Semi-rational engineering of cyclohexylamine oxidase for the deracemization of bulky secondary amines

Enantiomerically pure chiral amines are widely used as intermediates for agrochemicals and pharmaceuticals, and they are of increasing value in organic synthesis. Recently, we performed directed evolution on a cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans IH-35A and a few mutant enzymes were obtained to show activity toward 2-methyl-1, 2, 3, 4-tetrahydroquinoline. To further expand the range of substrate specificity and explore the relationship between the key residue sites and substrate specificity, the seven amino acid residues(T198, L199, M226, Y321, F351, F368 and Y459)related to the substrate-binding site were selected and mutated to amino acid residues of different property for creating the diversity mutant library. The mutant library was screened with a substrate library and it was found that mutant Y321I displayed enhanced activity toward 1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline. Since (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline was the intermediate of dextromethorphan, the enantioselective oxidation of 1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline catalyzed by mutant Y321I was coupled with the non-enantioselective reduction using BH₃·NH₃ to achieve the deracemization, giving (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline with high isolated yield (76.5%) and ee value (93.5%).