Journal articles

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively.

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphate and human urine urokinase from crude extracts.

The NADP+-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of M(r) 32 000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine.

A Love plate device, based on an SSBW piezoelectric substrate coated with a polymer (PMMA) layer, was used to excite a surface guiding shear horizontal wave. The surface mass sensitivity of the device was calculated as a function of the thickness of the polymer layer, usin a modelling theory derived for the polymer-Love plate. LB films were used to assess experimentally the surface mass sensitivity and results were compared with theoretical predictions. Investigation of protein interaction with the PMMA surface was performed by following the adsorption of IgG in the range 1-400 μg/ml.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10 000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 ± 3.3%.

An indirect competitive enzyme-linked immunosorbent assay capable of detecting the reactive triazine dye C.I. Reactive Blue 2 at concentrations down to 300 pM was developed, and representing a 3000-fold higher sensitivity over direct spectrophotometric analysis. An investigation of the cross-reactivity of the polyclonal antibody against various compounds structurally related to C.I. Reactive Blue 2 revealed that the immunoassay appeared to be specific for anthraquinone-containing dyes and was largely unaffected by substitutions at the triazine ring.

A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native M(r) of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native M(r) of 410,000 and probably existed as a tetramer.

This paper reports experimental developments in the construction and operation of a single-mode fibre-optic evanescent wave biosensor using an exposed core silica single-moded fibre embedded in a silica block. The device was able to monitor the concentration of a blue dye, Procion Blue MX-G, in overlayers of various refractive indices. The practicality of such a biosensor has been demonstrated with a colorimetric enzyme assay system.

We have constructed a bienzyme electrode for the detection of total cholesterol by incorporating cholesterol esterase and cholesterol oxidase in polypyrrole films. The in situ deposition of both enzymes during the electrochemical polymerisation of pyrrole provides a simple procedure for the immobilisation of biomolecules at electrodes. Our study shows that cholesterol oxidase entrapped in polypyrrole films grown at platinum electrodes gave a fast amperometric response to cholesterol and good storage stability.

A constitutive NAD+-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose. Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols. Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5. The Km values for propan-2-ol, NAD+ and acetone were 87, 413 and 143 μM respectively. The enzyme is a tetramer with subunit Mr of approximately 44 000.