Microbial oxidation of methane and methanol: Purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C₁-C₁₀ tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD⁺, NADP⁺, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.Non-Standard Abbreviations PMS phenazine methosulfate - DCPIP 2,6-dichlorophenol indophenol - DEAE diethylaminoethyl     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.     

Microbial oxidation of methanol: Purification and properties of formaldehyde dehydrogenase from a Pichia sp. NRRL-Y-11328

An NAD⁺-linked, reduced glutathione-dependent formaldehyde dehydrogenase was purified to homogeneity from soluble extracts of methanol-grown yeast, Pichia sp. Formaldehyde and methylglyoxal are oxidized in the presence of NAD⁺ as an electron acceptor. NADP⁺ could not replace NAD⁺. Other straight chain aldehydes (C₂–C₆ tested), branched-chain aldehydes (e.g., isobutyaldehyde), aromatic aldehydes (e.g., salicylal-dehyde, benzaldehyde), glutyraldehyde, glyceraldehyde, glycoaldehyde, and glyoxal-dehyde tested were not oxidized by the purified formaldehyde dehydrogenase. The product of formaldehyde oxidation by purified enzyme was demonstrated to be S-for-mylglutathione by measuring the absorption at 240 nm due to the formation of thioester of formaldehyde and reduced glutathione. The K_m values for NAD⁺, formaldehyde, and reduced glutathione were 0.12, 0.31, and 0.16 mm, respectively, for the forward reaction at pH 8.0. The purified formaldehyde dehydrogenase also catalyzed the reduction of S-formylglutathione in the presence of NADH. Formate was not reduced by the purified enzyme. The K_m values for S-formylglutathione and NADH were 0.60 and 0.25 mm, respectively, for the reverse reaction at pH 6.0. Formaldehyde dehydrogenase has a molecular weight of 84,000 as determined by gel filtration and subunit molecular weight of 41,000 as determined by sodium dodecyl sulfate-gel electrophoresis. S-Formylglutathione, a product of formaldehyde oxidation, was oxidized by the partially purified formate dehydrogenase from Pichia sp. Formate dehydrogenase has a higher affinity toward S-formylglutathione (K_m value 1.8 mm) than toward formate (K_m value 25 mm). Antiserum prepared against the purified formaldehyde dehydrogenase from Pichia sp. NRRL-Y-11328 forms strong precipitin bands with isofunctional enzymes from methanol-grown Pichia pastoris NRRL-Y-7556 and Torulopsis candida Y-11419 and weak precipitin bands with Hansenula polymorpha NRRL-Y-2214. No cross-reaction was observed with isofunctional enzyme derived from methanol-grown Kloeckera sp.     

Alcohol Dehydrogenase from Methylobacterium organophilum

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10⁻⁵ M for methanol and 8.2 × 10⁻⁵ M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD⁺. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.     

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii

From the yest Candida biodinili grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150 000 by sedimentation equilibirum as well as by flitration. The enzyme appears to be composed of four identical subunits (M_r = 38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD⁺ as an electron acceptor. The K_m values were found to be 0.099 mM for isopropanoi and 0.14 mM for NDA⁺. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidazed to the correponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45°C. The isolectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme productuion.     

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h^–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low K_m values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher K_m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V_max values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high K_m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde. Received: 31 August 1995 / Accepted: 4 December 1995     

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.     

Benzyl alcohol metabolism by Pseudomonas putida: a paradox resolved

Evidence is presented for the existence in Pseudomonas putida of two NAD-linked dehydrogenases that function sequentially to oxidize benzyl alcohol. Induction of muconate lactonizing enzyme, a 3-oxoadipate pathway enzyme, indicated that P. putida oxidized benzyl alcohol to benzoate. Polyacrylamide gel electrophoresis with activity staining and enzymatic assays for an NAD-dependent dehydrogenase both showed that cells contained a single, constitutive alcohol dehydrogenase capable of oxidizing benzyl alcohol. This enzyme was shown to have the same specificity in extracts of glucose-grown as in benzy alcoholgrown cells. An NAD-aldehyde dehydrogenase oxidized benzaldehyde but was most active with normal alkyl aldehydes. This aldehyde dehydrogenase was shown to be induced, by enzymatic assays and by activity staining of polyacrylamide gel electropherograms, not only in cells grown on benzyl alcohol, but also in cells grown on ethanol. These experiments suggested that the aldehyde dehydrogenase was induced by the alcohol being oxidized rather than the substrate aldehyde.In sum, the evidence from enzyme assays and polyacrylamide gel electrophoresis of extracts indicates that Pseudomonas putida catabolizes benzyl alcohol slowly when it is the sole carbon and energy source, by the action of a constitutive, nonspecific, alcohol dehydrogenase and an alcohol-induced, nonspecific aldehyde dehydrogenase to yield benzoate, which is further metabolized via the 3-oxoadipate (beta-ketoadipate) pathway.In memory of R. Y. Stanier     

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

NADP⁺:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD⁺ in place of NADP⁺ and has an absolute requirement for divalent cations. The apparent K_m values for dl-isocitrate, Mn²⁺, and NADP⁺ were found to be 8, 6, and 11 μm, respectively. The Mn²⁺-d_s-isocitrate complex is the most likely substrate for the mammary enzyme with a K_m of 3 μm. The properties of mammary NADP⁺:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.     

