PURIFICATION AND PROPERTIES OF METHANOL DEHYDROGENASE FROM METHYLOSINUS SP. WI-14

Similarly to the recently described methanol dehydrogenase (MDH) from Methylocystis sp. GB 25 (Grosse et al. 1997) MDH from Methylosinus sp. WI 14 is able to catalyse the oxidation of methanol to formate direct ly. The enzyme was purified about 9-fold to electrophoretic homogeneit y and is localised in the soluble fraction. The relative molecular mas s of the native enzyme has been determined to be 140 kDa. It is compos ed of two identical subunits of relative molecular mass 70 kDa. The am ino terminal sequence shows a strong similarity (a match of 80% over t he first 20 amino acids) to the MDH from Methylocystis sp. GB 25. pQQ could be detected as the prosthetic group of MDH in the purified enzym e fraction by using the apoenzyme of a membrane-bound glucose dehydrog enase from Pseudomonas aeruginosa. A PQQ ratio of 1.3 per mole MDH was estimated. The purified enzyme has an optimum activity at pH 9.0 and at 57 degrees C. MDH from Methylosinus sp. WI 14 oxidises only primary alcohols up to octanol and several aldehydes. The estimated K-m-value s vary between 0.18 mM for the sorbic alcohol and 6.3 mM for butanol a nd show no dependence upon the chain length.