PURIFICATION AND CHARACTERIZATION OF AN ETHER BOND-CLEAVING ENZYME INVOLVED IN THE METABOLISM OF POLYETHYLENE-GLYCOL

An ether bond-cleaving enzyme was purified as diglycolic acid (DGA) de hydrogenase associated with 3-(4,5-dimethyl-2-thioazolyl)-2,5-diphenyl -2H tetrazolium bromide and phenazine methosulfate. DGA dehydrogenase was inductively formed in a polyethylene glycol (PEG)-utilizing symbio tic mixed culture, E-1, composed of Sphingomonas terrae and Rhizobium sp. which were grown on PEG 6,000. DGA dehydrogenase was solubilized w ith 0.5% n-dodecyl-beta-D-maltoside from membrane fractions of the mix ed culture E-1 and purified almost to homogeneity, as determined by SD S-polyacrylamide gel electrophoresis, by DEAE-Toyopearl column chromat ography and Sepharose 6B gel filtration. The molecular weight of the e nzyme was determined to be about 40,000-41,000 Da by SDS-polyacrylamid e gel electrophoresis and 480,000 by gel filtration on Sepharose 6B in the presence of 0.5% n-dodecyl-beta-D-maltoside, The absorption spect rum of the purified enzyme showed two peaks at 345 nm and 430-450 nm i n addition to a major peak at 280 nm. The optimal pH and temperature w ere 8.9 and 40 degrees C, respectively. The enzyme was stable in the p H range of 7.8 to 9.0 and at below 30 degrees C. The enzyme was activa ted by neither metal ions nor ammonium ion, and strongly inhibited by 1,4-benzoquinone. The enzyme catalyzed the degradation of DGA, PEG dic arboxylic acids, glycolic acid and glyoxylic acid, but not primary alc ohols, ethylene glycol (EG), EG oligomers, PEG 6,000 and aldehydes.