Monday, November 9, 2009
P5
Fermentation scale-up of Bacillus megaterium and recombinant Escherichia coli for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine from their corresponding racemic amines
Thomas P. Tully, Ronald Hanson, Steven L. Goldberg, Michael A. Montana, Brian L. Davis, Steve Chen, and Ramesh Patel. Process Research & Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, NJ 08903
Chiral amines are important as pharmaceutical intermediates. Several bacterial strains possessing the desired transaminase activity were identified for the resolution of racemic amines by enantiospecific transamination. Scale-up studies were conducted and several large scale fermentations were performed in tanks to generate cells for racemic amine resolution. Fermentation of Bacillus megaterium SC6394 was carried out at the 750-L scale, and the cells were used to resolve two racemic amines to their enantiomers. Pyruvate and pyridoxal phosphate were required as amine acceptor and cofactor, respectively. In one example, racemic 1-cyclopropylethylamine was resolved to (R)-1-cyclopropylethylamine, while in a second example, (R)-sec-butylamine was prepared from racemic sec-butylamine. The N-terminal and internal amino acid sequences of the transaminase enzyme were determined, which ultimately led to the cloning and expression of the enzyme in E. coli. The rec E.coli strain SC16578 was grown at the 250-L scale,and the cell paste was used to resolve racemic sec-butyl amine to (R)-sec-butylamine. Details of the fermentation of both organisms, as well as the successful transamination reactions using the recovered cells, will be presented.
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