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US 8,916,364 B2Granted
Nitrilases
AssigneeC-LEcta GmbH
InventorsAndreas Vogel, Daniel Schwarze, Thomas Greiner-Stoeffele
Life of this patent
Estimating the term…
Abstract
The invention relates to a nitrilase having improved activity in the reaction of a nitrile to form the corresponding carboxylic acid, in particular with respect to reacting 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile, and benzonitrile. The nitrilase according to the invention is related to nitrilase from acidovorax facilis.
Claims16
A nitrilase comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO:
- 2, which, in comparison with the nitrilase having the amino acid sequence of SEQ ID NO:
- 2,
- (i) has an activity increased by at least 15% in the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile and benzonitrile;
- and/or
- (ii) has at least one amino acid substitution selected from the group consisting of:
- a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Arg, Glu, Ile, and Lys;
- b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ile, Met, Pro, and Thr;
- c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Tyr and Val;
- d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr and Val;
- e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Cys, Lys, Met, and Tyr;
- f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Gly, and Leu;
- g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Arg, Gly, His, Lys, Met, Phe, Ser, Thr, and Tyr;
- h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Leu and Met;
- and
- i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Asn, and Trp.
21 more elements
A nitrilase comprising an amino acid sequence having at least 98% homology with the amino acid sequence of SEQ ID NO:
- 8.
A nitrilase comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO:
- 2, which, in comparison with the nitrilase having the amino acid sequence of SEQ ID NO:
- 2,
- (i) has an activity increased by at least 15% in the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methyl glutaronitrile, 1-(cyanomethyl)cyclohex ane-1-carbonitrile and benzonitrile;
- and
- (ii) has at least one amino acid substitution selected from the group consisting of:
- a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Arg, Glu, Ile, and Lys;
- b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ile, Met, Pro, and Thr;
- c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Tyr and Val;
- d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr and Val;
- e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Cys, Lys, Met, and Tyr;
- f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Gly, and Leu;
- g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Ala, Arg, Gly, His, Lys, Met, Phe, Ser, Thr, and Tyr;
- h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Leu, and Met;
- and
- i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO:
- 2 by an amino acid selected from the group consisting of Asn and Tip.
21 more elements
Dependencies are inferred from the claim text.