpenoid from Chamaecyparis pisifera Endl

Isocitrate lyase was purified from the purple nonsulfur bacterium Rhodopseudomonas sp. No. 7. The purified enzyme was electrophoretically homogeneous. The molecular weights of the native enzyme and its subunit were estimated to be approximate 250,000 and 62,000 by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The optimum pH for its activity was 6.5. The optimum temperature was 45°C. The Km for dl-isocitrate was 0.136 mm in potassium phosphate buffer (pH 6.0). Mg²⁺ was required for full activity of the enzyme as a non-essential activator. The enzyme activity was inhibited by SH-blocking reagents. Non-competitive inhibitory effects on the enzyme were examined with malate and succinate. The Ki for malate and succinate were 2.7 and 0.24 mm, respectively.     

Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver

By means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, alpha-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final alpha-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except alpha-galactosidase activity, which amounted to 0.8% of the alpha-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80,000 at pH 4.2 and 40,000 at pH 7.2 by molecular sieve chromatography, and to be 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack alpha-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28 mM and 409 mumol/min X mg for p-nitrophenyl alpha-N-acetylgalactosaminide, and 0.0543 mM and 1.19 mumol/min X mg for ovine submaxillary asialoglycoprotein.     

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii

Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Purification of Acetyl Coenzyme A: Deacetylacephalosporin C O-Acetyltransferase from Acremonium chrysogenum

Acetyl CoA: deacetylcephalosporin C O-acetyltransferase, which catalyzes the final step of the biosythetic pathway to cephalosporin C, was stabilized by a buffer solution containing 7-aminocephalosporanic acid and purified over 1300-fold from Acremonium chrysogenum. The purified enzyme has a molecular weight of 55,000 as measured by gel filtration. SDS-polyacrylamide gel electrophoresis showed two subunit bands corresponding to molecular weights of 27,000 and 14,000. The enzyme has an isoelectoric point at pH 4.0 and optimum activity at pH 7.5.     

Production of Pentameric Hybrid Immunoglobulins Consisting of IgA and IgM

The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Purification and characterization of a Cu, Zn-superoxide dismutase from adult Paragonimus westermani.

In cytosolic fraction of adult Paragonimus westermani, superoxide dismutase activity was identified (4.3 units/mg of specific activity) using a xanthine-xanthine oxidase system. The enzyme was purified 150 fold in its activity using the ammonium sulfate precipitation, DEAE-Trisacryl M anion-exchange chromatography and Sephadex G-100 molecular sieve chromatography. The enzyme exhibited the enhanced activity at pH 10.0. The enzyme activity totally disappeared in 1.0mM cyanide while it remained 77.8% even in 10 mM azide. These findings indicated that the enzyme was Cu, Zn-SOD type. Molecular mass of the enzyme was estimated to be 34 kDa by gel filtration and 17 kDa on reducing SDS-polyacrylamide gel electrophoresis which indicated a dimer protein.     

Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.