ABSTRACT
A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Brevibacterium/enzymology , Hydrogen Peroxide/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/isolation & purification , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/isolation & purification , Phenylethyl Alcohol/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Brevibacterium/chemistry , Brevibacterium/genetics , Brevibacterium/metabolism , Cholesterol/metabolism , Enzyme Stability , Hot Temperature , Kinetics , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Sequence Alignment , Substrate SpecificityABSTRACT
Phenylacetaldehyde dehydrogenase (PADH) was purified and characterized from Brevibacterium sp. KU1309, which can grow on the medium containing 2-phenylethanol as the sole carbon source. This enzyme was a homotetrameric protein with a subunit of 61 kDa. The enzyme catalyzed the oxidation of aryl (benzaldehyde, phenylacetaldehyde, 3-phenylpropionaldehyde) and aliphatic (hexanal, octanal, decanal) aldehydes to the corresponding carboxylic acids using NAD(+) as the electron acceptor. The PADH activity was enhanced by several divalent cationic ions such as Mg(2+), Ca(2+), and Mn(2+). On the other hand, it was inhibited by SH reagents (Hg(2+), p-chloromercuribenzoate, iodoacetamide, and N-ethylmaleinimide). The substrate specificity of the enzyme is compared with those of various aldehyde dehydrogenases.
Subject(s)
Aldehyde Dehydrogenase/isolation & purification , Aldehyde Oxidoreductases/isolation & purification , Brevibacterium/enzymology , Phenylethyl Alcohol/chemistry , Phenylethyl Alcohol/metabolism , Aldehydes/metabolism , Brevibacterium/metabolism , Escherichia coli Proteins , Hydrogen-Ion Concentration , NAD/metabolism , Substrate SpecificityABSTRACT
A novel 2-phenylethanol dehydrogenase has been purified from a soil bacterium Brevibacterium sp. KU 1309. The enzyme was purified about 1400-fold to homogeneity, and found to be a monomeric enzyme of apparent 39 kDa. The enzyme had broad substrate specificity and catalyzes a reversible oxidation of various primary alcohols to aldehydes. The enzyme required NAD+, but not NADP+ as a cofactor. Thus, the enzyme was classified into a group of NAD+-dependent primary alcohol dehydrogenase. The activity was inhibited by Cu2+, Ni2+, Ba2+, Hg2+ and p-chloromercuribenzoate. The enzyme is expected to be applicable as an effective biocatalyst in the oxidation of various alcohols.
Subject(s)
Alcohol Dehydrogenase/chemistry , Bacterial Proteins/chemistry , Brevibacterium/enzymology , Phenylethyl Alcohol/metabolism , Alcohol Dehydrogenase/antagonists & inhibitors , Alcohol Dehydrogenase/isolation & purification , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/isolation & purification , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Ions/pharmacology , Metals/pharmacology , Molecular Sequence Data , Substrate Specificity , TemperatureABSTRACT
Microorganisms which can assimilate 2-phenylethanol were screened from soil for oxidation of various alcohols into carbonyl compounds. One strain, identified as Brevibacterium sp. KU1309, had high oxidative activity towards ArCH(CH(3))CH(2)OH and Ar(CH(2))(n)OH. When the substrate concentration was 0.1 approximately 0.4% (w/v), the oxidation reaction proceeded smoothly (6 approximately 96 h), and the corresponding carboxylates were obtained in good yields (68 approximately 87%).
