US 8,951,750 B2 · Malonate decarboxylases for industrial applications

Abstract

The present invention relates to a method for the enzymatic decarboxylation of malonic acid (propanedioic acid) derivatives catalyzed by enzymes structurally and/or functionally related to arylmalonate decarboxylase (AMDase) as isolated from microorganisms of the genus Bordetella. The present invention also relates to novel enzymes with a decarboxylase activity, useful for performing the claimed method, mutants thereof, corresponding coding sequences and expression systems, methods of preparing said novel enzymes, and screening methods for obtaining further suitable enzymes also having said decarboxylase activity.

Claims21

Dependent claims

A method for decarboxylating a compound of formula I

HOOC—C(R1)(R2)—COOH (I)
or a salt form thereof,
wherein R1 represents a linear or branched, mono-unsaturated C2-C8-alkenyl group;
and
R2 represents H, OH, SH, NH2, C1-C8-alkoxy, C1-C8-alkylthio, C1-C8-alkyl, C2-C8-alkenyl, C2-C8-alkynyl or hydroxyl C1-C8-alkyl, comprising
a) contacting the compound of formula I with a protein having decarboxylase activity;
and
b) isolating a decarboxylation product of the compound of the formula I,
wherein the protein having decarboxylase activity comprises an amino acid sequence having at least 95% sequence identity to residues 1 to 240 of the Bordetella bronchiseptica polypeptide of SEQ ID NO:
2 and comprises a functional mutation in one of the amino acid sequence positions 14, 189 and 190 of the polypeptide of SEQ ID NO:
2.

8 more elements

A method for the biocatalytic decarboxylation of a compound of formula I,

HOOC—C(R1)(R2)—COOH (I)
or a salt form thereof,
wherein R1 represents a linear or branched, mono-unsaturated C2-C8-alkenyl group;
and
R2 represents H, OH, SH, NH2, C1-C8-alkoxy, C1-C8-alkylthio, C1-C8-alkyl, C2-C8-alkenyl, C2-C8-alkynyl or hydroxyl C1-C8-alkyl,
wherein the method comprises
a) contacting a compound of the formula I with a protein having decarboxylase activity and comprising an amino acid sequence having at least 95% sequence identity to residues 1 to 240 of the Bordetella bronchiseptica polypeptide of SEQ ID NO:
2;
and
b) isolating a decarboxylation product of a compound of the formula I,
wherein the amino acid sequence comprises mutations in a combination of amino acid positions of the polypeptide of SEQ ID NO:
2 selected from the group consisting of positions:
13 and 14,
13 and 15,
14 and 15,
13, 14 and 15,
14, 15 and 189,
14, 15 and 190,
14, 15, 189 and 190,
13, 14, 15 and 189,
13, 14, 15 and 190,
13, 14, 15, 189 and 190
13, 14 and 191,
13, 15 and 191;
14, 15 and 191,
13, 14, 15 and 191,
14, 15, 189 and 191
14, 15, 190 and 191,
14, 15, 189, 190 and 191,
13, 14, 15, 189 and 191,
13, 14, 15, 190 and 191, and
13, 14, 15, 189, 190 and 191.

29 more elements

Dependencies are inferred from the claim text.

Malonate decarboxylases for industrial applications

Life of this patent

Malonate decarboxylases for industrial applications

Life of this patent

Abstract

Claims21