Oxidation of aromatic alcohols by purified methanol dehydrogenase from Methylosinus trichosporium.

Methanol dehydrogenase was found to be present in subcellular preparations of methanol-grown Methylosinus trichosporium and occurred almost wholly in the soluble fraction of the cell. The enzyme, purified by DEAE-Sephadex and Sephadex G-100 chromatography, showed broad specificity toward different substrates and oxidized the aromatic alcohols benzyl, vanillyl, and veratryl alcohols in addition to a range of aliphatic primary alcohols. No enzyme activity was found toward the corresponding aldehydes of the alcohols tested. The Km for methanol was 50 microM, and that for the aromatic alcohols was in the range of 1 to 2 mM. EDTA and p-nitrophenylhydrazine, which are inhibitors of methanol oxidation in whole cells of methylotrophs, had little effect on activity of the purified enzyme. The results now extend the range of substrates oxidized by methanol dehydrogenase to include the aromatic alcohols.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

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.     

Hydrogenase from Acetobacterium woodii

Hydrogenase from fructose-grown cells of Acetobacterium woodii has been purified 70-fold to a specific activity of 3,500 mol hydrogen oxidized per min per mg of protein measured at 35°C and pH 7.6 with methyl viologen as electron acceptor. At the same conditions with reduced methyl viologen as electron donor the enzyme catalyzes the evolvement of 440 mol of H₂ per min per mg of protein. The enzyme was found in the soluble portion of the cell, indicating that it is either not membrane-bound or is loosely associated with the membrane. The purified enzyme, which does not contain nickel, exhibits spectroscopic properties similar to the iron-sulfur hydrogenase of Clostridium pasteurianum. The enzyme is strongly inhibited by carbon monoxide, with 50% inhibition occurring at approximately 7 nM CO. Ferredoxin, flavodoxin, and carbon monoxide dehydrogenase are reduced in hydrogen-dependent reaction by the A. woodi hydrogenase.Abbreviations CO dehydrogenase carbon monoxide dehydrogenase - MV methyl viologen - SDS sodium dodecyl sulfate This paper is dedicated to Professor Dr. Hans G. Schlegel on the occasion of his 60-years birthday. Hans' contributions to the field of microbiology are many and it is a pleasure for us to commemorate him in this way. One of us, L. G. L., had the fortune as a Humboldt-Preis recipient to spend a year at the Institut für Mikrobiologie der Universität Göttingen. Besides the best possible working conditions memories involve pleasant evenings with a glass of Rhine-wine in the Schlegels' home to strenuous back-packing in the Austrian Alps.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

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.     

Isolation and some properties of lysineN 6-hydroxylase fromEscherichia coli strain EN222

Summary Lysine N⁶-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication ofEscherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium sulfate precipitation and fast protein liquid chromatography using Superose 12 and Mono Q columns. The molecular mass as determined by gel filtration was 200 kDa and 50 kDa by SDS/polyacrylamide gel electrophoresis. The enzyme binds 0.79 molecule FAD/50 kDa. The activity of the enzyme is strictly dependent on NADPH. Its properties are similar to other flavoprotein monooxygenases of the EC group 1.14.13.     

Purification and Stability during Storage of Phoshoenolpyruvate Carboxylase from Leaves of Amaranthus hypochondriacus,a NAD-ME Type C4 Plant

A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg^–1(protein) s^–1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40–60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, –20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen.     

Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

Broad-specificity quinate (shikimate) dehydrogenasefrom Pinus taeda needles

Two proteins having quinate dehydrogenase (QDH, quinate:NAD(P)⁺-oxidoreductase, EC 1.1.1.24) and shikimate dehydrogenase (SDH, shikimate:NADP⁺-oxidoreductase, EC 1.1.1.25) activities were purified about 3 000-fold from young loblolly pine (Pinus taeda L.) needles. A combination of ammonium sulfate solubilization, and chromatographies on DEAE-cellulose, 2′, 5′ ADP-Sepharose and Mono-Q was used. Throughout all purification steps, the QDH activity consistently co-purified with the activity of the first of three forms of SDH, and the ratio of QDH/SDH was constant (variation from 1.63 to 1.89). These data indicate that both QDH and SDH activities are catalyzed by a single broad-specificity quinate (shikimate) dehydrogenase. Gel chromatography on Superdex 75 was used to estimate the native molecular mass of two forms of the enzyme as 35 and 53 kDa.     

Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae

An isolate of Streptomyces tendae produced a extracellular protease which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 21 kDa. Optimum activity was at 70 degrees C and pH 6. It was stable at 55 degrees C for 30 min and between pH 4 and 9. It was resistant to neutral detergents and organic solvents such as Triton X-100, Tween 80, methanol, ethanol, acetone, and 2-propanol at 5% (v/v). The enzyme was completely inhibited by 5 mM PMSF, indicating it to be a serine protease. N-terminal amino acid sequence did not show any homology with other known proteolytic enzymes. The protease may therefore be a novel neutral serine protease, which is stable at high temperature and over a broad range of pH.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region

A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission. It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium. Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol is also present. Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl. Cells accumulate ectoine and glutamate as the main osmoprotectants. The G + C content of the DNA is 45.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).     

Archaeal alcohol dehydrogenase active at increased temperatures and in the presence of organic solvents

The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83°C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD⁺ as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t _1/2 = 42 min at 70°C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.     

Purification and characterization of methanol dehydrogenase of Methylobacterium nodulans rhizosphere phytosymbionts

Methanol dehydrogenase (MDH) of the facultative methylotrophic phytosymbiont Methylobacterium nodulans has been purified for the first time to an electrophoretically homogeneous state and characterized. The native protein with a molecular mass of 70 kDa consists of large (60 kDa) and small (6.5 kDa) subunits. The purified protein displayed a spectrum identical to that of pyrroloquinoline quinone (PQQ)-containing MDH, pI 8.7, pH optimum in the range 9–10. The enzyme was inactive in the absence of ammonium or methylamine and exhibited a wide substrate specificity with regard to C₁–C₅ alcohols with the high-est affinity to methanol (K _M = 70 μM), but it did not oxidize benzyl and secondary alcohols. The apparent K _M values to primary alcohols increased with the length of the carbon chain. The enzyme was characterized by a high stability level even in the absence of a substrate. An immobilized enzyme was used for amperometric methanol detection.     

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.     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.