Nobutake Hamada

Purification and properties of a polyvinyl alcohol-degrading enzyme produced by a strain of Pseudomonas.

Abstract An enzyme which degraded polyvinyl alcohol, a water-soluble synthetic polymer, was isolated as a single protein from a culture of a strain of Pseudomonas . The pink-colored enzyme had absorption maxima at 280, 370, and 480 nm, a molecular weight of about 30,000, and an isoelectric point at about pH 10.3. The enzyme was most active at pH values from 7 to 9 and at 40 dgC and was stable at pH values from 3.5 to 9.5 and at temperatures below 45 dgC. The viscosity of the reaction mixture decreased and the pH dropped when the enzyme acted on polyvinyl alcohol as a substrate. Furthermore, the enzyme required O 2 for the reaction and produced 1 mol of H 2 O 2 , per 1 mol of O 2 consumed. The molecules of polyvinyl alcohol were cleaved into small fragments with a wide distribution of molecular weights. Inorganic Hg ions markedly inactivated the enzyme, and the activity was immediately recovered by glutathione. Enzyme inhibitors tested, which included p -chloromercuribenzoic acid, KCN, o -phenanthroline, and H 2 O 2 , showed no effect on the activity. Polyvinyl alcohol oxidized by periodic acid was similarly oxidized by the enzyme. The enzyme did not oxidize most of a variety of low molecular weight hydroxy compounds examined, such as primary alcohols, secondary alcohols, tertiary alcohols, diols, triols, and polyols, except for some secondary alcohols, such as 4-heptanol.

Degradation mechanism of poly(vinyl alcohol) by successive reactions of secondary alcohol oxidase and β-diketone hydrolase from Pseudomonas sp.

The secondary alcohol oxidase from Pseudomonas sp. catalyzed the oxidation of various vinyl alcohol oligomers with the molecular weight of 220 to 1500 and of β-ketols such as 5-hydroxy-3-heptanone, 4-hydroxy-2-nonanone, 3-hydroxy-5-nonanone, 6-hydroxy-4-nonanone, 7-hydroxy-5-dodecanone, and 8-hydroxy-6-tridecanone. β-Diketone hydrolase from the same strain catalyzed the hydrolysis of various aliphatic β-diketones and some aromatic β-diketones such as 1-phenyl-1,3-butanedione and 1-phenyl-2,4-pentanedione. 4,6-Nonanediol, used as a low molecular weight model of poly(vinyl alcohol) (PVA), was oxidized to 4,6-nonanedione by way of 6-hydroxy-4-nonanone by secondary alcohol oxidase. 4,6-Nonanedione was hydrolyzed to 2-pentanone and n-butyric acid by β-diketone hydrolase. These reactions were stoichiometric.The presence of the β-diketone structure in PVA oxidized by secondary alcohol oxidase was confirmed by spectral experiments. The absorption due to β-diketone structure in the oxidized PVA decreased as it was...

Purification and Properties of Oxidized Poly(vinyl alcohol) Hydrolase with an Acidic Isoelectric Point

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg2+ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight c...

Conversion of naphthoates to cis-dihydrodiols by naphthalenesulfonate-assimilating Pseudomonas sp. TA-2

A naphthalenesulfonate-assimilating bacterium, Pseudomonas sp. TA-2, was found to convert 2-naphthoate to cis-1,2-dihydroxv-1,2-dihydronaphthalene-2-carboxylate (DDN2C) and cis-l,2-dihydroxy-l,2-dihydronaphthalene-3-carboxylate (DDN3C), and converted 1-naphthoate to as-l,2-dihydroxy-l,2-dihydronaphthalene-l-carboxylate (DDN1C). It was suggested that conversion of naphthoates was done by a dioxvgenase with relaxed regioselectivity.