A Fluorometric Assay for Detection of Lysyl Oxidase Enzyme Activity in Biological Samples

Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37 degrees C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments.     

L-cysteine oxidase activity in the membrane of Neisseria meningitidis.

Among the L-amino acids, only L-cysteine was oxidized by isolated washed membranes of group B Neisseria meningitidis SD1C. The cysteine oxidase in the membrane obeyed Michaelis-Menten kinetics and was heat labile. The pH optimum for the maximum velocity of the reaction was 9.8. Specific activity of the enzyme increased as cell growth progressed through the exponential phase toward the stationary phase of growth. The enzyme activity was markedly sensitive to inhibition by metal chelators, but was resistant to inhibitors of terminal oxidases with the exception of cyanide. All known cytochromes in the membrane, except b563, were reduced with L-cysteine. The additive nature of L-cysteine oxidase and succinate oxidase activities suggests that an unidentified oxidase is involved in the oxidation of cysteine.     

Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas.

A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 degrees C and grew over the range 0 degrees C-35 degrees C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 degrees C was lower than that of cells grown at 22 degrees C. However, the consumption of oxygen after heat treatment at 35 degrees for 35 min was reduced considerably by 2 degrees C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 degrees C and 22 degrees C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 degrees C produced an alanine oxidase with a temperature optimum of 35 degrees C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 degrees C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 degrees C contained an alanine oxidase system with an optimum temperature of 45 degrees C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 degrees C. The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 degrees C and 37 degrees C had a common optimum temperature of 45 degrees C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions.     

Immobilization of yeast pyridoxaminephosphate oxidase to halogenoacetyl polysaccharides

Pyridoxaminephosphate oxidase (EC 1.4.3.5, deaminating) that was partially purified about 40-fold from dry baker's yeast was immobilized to iodo- and bromoacetyl polysaccharides. The most effective carrier was an iodoacetyl cellulose, to which almost complete activity of pyridoxine 5'-phosphate oxidase was immobilized in 0.02M potassium phosphate buffer (pH 8.5) containing 2M ammonium sulfate at 4 degrees C. The immobilized enzyme was more stable than the purified, soluble enzyme against heat and pH change. It was confirmed that N-(5'-phosphopyridoxyl)-L-serine was degradedly oxidized to pyridoxal 5'-phosphate and L-serine by the immobilized enzyme as comparable rate as pyridoxine 5'-phosphate, whereas N-(5'-phosphopyridoxyl)-D-serine did not serve as substrate, as in the purified, soluble enzyme.     

Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.     

Fluorescent probe study of temperature-induced conformational changes in cytochrome oxidase in lecithin vesicle and solubilized systems

A protein-bound label, N-(1-anilinonaphthyl-4)-maleimide (ANM), was used to investigate conformational changes in bovine heart cytochrome oxidase. The fluidity of cytochrome oxidase vesicles was monitored by a lipophilic probe, 1,6-diphenyl-1,3,5-hexatriene. The fluroescence intensity and emission anisotropy of these probes were examined between 4 and 60 degrees C in enzyme--dipalmitoyllecithin vesicles, in enzyme--dimyristoyllecithin vesicles, in enzyme--dioleoyllecithin vesicles, and in the soluble enzyme. The temperature-dependent changes in these quantities indicated that there were two types of conformational changes in oxidized cytochrome oxidase: one was attributed to an intrinsic enzyme conformation change which occurred around 20 degrees C, and the other was attributed to a conformational change induced by the lipid phase transition. Although ANM-reactive subunits of cytochrome oxidase in these four lecithin vesicle and solubilized systems were different from each other, subunit I always reacted with ANM in preference to other subunits.     

Characterization of the oxidase activity in mammalian catalase

Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The Km for the substrate is 2.4 x 10(-4) m, and Vmax is 4.7 x 10(-5) m/s. Endogenous substrates, including the tryptophan precursor indole, the neurotransmitter precursor beta-phenylethylamine, and a variety of peroxidase and laccase substrates, as well as carcinogenic benzidines, were found to be oxidized by catalase or to inhibit this activity. Several dietary plant micronutrients that inhibit carcinogenesis, including indole-3-carbinol, indole-3-carboxaldehyde, ferulic acid, vanillic acid, and epigallocatechin-3-gallate, were effective inhibitors of the activity of catalase oxidase. Difference spectroscopy revealed that catalase oxidase/substrate interactions involve the heme-iron; the resulting spectra show time-dependent decreases in the ferric heme of the enzyme with corresponding increases in the formation of an oxyferryl intermediate, potentially reflecting a compound II-like intermediate. These data suggest a mechanism of oxidase activity involving the formation of an oxygen-bound, substrate-facilitated reductive intermediate. Our results describe a novel function for catalase potentially important in metabolism of endogenous substrates and in the action of carcinogens and chemopreventative agents.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

Substrate specificities of rat liver peroxisomal acyl-CoA oxidases: palmitoyl-CoA oxidase (inducible acyl-CoA oxidase), pristanoyl-CoA oxidase (non-inducible acyl-CoA oxidase), and trihydroxycoprostanoyl-CoA oxidase.

