Microbial degradation of phthalate esters. Part IX. Protocatechuate 3,4-dioxygenase from Nocardia erythropolis.

Abstract

Protocatechuate 3,4-dioxygenase was isolated from a gram-positive bacterium, Nocardia erythropolis, the enzyme participates in the phthalate ester metabolism in the bacterium. Cultural conditions for production of the enzyme, the purification procedure, and some properties of the enzyme were studied. A bouillon (beef) mediumwas the most effective amongthose tested for cell growth and enzyme formation. The effect was due to the ring closure type of creatine compounds. Protocatechuate 3,4-dioxygenase was purified from the cell-free extract ca. 1,400-fold and it gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be ca. 150,000. The optimal pH and temperature were ptt 8.0 and 40°C, respectively. The enzyme was stable in a pH range from 7.6 to 8.6 and below 42°C. The enzyme was inhibited by several metals such as Pb2+, Cd2+and Hg2+. The enzymewas active on a wide range of o-dihydroxyphenyl compounds, in contrast to the high specificity of similar enzymes from gram-negative bacteria (Pseudomonas). The enzyme had a broad absorption band in the visible region with a peak around 450nm, suggesting the presence of non-heme ion(s) bound to the enzyme as a co factor. The spectrum changed markedly upon addition of the substrate, possibly showing the formation of an enzyme-substrate complex. There have been many reports on the oxygenases for aromatic compounds. Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was first studied by Stanier and Ingraham in 1954,1} and since then a number of investigators have described various methods for purifying the enzyme from many kinds of microorganisms. Among microorganisms, much information is available on the dioxygenase for protocatechuic acid of the genus Pseudomonas, for example, as reported by