PURIFICATION AND PROPERTIES OF METHANOL DEHYDROGENASE FROM METHYLOCYSTIS SP GB-25

Methanol dehydrogenase (MDH) from Methylocystis sp. GB 25, which belon gs to the group II of methanotrophic bacteria, is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified 20 -fold by a 5 step procedure to electrophoretic homogeneity. After cell disruption by French press, about 95% of MDH-activity was found in th e soluble fraction. The relative molecular mass of the native enzyme h as been estimated to be 122 kDa by gel filtration and 115 kDa by the m ethod of HEDRICK and SMITH (1968). It seems to be composed of two iden tical subunits with a relative molecular mass of 62 kDa (estimated by SDS gel electrophoresis). The isoelectric point was found to be about 8.3. The amino terminal sequence shows a strong similarity to the alph a-chain of MDH from the facultative methylotrophic bacterium Methyloba cterium extorquens AM1. PQQ, the probable prosthetic group of MDH, cou ld be detected in the supernatant of the culture by using the apoenzym e of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginos a but not absolutely in the absorption spectra of the enzyme after DEA E-chromatography. The purified MDH has an optimum activity at pH 9.0 a nd at 45 degrees C. MDH of Methylocystis sp. GB 25 oxidises only prima ry alcohols from methanol to heptanol and aldehydes from formaldehyde to propionaldehyde and the glutaraldehyde, respectively The estimated K-m-values show no dependence upon the chain length of substrates.