A whole cell mediated deamination of 2, 3-dideoxyadenosine to didanosine has been demonstrated using actinomycetes strain, Brevibacterium pusillum DSM 20527. The reaction was carried out in co-solvent system consisting of water (pH adjusted to 7.4) and cyclohexane. Adenosine is used as the starting material which is converted to 2, 3 dideoxyadenosine which in turn is converted to 2, 3-dideoxyinosine (Didanosine) using biotransformation reaction. Impurity generation during the process of synthesis was found to get reduced under cosolvent system. Also, Brevibacterium pusillum showed tolerance towards various solvents. Our work shows biocatalytic synthesis of Didanosine with less impurity generation.