Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoateto 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K_mand V_max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate.The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Purification and characterization of a thermostable pullulanase fromThermoactinomyces thalpophilus

Summary Thermoactinomyces thalpophilus No. 15 produced an extracellular pullulanase in an aerobic fermentation with soluble starch, salts, and complex nitrogen sources. Acetone fractionation, ion-exchange chromatography, and gel filtration purified the enzyme from cell-free broth 16-fold to an electrophoretically homogeneous state (specific activity, 1352 U/mg protein; yield, 4%). The purified enzyme (estimated MW 79 000) was optimally active at pH 7.0 and 70°C and retained 90% relative activity at 80°C (30 min) in the absence of substrate. The enzyme was activated by Co²⁺, inhibited by Hg²⁺, and exhibited enhanced stability in the presence of Ca²⁺. The enzyme hydrolyzed pullulan (K _m 0.32%, w/v) forming maltotriose, and hydrolyzed amylopectin (K _m 0.36%, w/v), amylopectin beta-limit dextrin (K _m 0.45%, w/v) and glycogen beta-limit dextrin (K _m 1.11%, w/v) forming maltotriose and maltose.     

Rapid process for purification of an extracellular β-xylosidase by aqueous two-phase extraction

A rapid process for purification of an extracellular β-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na₂HPO₄. The aqueous two-phase extraction was performed with PE62 (10%)–NaH₂PO₄ (15%) as phase-forming reagent. SDS–PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.     

Determination of the partial specific volume of cytochrome P450 LM2

The partial specific volume (v) of highly purified membrane protein cytochrome P450 LM2 monooxygenase from rabbit liver endoplasmatic reticulum has been estimated by various independent methods. The values of v obtained through our experiments are practically equal to the value calculated from the amino acid composition of the protein (0.75 cm3/g).     

Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning

Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH(2)PO(4)) in aqueous solution. beta-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH(2)PO(4) were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

Purification and kinetic properties of a membrane-bound phosphatidylinositol kinase of the bovine adrenal medulla

Phosphatidylinositol (PI) kinase (EC 2.7.1.67), an integral membrane protein of chromaffin granule ghosts of the bovine adrenal medulla, was found to phosphorylate PI in the 4-position of the inositol ring. The PI kinase was purified about 200-fold from a membrane fraction containing chromaffin granules and microsomes by extraction with Triton X-114, followed by phase partition (clouding) and heparin Sepharose chromatography. The PI kinase preparation (specific activity of 5.1 nmol PIP/mg protein per min) was free from other enzymatic activities that metabolize polyphosphoinositides. Km values of 55 microM and 40 microM for ATP and PI, respectively, were estimated for the purified enzyme. Concentrations of Triton X-100 above the critical micellar concentration (0.01%, w/v) were necessary to support significant enzyme activity, which was optimal at about 0.1% (w/v). Its dependence of pH was similar to that of the membrane-bound enzyme, with a broad optimum around pH 7. Mes in the millimolar concentration range was found to strongly inhibit the activity of the purified PI kinase (I50 at about 4 mM). The enzyme was almost totally inhibited by low micromolar concentrations of free calcium, and stimulated by hydrophilic cations, e.g., Mg2+ and poly(L-lysine), with the same potencies as for the membrane-bound enzyme. The amphiphilic cation trifluoperazine, however, stimulated the activity of purified PI kinase less effectively than the membrane-bound enzyme (Husebye, E.S. and Flatmark, T. (1988) Biochem. Pharmacol. 37, 449-456), whereas the inhibitory effect of near millimolar concentrations of trifluoperazine was the same for the two forms of the enzyme. It is concluded that the membrane-bound PI kinase of this tissue is of type II according to the classification of Cantley and co-workers (Whitman et al. (1987) Biochem. J. 247, 165-174).     

Cloning, high-level expression, purification, and properties of a novel endo-beta-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris

A novel gene coding for an endo-beta-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The beta-mannanase showed an identity of 90.2-92.9% (< or =95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified beta- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant beta-mannanase had an optimum temperature of 45 degrees C and optimum pH of 6.5. The enzyme was stable at temperatures up to 50 degrees C (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant beta-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.     

Purification and Properties of 3 Types of Monohydroxybenzoate Oxygenase from Rhodococcus erythropolis S-1

Three types of monohydroxybenzoate oxygenase, salicylate 5-oxygenase (SAL5O) forming gentisate from salicylate, m-hydroxybenzoate 6-oxygenase (MHB6O) forming gentisate from m-hydroxybenzoate, and p-hydroxybenzoate 3-oxygenase (PHB3O) forming protocatechuate from p-hydroxybenzoate, were purified from a cell-free extract of Rhodococcus erythropolis S-1, a Gram-positive bacterium. Each purified enzyme was homogenous on native PAGE. Each enzyme was a tetramer having identical subunits, a flavoporotein containing FAD, and a NADH-dependent monooxygenase. The three enzymes were much alike in general enzymatic properties, but very different in substrate specificity.     

