US2012270285A1PendingUtilityA1
Stereospecific carbonyl reductases
Individually held — no corporate assignee on recordPriority: Jun 23, 2009Filed: Jun 23, 2010Published: Oct 25, 2012
Est. expiryJun 23, 2029(~2.9 yrs left)· nominal 20-yr term from priority
Y02P20/52C12P 41/002C12N 9/0006C12P 7/04C12P 7/22C12P 7/62
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Claims
Abstract
Stereospecific carbonyl reductases SCR1, SCR2, and SCR3 are described herein as are nucleotide sequences that encode these reductases. These stereospecific carbonyl reductases have anti-Prelog selectivity and have specificities that are useful for fine biochemical synthesis.
Claims
exact text as granted — not AI-modified1 . A purified polypeptide, the sequence of which comprises an amino acid sequence that has at least 70% identity to a Candida parapsilosis stereospecific carbonyl reductase, wherein the polypeptide has carbonyl reductase activity and does not comprise SEQ ID NO:1.
2 . The purified polypeptide of claim 1 , wherein the amino acid sequence has at least 70% identity to at least one of the Candida parapsilosis stereospecific carbonyl reductases represented by SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
3 . The purified polypeptide of claim 2 , wherein the amino acid sequence has at least 70% identity to SEQ ID NO:2.
4 . The purified polypeptide of claim 2 , wherein the amino acid sequence has at least 70% identity to SEQ ID NO:3.
5 . The purified polypeptide of claim 2 , wherein the amino acid sequence has at least 70% identity to SEQ ID NO:4.
6 . The purified polypeptide of claim 1 , wherein the amino acid sequence has at least 75% identity to the Candida parapsilosis stereospecific carbonyl reductase.
7 . The purified polypeptide of claim 6 , wherein the amino acid sequence has at least 80% identity to the Candida parapsilosis stereospecific carbonyl reductase.
8 . The purified polypeptide of claim 7 , wherein the amino acid sequence has at least 85% identity to the Candida parapsilosis stereospecific carbonyl reductase.
9 . The purified polypeptide of claim 8 , wherein the amino acid sequence has at least 90% identity to the Candida parapsilosis stereospecific carbonyl reductase.
10 . The purified polypeptide of claim 9 , wherein the amino acid sequence has at least 95% identity to the Candida parapsilosis stereospecific carbonyl reductase.
11 . The purified polypeptide of claim 10 , wherein the amino acid sequence has at least 99% identity to the Candida parapsilosis stereospecific carbonyl reductase.
12 . The purified polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
13 . The purified polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.
14 . The purified polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO:2.
15 . The purified polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO:3.
16 . The purified polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO:4.
17 . The purified polypeptide of claim 1 , wherein the carbonyl reductase activity of the polypeptide is NADPH-dependent.
18 . The purified polypeptide of claim 1 , wherein the polypeptide is an anti-Prelog-type stereospecific carbonyl reductase.
19 . A composition comprising the polypeptide of claim 1 .
20 . An isolated nucleic acid sequence comprising a sequence that encodes the polypeptide of claim 1 .
21 . The isolated nucleic acid of claim 20 , wherein the sequence comprises SEQ ID NO:6 or of a degenerate variant of SEQ ID NO:6.
22 . The isolated nucleic acid of claim 20 , wherein the sequence comprises SEQ ID NO:7 or of a degenerate variant of SEQ ID NO:7.
23 . The isolated nucleic acid of claim 20 , wherein the sequence comprises SEQ ID NO:8 or of a degenerate variant of SEQ ID NO:8.
24 . The isolated nucleic acid of claim 20 , wherein the sequence encodes SEQ ID NO:2.
25 . The isolated nucleic acid of claim 20 , wherein the sequence encodes SEQ ID NO:3.
26 . The isolated nucleic acid of claim 20 , wherein the sequence encodes SEQ ID NO:4.
27 . An expression vector comprising an expression cassette operably linked to the nucleic acid molecule of claim 20 .
28 . A host cell comprising the vector of claim 27 .
29 . A method of reducing a carbonyl substrate, comprising contacting the substrate with the purified polypeptide of claim 1 , in conditions suitable to catalyze the reduction of the carbonyl substrate.
30 . The method of claim 29 , wherein the reduction takes place in the presence of a coenzyme.
31 . The method of claim 30 , wherein the coenzyme is NADPH.
32 . The method of claim 29 , wherein the carbonyl substrate comprises an α-ketoester, a β-ketoester, an aryl ketone or an aliphatic ketone.
33 . The method of claim 32 , wherein the carbonyl substrate comprises an α-ketoester.
34 . The method of claim 33 , wherein the α-ketoester is methyl pyruvate, methyl phenylglyoxylate, ethyl pyruvate or ethyl benzoylformate.
35 . The method of claim 32 , wherein the carbonyl substrate comprises β-ketoester.
36 . The method of claim of claim 35 , wherein the β-ketoester is ethyl trifluoroacetoacetate, methyl acetoacetate, methyl 3-oxovalerate, methyl 4-fluorobenzoylacetate, ethyl acetoacetate, ethyl 3-oxovalerate, ethyl 4-chloroacetoacetate, ethyl benzoylacetate, or ethyl 3,4-dimethoxybenzoylacetate.
37 . The method of claim 32 , wherein the carbonyl substrate comprises an aryl ketone.
38 . The method of claim 37 , wherein the aryl ketone is 2-hydroxyacetophenone, or a derivative thereof.
39 . The method of claim 38 , wherein the aryl ketone is 2′-chloro-2-hydroxyacetophenone, 3′-chloro-2-hydroxyacetophenone, 4′-chloro-2-hydroxyacetophenone or 4′-methoxy-2-hydroxyacetophenone.
40 . The method of claim 32 , wherein the carbonyl substrate comprises an aliphatic ketone.
41 . The method of claim 40 , wherein the aliphatic ketone is 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone or 2-methyl-3-pentanone.
42 . The method of claim 29 , wherein the carbonyl substrate is ethyl 4-chloro-3-oxobutyrate.
43 . The method of claim 29 , wherein the reduction takes place at pH ranging from 5.0 to 6.0.Join the waitlist — get patent alerts
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