BIOCATALYZED SYNTHESIS OF TESTOSTERONE

We have recently developed and submitted for a patent a new industrial process for the production of testosterone making use of biocatalysis. In particular, a non commercial enzyme was proposed to overcome a critical steps in the organic synthesis of the hormone: the regio- and stereo-specific conversion of 4-androstene-3,17-dione to testosterone. Design of the process was based on a strategy starting with the identification of the enzyme by crossed bioinformatics and literature screening: the chosen candidate was the 17P-hydrosteroid dehydrogenase type 5 from mouse. The coding sequence for the enzyme was cloned in a vector suitable for its expression, in fusion with an His-tag, in E.coli. Small scale preparations of the recombinant enzyme were initially set up and activity tests on the desired substrate were performed, using the co-factor NADPH or NADH. The small scale purifications allowed verification of the product obtained in terms of regio- and stereo-selectivity and yield, evaluation of thermal stability of the enzyme, kinetic analysis of the reaction and choice of an adequate partial organic mixture to work with. A protocol for purification of the enzyme from medium scale cultures was then drawn and performed. The amount of enzyme obtained from these preparations, in combination with the commercially available D-glucose dehydrogenase required for the recycling of the cofactor NADH, allowed the conversion of milligrams of androstendione. Conditions for higher scale synthesis of testosterone are going to be developed.