Bioconversion of hydrophobic compounds by enzyme systems.
Explore the source record for details and available documents.
Alcohol Dehydrogenase↗
Biomedical subjects
M D Legoy
Publications and source records attributed to M D Legoy.
Inactivation of 3 alpha-hydroxysteroid dehydrogenase by superoxide radicals. Modification of histidine and cysteine residues causes the conformational change.
3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity.
3-Hydroxysteroid Dehydrogenases↗
3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site.
3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.
17-Hydroxysteroid Dehydrogenases↗
Study and use of spontaneous cofactor regeneration in an immobilized enzyme-coenzyme system.
Explore the source record for details and available documents.
17-Hydroxysteroid Dehydrogenases↗
Steroid modifications with immobilized biocatalysts--use of immobilized enzyme-requiring cofactor regeneration and of immobilized mycelium.
Two biological approaches have been investigated for specific modifications of steroids. The first one uses the purified enzyme for the specific dehydrogenation of androsterone to androstanedione. The enzyme used is 3 alpha-hydroxysteroid dehydrogenase which requires a cofactor (NAD). A cofactor regeneration is needed so that the process could work continuously. The conjugation of two points (immobilization of the enzyme and optimization of the ratio methanol-water) allows a continuous work of the enzyme during 25 days. Moreover, we propose a chemical regeneration of the cofactor using methoxy derivative of phenazine methosulphate. Right now it is the limiting step of the process. The second approach of steroid modification uses a whole mycelium of Aspergillus phoenicis for the specific hydroxylation of progesterone to 11 alpha-hydroxy progesterone. The transformation of 90% of the progesterone is obtained with calcium alginate immobilization and the lowest number of products is obtained at pH lower than 2.5 with carrageenan and polyurethane immobilization. It seems promising to apply immobilized biocatalysts to the bioconversion of hydrophobic compounds in organic solvents system.
3-Hydroxysteroid Dehydrogenases↗
Chemical grafting of functional NAD in the active site of a dehydrogenase: regeneration in situ.
A functional NAD molecule was immobilized at the active site of Alcohol dehydrogenase within a proteic membrane. The presence and the functionality of the cofactor was checked by fluorescence analysis. The dehydrogenase NAD membrane does not require addition of soluble cofactor for its activity. The system represents a new worthwhile approach because both problems of retention and regeneration of cofactor are solved. The method can be used not only for industrial and analytical applications but also to try to get a better understanding of the kinetics and mechanisms of the catalytic action of dehydrogenase.
Alcohol Oxidoreductases↗
Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media.
Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.
Ascorbic Acid↗