Isolation of organisms and characterization of o-aminotransferases for the asymmetric synthesis of amines.

摘要

Microbes containing o-aminotransferases were isolated from environmental samples by virtue of their ability to grow with one of nine amine compounds as the sole nitrogen source. A total of 209 pure cultures were isolated and 57 contained putative aminotransferases. Aminotransferase activity was confirmed by the action of inhibitors and the requirement for two substrates (a ketone and an amino acid) in each of 15 microbes examined.; Based on whole cell biotransformations, the putative aminotransferases produced high activity for reactions involving alpha-methylbenzylamine, 1-methylheptylamine, amphetamine, and endo-2-aminonorbornane. Less activity was detected with 2-amino-1-butanol and 1,2,3,4-tetrahydro-1-naphthylamine and no activity was detected with diphenyl compounds. Of amino acids investigated for used as the amine donor, activity was only observed with alanine, and the L-enantiomer was preferred. Among 36 microbes examined, all produced the (S)-enantiomer of alpha-methylbenzylamine. Only 11 microbes produced alpha-methylbenzylamine, 1,2,3,4-tetrahydro-1-naphthylamine, and amphetamine with an enantiomeric excess greater than 99%. The kinetic resolution of alpha-methylbenzylamine, using the aminotransferase to deaminate the (S)-enantiomer, was used to obtain (R)-alpha-methylbenzylamine with an enantiomer excess greater than 99.9%. A model for substrate binding was proposed that explained both the reaction specificity and chirality.; In a cell free extract, the pH optimum for three enzymes was approximately 8.0. The Kappm for phenylacetone was in the range of 0.2 to 0.8 mM and the Kappm for L-alanine was in the range of 6 to 33 mM. With phenylacetone, substrate inhibition was observed with a Kappis in the range of 1.4 to 6.4 mM.; An aminotransferase from Fusarium solani was selected for further study. Culture kinetics showed the specific activity of this enzyme varied with culture age and the nitrogen source in the medium. The aminotransferase was purified to homogeneity by a sequence of PEG precipitation and five chromatography steps. A 130 fold purification was obtained and the yield was 28.4%. A N-terminal amino acid sequence was obtained. The sequence suggested that the isolated enzyme was a member of the o-aminotransferase family.