Oxidation of n-alkanes: Isolation of alkane hydroxylase from Pseudomonas putida

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The disruption of Pseudomonas putida cells capable of n-alkane assimilation was investigated by enzymic lysis and mechanical disruption in a high pressure-homogeniser, with a view to the isolation of alkane hydroxylase activity. Examination of the conditions for enzymic lysis showed that disruption with lysozyme/EDTA could be replaced effectively with lysozyme alone in phosphate buffer, pH 8.0 (I=0.05). This allowed inclusion of DNase during the lysis procedure for high bacterial concentrations and gave improved cell disruption. Mechanical disruption resulted in the solubilisation of alkane hydroxylase activity. In contrast enzymic lysis allowed the isolation of an insoluble fraction containing alkane hydroxylase activity, and although some solubilisation of the enzyme system did occur much of the activity was retained in the insoluble fraction. This fraction also contained a high level of n-alkane or diethoxymethane inducible, NAD-independent alcohol dehydrogenase activity.