Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Purification and characterization of extracellular cholesterol oxidase from <Emphasis Type="Italic">Enterobacter</Emphasis> sp.

The objective of this work is to obtain an abundant source of cholesterol oxidases for industrial and medicinal needs. Thirteen bacterial strains that express high level of inducible extracellular cholesterol oxidase (COX) were isolated from carnivore feces. One of these strains, named COX8-9, belonging to the genus Enterobacter, was found to produce the highest level of cholesterol oxidase. COX from strain COX8-9 was purified from the culture supernatant by ultrafiltration followed with two consecutive Q-Sepharose chromatographies at different pH values, and then by Superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The K _m value for oxidation of cholesterol by this enzyme was 1.2 × 10⁻⁴ M, with optimum activity at pH 7.0. Enzymatic activity of COX was enhanced 3-fold in the presence of metal ion Cu²⁺, and the enzyme was stable during long-term aqueous storage under various temperatures, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way. Deping Ye and Jiahong Lei are contributed equally to this work.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Existence of aa 3-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 α-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 °C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A₁ 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.     

Purification and characterization of oxalate oxidase from wheat seedlings

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat (Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K _m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

Purification and Molecular Analysis of a Monoamine Oxidase Isolated from Narcissus tazetta

Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS-520 HQ column, was estimated to be 135 kDa. SDS–PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The K _m value for n-propylamine was 5.9 × 10^?5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.     

Microbial oxidases of acidic -amino acids

Two microbial oxidases of acidic -amino acids have been purified to homogeneity. One is a -aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with -aspartate and is far more active toward -aspartate than -glutamate. The other is a -glutamate oxidase of Candida boidinii 2201 that preferred -glutamate to -aspartate as a substrate in terms of k_cat/K_m, but was not induced very effectively by -glutamate. The most potent competitive inhibitor of the C. humicolus -aspartate oxidase was malonate, and that of the C. boidinii -glutamate oxidase was -malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus -aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus -aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K_d for the apoprotein-FAD complex was very low (8.2×10⁻¹² M) consistent with the observed tight binding. The C. humicolus -aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral -amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.     

Overexpression,one-step purification,and characterization of a type II cholesterol oxidase from a local isolate <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633

A 1.6 kb gene encoding a cholesterol oxidase (choR) from a local isolate, Rhodococcus sp. PTCC 1633 was cloned into pET23a and the highly expressed recombinant enzyme was purified from the cell lysate of IPTG-induced Escherichia coli BL21(DE3)pLysS with one-step absorption on cholesterol. The purified protein had a molecular mass of 55 kDa, isoelectric point at about pH 9.0 and absorption peaks at 280, 380 and 460 nm, indicating that the enzyme is a flavoprotein. The optimum pH and temperature for the recombinant enzyme were 7.0 and 50°C, respectively. Steady-state kinetic revealed that the cholesterol oxidase had a K _m of 32 μM. This study is the first report concerning expression and one-step purification of a gene encoding cholesterol oxidase from Rhodococcus spp. This study revealed that this enzyme is a type II cholesterol oxidase.     

Isolation and purification of methyl mercaptan oxidase fromRhodococcus rhodochrous for mercaptan detection

Methyl mercaptan oxidase was successfully induced fromRhodococcus rhodochrous IGTS8 using methyl mercaptan gas and purified to homogeneity for the detection of mecrcaptans. The purification procedure involved DEAE-Sephacel and Superose 12 column chromatography with recovery yields of 85.8 and 83.3%, and a specific activity of 92.7 and 303.4 units/mg-protein, respectively. The molecular weight of purified methyl mercaptan, oxidase was determined to be 64.5 kDa by SDS-PAGE. The extract from gel filtration chromatography oxidizes methyl mercaptan to produce formaldehyde, which can be easily detected by the purpald-coloring method. Optimum temperature for activity was achieved at 60°C. This enzyme was inhibited by both K₂SO₄ and NaCl at concentration of less than 100 mM and recovered to original activity at concentration of 200 mM. In the presence of methanol, the activity decreased by 33%.     

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.     

Purification and characterization of an H<Subscript>2</Subscript>O-forming NADH oxidase from <Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria

Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O₂. When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O₂-free N₂ carrier-gas) to microoxic (sparged with 3% O₂/97% N₂ mixed carrier-gas) growth conditions in the mid exponential phase (OD₆₆₀=1.0). When the strain grew under 3% O₂/97% N₂, the medium remains anoxic. Thirty minutes after beginning aeration with 3% O₂, the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H₂O, and the estimated K_m for oxygen was 61.9 M. These data demonstrate that an O₂-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.Abbreviations NRIC NODAI Research Institute-Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Characterization of aldehyde oxidase from <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. MEY43 and its application to oxidative removal of glutaraldehyde

Bacterium MEY43, an isolate from soil, produced aldehyde oxidase when it was cultivated in a medium containing methanol as a sole source of carbon and energy. The methylotrophic bacterium was identified as Brevibacillus sp. Its cultivation in media containing other substrates, such as ethanol and glucose, resulted in little production of the enzyme. Aldehyde oxidase purified from a cell-free extract of the bacterium was a hetero-trimeric protein comprised of large, medium, and small subunits with molecular masses of 87, 35, and 19 kDa, respectively. Its UV/visible spectrum and the presence of molybdenum, 5′-CMP, flavin adenine dinucleotide, iron, and acid-labile sulfur suggested that the enzyme belonged to the xanthine oxidase family. The enzyme acted on a wide range of aliphatic and aromatic aldehydes. The K _m value for formaldehyde was 32 mM, whereas those for the other aldehydes tested were below 0.2 mM. When 10 mM glutaraldehyde was treated with 2.0 units of the enzyme ml⁻¹ in the presence of 100 units ml⁻¹ catalase for 120 min, the concentration of the aldehyde decreased to below a detectable level.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase fromSynechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacteriumSynechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6–8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min^–1 at 40° C. The activation energy was 12.4 Kcal for t>29° C and 20.6 Kcal for t<29° C. The specific absorption coefficient, A _{280 mm} ^{1% 1cm} of the pure enzyme in phosphate buffer at pH 6.8 was 15.2.By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer.When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998