Purification and characterization of an NADH-linked aldehyde reductase from bovine brain

Abstract— The presence of a nonspecific NADH-linked aldehyde reductase was demonstrated in various regions of bovine brain in vitro. With m-nitrobenzaldehyde as substrate, the rate of NADH oxidation was approximately 4 nmol.min^-1.(mg of protein)^-1 in the cerebellum, pons and medulla; but somewhat lower rates [2–3 nmol.min^-1.(mg of protein)-^l] were obtained in the other areas of the brain examined. The enzyme was localized primarily in the soluble, supernatant fraction of rat brain homogenates. The enzyme from the supernatant fluid fraction of bovine brain was purified approximately 350-fold by ammonium sulphate fractionation and chromatography on calcium phosphate-gel, DEAE-cellulose and Sephadex G200 columns. The partially purified enzyme catalysed the reduction of a number of aldehydes, including substituted benzaldehydes and aliphatic aldehydes of intermediate chain lengths. Short chain aliphatic aldehydes, such as acetaldehyde, were not reduced by the enzyme and butyraldehyde was a poor substrate. With m-nitrobenzaldehyde as substrate, NADH was oxidized at an approximately 10-fold faster rate than NADPH. The pH optimum for the enzyme was 6.75 for aldehyde reduction, whereas the rate of oxidation of m-nitrobenzylalcohol was optimal at pH 10.0 with NAD as the co-substrate. K_m and K₃ values ranged from 10 μM to 10 mM for various aldehydes and from 10 to 30 μM for the cofactors. Oxidation of NADH by the partially purified enzyme was not inhibited by 10m pyrazole or by 1 mM phenobarbital. However, the enzyme activity was inhibited by approximately 60 percent by 1 mM chlorpromazine or by 5 mM 1,10-orthophenanthroline. Our data demonstrate that the enzyme is not only separable from the NADPH-linked aldehyde reductase described previously by TABAKOFF and ERWIN, but also is quite different in substrate specificity and inhibitor sensitivity from the ‘classical’, pyrazole-sensitive, NAD- linked alcohol dehydrogenase (EC 1.1.1.1).     

Purification and properties of NADPH-dependent aldehyde reductase from human liver.

An aldehyde reductase (EC 1.1.1.2) from human liver has been purified to homogeneity. The enzyme is NADPH-dependent, prefers aromatic to aliphatic aldehydes as substrates, and is inhibited by barbiturates and hydantoins. The following physicochemical parameters were determined: molecular weight, 36,200; sedimentation coefficient, 2.9 S; Stokes radius, 2.65 nm; isoelectric point, pH 5.3; extinction coefficient at 280 nm, 54,300 M-1 cm-1. Results from polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, gel filtration, and ultracentrifugation suggest a monomeric structure. On molecule of NADPH binds to the enzyme causing a red shift of the coenzyme absorption maximum from 340 to 352 nm. The amino acid composition has been determined and a partial specific volume of 0.74 was computed from these data. An alpha-helicity of 7 and 18% was estimated from the ellipticities at 208 and 222 nm, respectively. Combination of the most reactive thiol group with p-mercuribenzoate does not cause loss of catalytic activity. Inactivation occurs when more than one thiol group is modified. The presence of NADPH or NADP+ prevents loss of activity by thiol modification. The comparison of structural features of aldehyde reductase with other monomeric and oligomeric dehydrogenases suggest similarities of aldehyde reductase with octopine dehydrogenase.     

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α₂ structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The K_m values for coniferyl aldehyde and NAD⁺ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH₂-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD⁺-dependent aldehyde dehydrogenases from different sources.     

Heterogeneity of the Polypeptide in Pig Serum Low-density Lipoproteins

Aldehyde dehydrogenase catalyzes the irreversible conversion of aldehydes into their corresponding acids. NAD-dependent aldehyde dehydrogenase purified from bovine liver mitochondria was used to remove the green beany flavor of soybean products. Incubation of the enzyme, in the presence of NAD⁺, with defatted soybean extracts or with soybean milk, resulted in the almost complete disappearance or in a great reduction of the flavor. It was found from experiments with pyrazole, an inhíbitor of alcohol dehydrogenase, was used, that alcohols contributing to the beany flavor were converted into acids by the cooperative action of alcohol dehydrogenase and aldehyde dehydrogenase. The protein isolate prepared from the soybean extract after treatment with these enzymes produced no substantial beany flavor after storage in powdered form. Aldehyde dehydrogenase improved flavor in extract of mutton.     

Purification and characterization of phosphoenolpyruvate carboxylase from maize leaves

Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s_20,w) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.     

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD⁺-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.