Evaluation of immobilized cytochrome P-448 from Saccharomyces cerevisiae using permeabilized whole cell, microsomal fraction and highly purified reconstituted forms, with benzopyrene-3-monooxygenase activity |
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Authors: | Mahmood R Azari Alan Wiseman |
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Institution: | Biochemistry Division, Department of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH, UK |
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Abstract: | Cytochrome P-448 from Saccharomyces cerevisiae in permeabilized whole cell, microsomal fraction and in a highly purified reconstituted benzopyrene-3-monooxygenase (EC 1.14.14.1) system have been immobilized on various supports. Calcium alginate was found to be especially useful and the kinetics of hydroxylation were close to that of the free enzyme system with all three forms of enzyme, even with permeabilized whole yeast cells (V max of 664 pmol 3-hydroxybenzo(a)pyrene produced per h per nmol cytochrome P-448 compared with 1000 for free highly purified reconstituted enzyme system). Only the highly purified reconstituted form was successfully immobilized by BrCN-activated Sepharose-4B or by acrylamide. Both of these supports stabilized the highly purified reconstituted cytochrome P-448 benzopyrene-3-monooxygenase activity in prolonged storage at 4°C. Applications for various immobilized enzymes and cells are assessed. |
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Keywords: | Chemical kinetics cytochrome P-448 Saccharomyces cerevisiae benzopyrene-3-monooxygenase immobilized enzyme immobilized cells |
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