Thermostable omega-transaminases
View Patent ↗Thermostable omega-transaminases, particularly thermostable omega-transaminases which have a high reaction rate and which are tolerant to high concentrations of donor amine, can be used to enrich enantiomerically a mixture of chiral amines or to synthesize stereoselectively one of a pair of chiral amines in which the amino group is bound to a non-terminal, chirally substituted, carbon atom.
1. An isolated thermostable omega transaminase which comprises the amino acid sequence SEQ ID NO:12.
2. An isolated polynucleotide which encodes the amino acid sequence SEQ ID NO:12.
3. The isolated polynucleotide of claim 2 which comprises the nucleotide sequence of SEQ. ID NO:11.
4. An expression construct comprising the polynucleotide of claim 2 .
5. A method for enantiomeric enrichment of a mixture of two enantiomeric chiral amines comprising contacting:
(a) a mixture comprising a first chiral amine and a second chiral amine which is an enantiomer of the first chiral amine, wherein the first chiral amine has the structural formula:
and wherein the second chiral amine has the structural formula:
wherein each of R 1 and R 2 , when taken independently, is alkyl, arylalkyl, or aryl and may be unsubstituted or substituted with one or more enzymatically non-inhibiting groups, and wherein R 1 and R 2 render the molecule chiral;
(b) the thermostable omega transaminase of claim 1 ; and
(c) an amino acceptor for a time sufficient to convert the first chiral amine to a ketone such that the amount of the second chiral amine in the mixture is at least about 90% relative to the amount of the first chiral amine.
6. The method of claim 5 wherein the contact is maintained at least until the amount of the second chiral amine in the mixture is at least about 99% relative to the amount of the first chiral amine.
7. A method for stereoselective synthesis of one of two chiral forms of an amine comprising contacting:
(a) a ketone having the structural formula:
wherein each of R 1 and R 2 , when taken independently, is alkyl, arylalkyl, or aryl and may be unsubstituted or substituted with one or more enzymatically non-inhibiting groups;
(b) the thermostable omega transaminase of claim 1 ; and
(c) an amine donor for a time sufficient to form a substantially greater amount of a first chiral form of the amine than the amount of a second chiral form of the amine.
8. The expression construct of claim 4 wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:11.
9. The method of claim 5 wherein the one or more enzymatically non-inhibiting groups are selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, mono-(lower alkyl)-substituted carbamoyl, di-(lower alkyl)-substituted carbamoyl, trifluoromethyl, phenyl, nitro, amino, mono-(lower alkyl) substituted amino, di-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl, alkylcarboxamido, arylcarboxamido, 2-methylbutane-1,4-diyl, pentane-1,4-diyl, hexane-1,4-diyl, hexane-1,5-diyl, and 2-methylpentane- 1,5-diyl.
10. The method of claim 7 wherein the one or more enzymatically non-inhibiting groups are selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, mono-(lower alkyl)-substituted carbamoyl, di-(lower alkyl)-substituted carbamoyl, trifluoromethyl, phenyl, nitro, amino, mono-(lower alkyl) substituted amino, di-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl, alkylcarboxamido, arylcarboxamido, 2-methylbutane- 1, 4-diyl, pentane- 1, 4-diyl, hexane-1,4-diyl, hexane-1,5-diyl, and 2-methylpentane-1,5-diyl.