A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties.

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.

[1]  A. Steinbüchel,et al.  Fermentation Enzymes in Strictly Aerobic Bacteria: Comparative Studies on Strains of the Genus Alcaligenes and on Nocardia opaca and Xanthobacter autotrophicus , 1983 .

[2]  A. Steinbüchel,et al.  NAD-linked L(+)-lactate dehydrogenase from the strict aerobe Alcaligenes eutrophus. 1. Purification and properties. , 1983, European journal of biochemistry.

[3]  A. Steinbüchel,et al.  NAD-linked L(+)-lactate dehydrogenase from the strict aerobe alcaligenes eutrophus. 2. Kinetic properties and inhibition by oxaloacetate. , 1983, European journal of biochemistry.

[4]  D. A. Small,et al.  Triazine-dye affinity; chromatography. , 1981, Biochemical Society transactions.

[5]  R. Martín,et al.  Purification and some properties of L-glycol dehydrogenase from hen's muscle. , 1981, Biochimica et biophysica acta.

[6]  H. Schlegel,et al.  Die relative Respirationsrate (RRR), ein neuer Belüftungsparameter , 1981 .

[7]  M. Freeling,et al.  Mutants and Variants of the Alcohol Dehydrogenase-1 Gene in Maize , 1981 .

[8]  H. Schlegel,et al.  Formation of the Dehydrogenases for Lactate, Ethanol and Butanediol in the Strictly Aerobic Bacterium Alcaligenes eutrophus , 1980 .

[9]  R. Favilla,et al.  Polyfunctionality of Horse Liver Alcohol Dehydrogenase , 1980 .

[10]  J. Jeffery.  The specificity of dehydrogenases. , 1980, Experientia. Supplementum.

[11]  C. Pace,et al.  Purification and partial characterization of alcohol dehydrogenase from wheat. , 1979, Archives of biochemistry and biophysics.

[12]  R. Pietruszko.  Nonethanol Substrates of Alcohol Dehydrogenase , 1979 .

[13]  R. Prince.  Some Aspects of the Bioinorganic Chemistry of Zinc , 1979 .

[14]  E. Noble,et al.  Biochemistry and pharmacology of ethanol , 1979 .

[15]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[16]  K. Dalziel.  Dynamic aspects of enzyme specificity. , 1975, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[17]  H. Eklund,et al.  3 Alcohol Dehydrogenases , 1975 .

[18]  R. Martín,et al.  Pigeon liver diacetyl reductase: purification and some properties. , 1974, Biochimica et biophysica acta.

[19]  H. Schulz,et al.  Purification and Properties of a Diacetyl Reductase from Escherichia coli , 1974, Journal of bacteriology.

[20]  J. Knabe.  Methoden der enzymatischen Analyse, 3. Auflage. Herausgeb. v. H. U. Bergmeyer, LXXXI, 2353 Seiten in 2 Bänden. Preis DM 460,–, Verlag Chemie, Weinheim/Bergstr., 1974 , 1974 .

[21]  H. Francksen,et al.  Potato Proteins: Genetic and Physiological Changes, Evaluated by One-and Two-dimensional PAA-Gel-techniques , 1973, Zeitschrift fur Naturforschung. Teil C: Biochemie, Biophysik, Biologie, Virologie.

[22]  F. C. Størmer,et al.  Diacetyl (acetoin) reductase from Aerobacter aerogenes. Kinetic studies of the reduction of diacetyl to acetoin. , 1973, European journal of biochemistry.

[23]  R. Martín,et al.  Purification and some properties of diacetyl reductase from beef liver. , 1972, Biochimica et biophysica acta.

[24]  J. Pringle,et al.  Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. , 1972, Methods in enzymology.

[25]  U. Al-Khalidi,et al.  Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl. , 1971, The Biochemical journal.

[26]  O. Adachi,et al.  Catalytic Properties of Alcohol Dehydrogenase from Leuconostoc mesenteroides , 1971 .

[27]  O. Adachi,et al.  NAD-Dependent and NADP-Dependent Alcohol Dehydrogenases in Escherichia coli , 1971 .

[28]  B. Olsen,et al.  Diacetyl (acetoin) reductase from Aerobacter aerogenes. Structural properties. , 1971, European journal of biochemistry.

[29]  O. Hetland,et al.  The reduction of diacetyl and acetoin in Aerobacter aerogenes. Evidence for one enzyme catalyzing both reactions. , 1971, European journal of biochemistry.

[30]  K. Weber,et al.  The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. , 1969, The Journal of biological chemistry.

[31]  H. Ursprung,et al.  Drosophila alcohol dehydrogenase. Purification and partial characterization. , 1968, The Journal of biological chemistry.

[32]  K. Kersters,et al.  [64] Primary and secondary alcohol dehydrogenases from Gluconobacter , 1966 .

[33]  P. Andrews,et al.  Estimation of the molecular weights of proteins by Sephadex gel-filtration. , 1964, The Biochemical journal.

[34]  B. Ames,et al.  A method for determining the sedimentation behavior of enzymes: application to protein mixtures. , 1961, The Journal of biological chemistry.

[35]  R. Abeles,et al.  The dismutation of formaldehyde by liver alcohol dehydrogenase. , 1960, The Journal of biological chemistry.

[36]  E. Racker.  [79] Alcohol dehydrogenase from baker's yeast: RCH2OH + DPN+ ⇄ RCHO + DPNH + H+ , 1955 .

[37]  I. Harary,et al.  Bacterial butylene glycol dehydrogenase and diacetyl reductase. , 1954, The Journal of biological chemistry.

[38]  A. N. Ramanathan,et al.  Liver alcohol dehydrogenase and ester formation. , 1952, The Biochemical journal.

[39]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[40]  E. Racker.  Spectrophotometric measurement of hexokinase and phosphohexokinase activity. , 1947, The Journal of biological chemistry.