Stuart Harbron

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

SummaryThe 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.

Large scale preparation and purification of apo-D-aminoacid oxidase for use in novel amplification assays

Dissociation of FAD from D-aminoacid oxidase occurred most rapidly at pH 6.0 in the presence of 1 M KBr. Diafiltration of 0.6 g of enzyme under these conditions yielded apoenzyme containing 1.3% of residual holoenzyme activity, which was subsequently reduced to less than 0.01% by chromatography on Blue Sepharose and ion exchange, giving material containing <1 ppb of contaminating phosphatase and nucleotidase.

A model for prosthetogenic enzyme amplification assays

A mathematical model describing the behaviour of a new class of prosthetogenic enzyme amplification assays is described. The predictions of the model are favourably compared with an enzyme amplification assay for alkaline phosphatase. The model is used to kinetically characterise and optimise the enzyme amplification assay.

Section Review: Recombinant antigens in immunoassay:Biologicals & Immunologicals

Recombinant antigens are increasingly being used in immunoassay for the detection of antibodies produced in response to infection. They can offer increased specificity, illustrated by their use in the diagnosis of parasite infections, or broader specificity, exemplified by problems involved in identifying new strains of Norwalk virus. Recombinant antigens may also be easier, safer and cheaper to produce, The current state of the art with respect to the early diagnosis of HCV infection and Lyme disease is discussed.