METHYLOSINUS-TRICHOSPORIUM OB3B WHOLE-CELL METHANE MONOOXYGENASE ACTIVITY IN A BIPHASIC MATRIX

Reconstituted whole-cell preparations of lyophilized Methylosinus tric hosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqu eous phase and 2,2,4-trimethylpentane (isooctane), The rate of convers ion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable solub le methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although th e initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the spec ific rate and yields in the biphasic system were much less sensitive t o increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme syste m was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time, V-(app .) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K-m(app.) for propy lene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding rati os of V-(app.)to K-m(app.).