Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10⁵, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10⁴. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q₁ and Q₂. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K_m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C₃–C₁₂, the corresponding aldehydes of C₃–C₇, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.     

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.     

Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus (Texas Strain) and Pseudomonas Species M27

A primary alcohol dehydrogenase has been purified from Methylococcus capsulatus (Texas strain). The purified enzyme catalyzes the oxidation of methanol and formaldehyde to formate; other primary alcohols are oxidized to their corresponding aldehydes. Ammonium ions are required for enzyme activity. The enzyme has a molecular weight of 120,000 daltons and consists of two 62,000 molecular-weight subunits which dissociate at acidic pH. The enzyme is similar to an alcohol dehydrogenase enzyme isolated from Pseudomonas sp. M27.     

A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols.     

Microbial oxidation of methane and methanol: crystallization and properties of methanol dehydrogenase from Methylosinus sporium.

Obligate methylotrophs are divisible into two types on the basis of ultrastructural biochemical characteristics. Both groups possess a soluble phenazine methosulfate (PMS)-dependent methanol dehydrogenase. In addition, particulate PMS-dependent methanol dehydrogenase and PMS-independent methanol oxidase have been found in the type I membrane group. A procedure was developed for the crystallization of methanol dehydrogenase from the soluble fraction of the type II obligate methylotroph Methylosinus sporium. This is the first report of a crystalline methanol dehydrogenase from a methylotrophic bacterium. The crystallized enzyme is homogeneous as judged by ultracentrifugation and by acrylamide gel electrophoresis. In the presence of an electron acceptor (phenazine or phenazinium compound) and an activator (ammonium compound), the crystallized enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight of the enzyme as estimated by gel filtration is approximately 60,000, and as estimated by sedimentation equilibrium analysis it is 62,000. The sedimentation constant (S20,W) is 2.9. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 60,000. The amino acid composition and spectral properties of the enzyme are also presented. Antisera prepared against the crystalline enzyme are nonspecific, they cross-reacted and inhibited isofunctional enzymes from other obligate methylotrophic bacteria.     

The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol.

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.     

Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1

An NAD-dependent secondary alcohol dehydrogenase (ADH) produced by Rhodococcus sp. GK1 was purified about fivefold with a yield of 82% by hydrophobic interaction chromatography. This enzyme reduced monoketones, diketones and α-dicarbonyl compounds ; it oxidized secondary alcohols but not primary alcohols. Optimum pH was 7·0 or 8·5 for reduction or oxidation of substrates, respectively, and optimal temperature for activity was 55 °C. The apparent molecular mass of ADH was about 60 kDa by gel filtration chromatography.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme.     

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.     

Aliphatic alcohol dehydrogenase from potato tubers

Potato tubers are shown to contain at least 3 alcohol dehydrogenases, one active with NAD and aliphatic alcohols, one active with NADP and terpene alcohols and one active with NADP and aromatic alcohols. The purification of the aliphatic alcohol dehydrogenase is described and its activity with a wide range of substrates is reported. On the basis of substrate specificity, the enzyme is shown to resemble yeast alcohol dehydrogenase rather than liver alcohol dehydrogenase. The enzyme shows high activity with and high affinity for ethanol, activity and affinity decline as the chain length is increased from ethanol to butanol, but a further increase in chain length leads to increased affinity for the alcohol. The physiological significance of the results is briefly discussed.     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.     

Oxidation of Lignin-Related Aromatic Alcohols by Cell Suspensions of Methylosinus trichosporium

Cell suspensions of Methylosinus trichosporium oxidized the aromatic alcohols benzyl alcohol, vanillyl alcohol, and veratryl alcohol to the corresponding aldehydes, and with the exception of vanillyl alcohol, the aldehydes were further oxidized to the corresponding aromatic acids. No other transformation was observed, and the methoxyl moieties attached to the aromatic nucleus remained intact. More than 70% of the alcohol oxidized could be accounted for by aldehyde and/or acid. Investigation of the inhibitor kinetics of EDTA or p-nitrophenylhydrazine (specific for NAD⁺-independent methanol dehydrogenase in methylotrophs) on aromatic alcohol oxidation revealed noncompetitive inhibition in which the V_max was decreased but the K_m remained unchanged. The pattern of inhibition of aromatic alcohol oxidation matched that of methanol oxidation, and the K_m values for all of the substrates were similar (12 to 16 mM). The results indicate that the initial step in the oxidation of aromatic alcohols was similar to that for methanol, and because oxidation was incomplete (i.e., only the corresponding aldehyde or acid was produced), there may be some biotechnological advantages in using whole cells of methylotrophs to facilitate aromatic biotransformations.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver

Two acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36,000-39,000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP- and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20 alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3 alpha-hydroxysteroids at low rates and was inhibited by cyclopentane-1,1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3 alpha-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10(-6) to 10(-7) M) were lower than those for xenobiotic alcohols.     

Microbial Oxidation of Methane and Methanol: Crystallization of Methanol Dehydrogenase and Properties of Holo- and Apo-Methanol Dehydrogenase from Methylomonas methanica

Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.     

Purification and partial characterization of dimeric dihydrodiol dehydrogenase from monkey kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of several monkey tissues, among which kidney exhibited the highest activity and contained a high-molecular weight (Mr approximately 65,000) enzyme species. The enzyme species was purified to apparent homogeneity and showed a subunit molecular weight of 39,000. The enzyme oxidized benzene dihydrodiol (Km = 0.9 mM) at a pH optimum of 9.8, and highly reduced vicinal diketones such as camphorquinone (Km = 0.1 mM) and diacetyl (Km = 0.8 mM) around pH 7.5, but alicyclic alcohols, hydroxysteroids and ketosteroids were inactive substrates for this enzyme. Quercitrin, SH-reagents, stilbestrol were inhibitory to the enzyme activity, but other synthetic estrogens, anti-inflammatory agents and 3-ketosteroids were not.     

Microbial Oxidation of Methane and Methanol: Isolation of Methane-Utilizing Bacteria and Characterization of a Facultative Methane-Utilizing Isolate

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.     

A novel type of formaldehyde-oxidizing enzyme from the membrane of Acetobacter sp. SKU 14

Membrane-bound NADP-independent formaldehyde-oxidizing enzyme was purified to homogeneity from the membrane of Acetobacter sp. SKU 14 isolated in Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Tween 20 at pH 2.85, and purified to homogeneity through the steps of column chromatographies on DEAE-Sephadex A-50 and Q-Sepharose in the presence of 0.1% Tween 20 and 0.1% Triton X-100. The enzyme purified together with a cytochrome c showed a single protein band on native-PAGE, and was dissociated into three different subunits upon SDS-PAGE with molecular masses of 78 kDa, 55 kDa, and 18 kDa. The purified enzyme was finally characterized as a quinoprotein alcohol dehydrogenase (QADH), and this is the first indication that QADH highly oxidizes formaldehyde. The substrate specificity of the enzyme was found to be broad toward aldehydes and alcohols, and alcohols, especially n-butanol, n-propanol, and ethanol, were oxidized more rapidly than formaldehyde.