Catalyst and use thereof
Disclosed is a method that includes use of a catalyst in a method of reducing a substrate, the method including contacting a substrate with a catalyst, optionally in the presence of a co-substrate, thereby to generate a reduced substrate. The catalyst is a polypeptide including an amino acid sequence having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 9. In the method the substrate concentration is at least 50 mM.
1. A method of reducing an ethylene group in an unsaturated substrate, the method comprising contacting a solution comprising the unsaturated substrate with a catalyst, optionally in the presence of a co-substrate, thereby forming a reduced substrate;
wherein the ethylene group in the unsaturated substrate is conjugated to an aldehyde, a ketone carbonyl, a carboxy group, an alkoxy carbonyl group, an amido group, or a nitro group;
wherein the catalyst is a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1; and
wherein the unsaturated substrate is initially present in the solution in a concentration in a range of from 50 mM to 1500 mM.
2. The method of claim 1 wherein the substrate concentration in the solution is at least 100 mM.
3. The method of claim 2 wherein the substrate concentration in the solution is at least 300 mM.
4. The method of claim 1 wherein the catalyst is a polypeptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 1.
5. The method of claim 4 wherein the catalyst is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1.
6. The method of claim 1 , wherein the ethylene group is conjugated to an acyl, carboxy, acyloxy, acylamino, or nitro group.
7. The method of claim 1 , wherein the substrate is a compound of formula (I):
where:
—R 1 is acyl, carboxy, acyloxy, nitro, acylamino, or nitrile;
—R 2 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, amino, hydroxyl, or oxy;
—R 3 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, amino, hydroxyl, oxy, carboxy, acyloxy, nitrile, or acylamino,
or —R 1 and —R 2 or R 1 and R 3 form a ring containing an acyl, acyloxy or acylamino group, and
—R 4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, amino, hydroxyl, oxy, carboxy, acyloxy, nitrile, or acylamino,
and each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, and aryl group is optionally substituted;
or —R 2 and —R 4 together form an unsubstituted or substituted ring.
8. The method of claim 1 wherein a co-substrate is present and is a cofactor.
9. The method of claim 8 , wherein the cofactor is NADH.
10. The method of claim 1 wherein the substrate concentration in the solution is at least 500 mM.
11. The method of claim 1 , wherein the substrate concentration in the solution is at least 750 mM.
12. The method of claim 1 , wherein the catalyst is a polypeptide characterized as having high tolerance to substrate concentration.
13. The method of claim 1 , wherein the catalyst is a polypeptide having an ability to catalytically reduce at least 70% of 2-methylcyclopent-2-en-1-one when contacted with the 2-methylcyclopent-2-en-1-one for 18 hours at 35° C., when the 2-methylcyclopent-2-en-1-one is initially present in the solution at a concentration of 50 mM.