Sedimentation study of a catalytically active form of rabbit muscle phosphofructokinase at pH 8.55

The enzymatic active form of rabbit muscle phosphofructokinase (PFK) was observed directly by using the method of reacting or active enzyme centrifugation (AEC). These studies were performed in two assay systems: a coupled enzyme and a pH-dependent dye-linked system in glycylglycine buffer at pH 8.55 and 23 +/- 1 degree C. The sedimenting band of PFK was stabilized by three solvent systems: 50% (v/v) D2O, 10% (w/v) sucrose, and 4% (v/v) or 10% (v/v) glycerol. The active PFK species sediments as a single component with a sedimentation coefficient of 12.4 +/- 0.5 S, after correcting for protein--solvent interactions. Although PFK may undergo association--dissociation, there is no observable change in the value of s20,w over a 57-fold range of protein concentration. Throughout this range only a single active species of PFK was observed, and within an experimental uncertainty of +/- 10%, the enzymatic activity observed in the sedimentation studies accounts for the total enzymatic activity observed in the steady-state kinetics. Partially purified PFK was subjected to AEC analysis. Results reveal the presence of again a single active form sedimenting at the same rate as the purified enzyme. Results from sedimentation velocity studies indicate that the stabilizing solvents employed in AEC enhance the self-association of PFK. However, such an enhancement alone cannot account for the observation of a single active species with a sedimentation coefficient of 12.4 S. The interactions between solvent additives and PFK were studied by density measurements and by the application of multicomponent theory. Results from such a preferential solvent interaction study indicate that PFK is preferentially hydrated in the presence of sucrose or glycerol. The enhancement of PFK self-association is most likely due to a nonspecific solvent--protein interaction.     

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.     

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Purification to homogeneity and initial physical characterization of secondary amine monooxygenase

Secondary amine monooxygenase from Pseudomonas aminovorans grown on trimethylamine has been purified 265-fold to apparent homogeneity. The purified enzyme exhibits a specific activity of 14.7 mumol of NADPH oxidized per min per mg of protein, a native molecular weight of 210,000, and nondisulfide-linked subunits of molecular weight 42,000, 36,000, and 24,000, each of which is required for activity. The enzyme is extremely labile during purification; rapid handling and the presence of 5% ethanol are essential to enzyme stability. Storage at 77 K in the presence of NADH (1 mM) also confers considerable stability to the purified enzyme. The heme prosthetic group in the enzyme has been identified as protoporphyrin IX. The quantification of prosthetic group components reveals the presence of 1.6 mol of flavin as FMN, 2.0 mol of heme iron, 4.0 mol of acid-soluble (nonheme) iron, and 3.6 mol of free sulfide/210,000 molecular weight enzyme. Ferric and ferrous-CO secondary amine monooxygenase exhibit electronic absorption spectra that are very similar to those of analogous myoglobin derivatives and, therefore, quite distinct from parallel forms of cytochrome P-450, the most extensively studied heme iron-containing monooxygenase. Like myoglobin and, again, in contrast to P-450, this enzyme forms a very stable dioxygen complex. In fact, it is this oxygen-bound form of the enzyme that is obtained from the purification procedure. Once again, the absorption spectrum of oxygenated secondary amine monooxygenase is nearly identical to that of oxymyoglobin. The spectroscopic similarities between secondary amine monooxygenase and myoglobin suggest the presence of an endogenous histidine fifth ligand to the heme iron of the enzymes.     

Purification and properties of methanol dehydrogenase from Methylophaga marina

Purification of methanol dehydrogenase from Methylophaga marina, in order to avoid the instability observed in crude extracts, was achieved initially by a rapid procedure using mainly an aqueous two-phase partition system composed of polyethylene glycol 1000 (50%, v/v) and potassium phosphate (50%, w/v). The purified enzyme gave a single band of protein after SDS-polyacrylamide gel electrophoresis. Antiserum raised against purified methanol dehydrogenase was used to detect possible protein contaminants in the enzyme preparation. The enzyme is an NAD-independent dehydrogenase containing pyrrolquinoline quinone (PQQ) as a prosthetic group. It is made of two apparently identical subunits giving a total MW of 145,000 for the native enzyme. The isoelectric point is 6.4. In addition to methanol and formaldehyde, multicarbon primary alcohols and aldehydes as well as secondary alcohols can be used as substrates. Except for ammonium chloride, which is a necessary activator in vitro, no effector was found which could modify the rate of enzyme activity under standard conditions of the assay. Although the main properties of this methanol dehydrogenase are similar to those already described in the literature, it does not belong in any of the five categories described by Anthony.