Rat liver peroxisomes contain three acyl-CoA oxidases:palmitoyl-CoA oxidase, pristanoyl-CoA oxidase, and trihydroxycoprostanoyl-CoA oxidase. The three oxidases were separated by anion-exchange chromatography of a partially purified oxidase preparation, and the column eluate was analyzed for oxidase activity with different acyl-CoAs. Short chain mono (hexanoyl-) and dicarboxylyl (glutaryl-)-CoAs and prostaglandin E2-CoA were oxidized exclusively by palmitoyl-CoA oxidase. Long chain mono (palmitoyl-) and dicarboxylyl (hexadecanedioyl-)-CoAs were oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the former enzyme catalyzing approximately 70% of the total eluate activity. The very long chain lignoceroyl-CoA was also oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the latter enzyme catalyzing approximately 65% of the total eluate activity. Long chain 2-methyl branched acyl-CoAs (2-methylpalmitoyl-CoA and pristanoyl-CoA) were oxidized for approximately 90% by pristanoyl-CoA oxidase, the remaining activity being catalyzed by trihydroxycoprostanoyl-CoA oxidase. The short chain 2-methylhexanoyl-CoA was oxidized by trihydroxycoprostanoyl-CoA oxidase and pristanoyl-CoA oxidase (approximately 60 and 40%, respectively, of the total eluate activity). Trihydroxycoprostanoyl-CoA was oxidized exclusively by trihydroxycoprostanoyl-CoA oxidase. No oxidase activity was found with isovaleryl-CoA and isobutyryl-CoA. Substrate dependences of palmitoyl-CoA oxidase and pristanoyl-CoA oxidase were very similar when assayed with the same (common) substrate. Since the two oxidases were purified to a similar extent and with a similar yield, the contribution of each enzyme to substrate oxidation in the column eluate probably reflects its contribution in the intact liver.     

Repeat polypeptide models of elastin as substrates for lysyl oxidase

Synthetic repeat polypeptides analogous in sequence to the valine-rich regions of elastin have been tested as substrates for purified bovine aorta lysyl oxidase. These polypeptides, HCO(phi-Pro-Gly-Gly)n-Val-OMe, HCO(Val-Pro-Gly-phi-Gly)n-Val-OMe, and HCO-Val-(Ala-Pro-Gly-phi-Gly-Val)n-OMe, where phi = Val or Lys at approximately a 4:1 ratio and where n greater than or equal to 40, are models of the tetra-, penta-, or hexapeptide repeat sequences found in elastin. alpha-Aminoadipic delta-semialdehyde is generated in each of these upon incubation with lysyl oxidase at 37 degrees C, whereas the aldol and anhydrolysinonorleucine bifunctional cross-linkages were formed only in the incubation of enzyme with polypentapeptide. Incubation of the polypentapeptide at 55 degrees C, which enhances coacervation of the peptide, increases aldehyde formation and generates a much higher ratio of cross-linkages to aldehyde than occurred at 37 degrees C. These results demonstrate that lysyl oxidase can oxidize lysine in synthetic polypeptides and suggest important conformational aspects of lysyl oxidase substrates which may control substrate potential as well as the ability of peptidyl aldehyde, once formed by the enzyme, to condense to cross-linkage products.     

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methane- and methanol-grown microbes.

Cultures of methane- or methanol-utilizing microbes, including obligate (both types I and II) and facultative methylotrophic bacteria, obligate methanol utilizers, and methanol-grown yeasts were isolated from lake water of Warinanco Park, Linden, N.J., and lake and soil samples of Bayway Refinery, Linden, N.J. Resting-cell suspensions of these, and of other known C1-utilizing microbes, oxidized secondary alcohols to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Succinate-grown cells of facultative methylotrophs did not oxidize secondary alcohols. Among the secondary alcohols, 2-butanol was oxidized at the highest rate. The optimal conditions for in vivo methyl ketone formation were compared among five different types of C1-utilizing microbes. Some enzymatic degradation of 2-butanone was observed. The product, 2-butanone, did not inhibit the oxidation of 2-butanol. The rate of the 2-butanone production was linear for the first 4 h of incubation for all five cultures tested. A yeast culture had the highest production rate. The optimum temperature for the production of 2-butanone was 35 degrees C for all the bacteria tested. The yeast culture had a higher temperature optimum (40 degrees C), and there was a reasonably high 2-butanone production rate even at 45 degrees C. Metal-chelating agents inhibit the production of 2-butanone, suggesting the involvement of metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble extract of sonically disrupted cells. The cell-free system requires a cofactor, specifically nicotinamide adenine dinucleotide, for its activity. This is the first report of a nicotinamide adenine dinucleotide-dependent, secondary alcohol-specific enzyme.     

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.     

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.     

Enzymatic Characterization and Functional Groups of Polyphenol Oxidase from the Pupae of Blowfly (Sarcophaga bullata)

Polyphenol oxidase (EC 1.14.18.1) was purified from the pupae of blowfly (Sarcophaga bullata) by a procedure involving ammonium sulfate fractionation and chromatography on DEAE-cellulose and Sephadex G-100. Kinetic characteristics of the enzyme were determined using L-DOPA as substrate. The specific activity of the enzyme was 770 U/mg, and the Michaelis constant (Km) was 1.5 +/- 0.1 mM (pH 6.8, 30 degrees C). Activity was maximal at 40 degrees C, pH 6.5. Chemical modification experiments demonstrated that cysteine and tryptophan residues are essential and arginine residues are not essential to the enzyme function. The enzyme is inhibited by quercetin with an IC50 of 0.20 +/- 0.06 mM. The inhibition is of competitive type, and the inhibition constant was determined to be 88 micro M.     

Purification and characterization of <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase with thermal,organic solvent,and detergent tolerance

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.     

Aspergillus niger sulfhydryl oxidase

A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)