ANDROSTENEDIONE PRODUCTION BY BIOTRANSFORMATION OF PHYTOSTEROL by ALOK KUMAR MALAVIYA

This thesis embodies the work carried out using free Mycobacterium sp. cells as whole cell biocatalysts for androstenedione (AD) production from (3-sitosterol by selective side chain cleavage. While studying sitosterol to AD biotransformation in aqueous medium, different strategies were tried to improve the production of AD. In the first step, a mutant strain of Mycobacterium sp DSMZ 2966 was developed. A two stage strain improvement program was applied for this purpose. Acclimatization to stage wise increasing concentration of sitosterol was applied and a strain (AS-08) capable of growing on 20 gl-1 sitosterol was obtained. A total of 2.2 fold increase in AD accumulation ability was exhibited by AS-08. AS-08 was further subjected to chemical mutagenesis resulting in isolation of an improved strain (PL-17). This strain was resistant to 1 gl-1 AD and exhibited a 3.3 fold increase in AD accumulation capacity of the parent Mycobacterium sp. While studying the effect of operational parameters, maximum AD formation was observed at 28°C, 300 rpm and at 7 pH in aqueous medium. Addition of cyclodextrin (CD) in the fermentation medium at substrate to CD ratio of 1:3 resulted in 2.20 fold increase in AD formation. Mycobacterial cell wall is rigid and offers a high resistance to the transport of sitosterol into the cytosol. Therefore, the effect of cell wall permeabilizing antibiotics, such as ethambutol, penicillin, polymixin and bacitracin, on biotransformation of sitosterol to androstenedione by modification of cell wall permeability was examined. Drug sensitivity assay results established that bacitracin increased the permeability of the cell wall to hydrophobic compounds. Growth inhibitory study of bacitracin and rifamycin individually as well as in combination showed that these two antibiotics act