Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase.

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.     

Purification and characterization of chicken erythrocyte ferrochelatase.

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.     

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.     

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.     

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA). Both Mg(2+) and Cu(2+) stimulated PO activity when compared with controls. The beta-sheet content of PPO treated with Mg(2+) and Cu(2+) increased significantly (P<0.05). The purified PPO has magnesium level of 5.674+/-2.294 microg/mg and copper level of 1.257+/-0.921 microg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.     

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.     

Purification and properties of a manganese-stimulated endonuclease from Bacillus subtilis.

An endonuclease stimulated by manganese or calcium ions was isolated from Bacillus subtilis. This enzyme attacked double- or single-stranded deoxyribonucleic acid from a variety of sources, including B. subtilis, and was purified from the material released into the medium during protoplast formation. The enzyme appeared as a single peak after glycerol gradient centrifugation and comprised approximately 30 to 35% of the protein in the most purified preparations, as estimated by gel electrophoresis. It had a molecular weight of about 46,000. The mode of action of the enzyme was endonucleolytic, and circular deoxyribonucleic acid was readily cleaved. The enzyme introduced a limited number of both double- and single-strand breaks into native deoxyribonucleic acid, generally yielding products of 1 X 10(6) daltons or more in size. The reasons for this limitation of cleavage were not clear. The activity of the enzyme was inhibited by low levels of Cu2+, Co2+, Hg2+, and Zn2+. It was also inhibited by high concentrations of NaCl. A role for this enzyme in bacterial transormation is suggested.     

Modifiers of mercaptopyruvate sulfurtransferase catalyzed conversion of cyanide to thiocyanate in vitro

The enzyme mercaptopyruvate sulfurtransferase appears to play an important role in the in vivo detoxification of cyanide. It does so by transferring sulfur to cyanide to produce thiocyanate, which is less toxic and may be excreted through the kidney. Several compounds were tested for their ability to affect the rate of enzyme catalyzed thiocyanate formation in vitro. The studies were carried out using both a partially purified bovine kidney extract and a highly purified enzyme preparation. Hypotaurine and methanesulfinic acid doubled sulfurtransferase activity in the partially purified extract at 30 mM, but inhibited the purified enzyme to 57% (hypotaurine) and 27% (methanesulfinic acid) of control activity at the same concentration. Pyruvate, phenylpyruvate, oxobutyrate, and oxoglutarate each inhibited the extract and purified forms of mercaptopyruvate sulfurtransferase. Phenylpyruvate was the most effective inhibitor, reducing activity to 0.2% of control values in the extract, and 11% of control values for purified MPST when added to the reaction at 30 mM. Other compounds tested (see Table 1) had a negligible effect on sulfurtransferase activity. A heat stable cofactor was found in boiled kidney extract which stimulated sulfurtransferase activity in the extract but inhibited sulfurtransferase activity in the purified enzyme, as was observed for hypotaurine and methanesulfinate. The boiled extract had no thiocyanate forming activity of its own. The cofactor operated in synergy with methanesulfinate, but independently of hypotaurine.     

Purification of NADPH-linked alpha,beta-ketoalkene double bond reductase from rat liver

NADPH-linked alpha,beta-ketoalkene double bond reductase was purified from rat liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose. AF-Blue Toyopearl and hydroxyapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 39,500 by the electrophoresis and by HPLC gel filtration on a TSK gel G3000 SWXL column. The double bond of 2-alkenals was also reduced by the enzyme, but to a lesser extent. The enzyme activity was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, N-ethylmaleimide, iodoacetamide, dicumarol, quercitrin, and disulfirum. However, the enzyme was insensitive to oxygen.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.

Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.     

Characterization of bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase from aspen.

Bispecific O-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremuloides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-L-homocysteine agarose using substrate elution. The enzyme has a M(r) of 40,000 as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both purified and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.     

Purification and characterization of homogeneous sunflower seed acid phosphatase

Acid phosphatase (EC 3.1.3.2) from sunflower seed was purified 1800-fold to homogeneity using both conventional and affinity chromatographic methods. The purified enzyme was a mixture of two enzyme forms distinguishable by polyacrylamide gel electrophoresis (PAGE). Gel exclusion chromatography, which did not distinguish between the two forms, gave an apparent M, of 103 000. Preparative PAGE permitted the separation of the two forms, and SDS-PAGE showed that they contained equivalent peptide subunits of apparent M, 56 000 and 52 000. Amino acid analysis indicated that both enzyme forms have similar amino acid compositions. Data on substrate specificity and pH dependence is presented. The kinetic constants for hydrolysis of p-nitrophenyl phosphate as catalysed by sunflower seed acid phosphatase were independent of pH in the range 3-5. The enzyme was competitively inhibited by inorganic phosphate and non-competitively inhibited by phosphomycin.     

Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties

Arachidonic acid 15-lipoxygenase was purified from rabbit peritoneal polymorphonuclear leukocytes. The enzyme was recovered in the cytosol fraction after sonication and purified about 250-fold by acetone precipitation, column chromatography on CM52, Sephadex G-150, and hydroxyapatite. The enzyme catalyzed the conversion of arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which then decomposed to a mixture of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11,13-eicosatetraenoic acid, 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid. The enzyme was specific for oxygenation at carbon 15 of arachidonic acid. The apparent molecular weight of the enzyme was about 61,000 as measured by Sephadex G-150 gel filtration chromatography. The enzyme was sensitive to sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid. The enzyme activity was inhibited by eicosatetraynoic acid (ETYA) or 3-amino-1-(m-(trifluoromethyl)-phenyl)2-pyrazoline (BW755C), but not by indomethacin up to 200 micrograms/ml.     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.     

Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.     

Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils

Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.     

Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2

A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.