An improved purification procedure for cyclosporin synthetase.
We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation-->gel filtration-->hydrophobic interaction chromatography-->anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24-29 degrees C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.
Velkov
Tony
T
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Vic. 3800, Australia.
Singaretnam
Lloyd George
LG
Lawen
Alfons
A
D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010453:Peptide Synthases / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D005656:Fungal Proteins; D009097:Multienzyme Complexes; C048525:cyclosporin A synthetase; D016572:Cyclosporine; D010453:Peptide Synthases