Laccases, nucleic acids encoding them and methods for making and using them
View Patent ↗The invention provides laccases, polynucleotides encoding these enzymes, the use of such polynucleotides and polypeptides. In one aspect, the invention relates to the enzymatic production of nootkatone by way of the conversion of valencene using proteins having a laccase activity, e.g., a novel laccase of the invention. In one aspect, the invention provides methods of depolymerizing lignin, e.g., in a pulp or paper manufacturing process, using a polypeptide of the invention. In another aspect, the invention provides methods for oxidizing products that can be mediators of laccase-catalyzed oxidation reactions, e.g., 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol, and the like.
1. An isolated, synthetic, or recombinant nucleic acid comprising:
(a) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:23, wherein the nucleic acid encodes a polypeptide having a laccase activity, or
(b) a nucleic acid sequence completely complementary to (a).
2. An isolated, synthetic, or recombinant nucleic acid comprising a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO.:23, wherein the nucleic acid encodes a polypeptide having a laccase activity.
3. An isolated, synthetic, or recombinant nucleic acid comprising a nucleic acid sequence having at least 99% sequence identity to SEQ ID No.:23, wherein the nucleic acid encodes a polypeptide having a laccase activity.
4. An isolated, synthetic, or recombinant nucleic acid comprising the sequence of SEQ ID NO:23.
5. The isolated, synthetic, or recombinant nucleic acid as in any one of the preceding claims, wherein the nucleic acid comprises a sequence that encodes a polypeptide having laccase activity and having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:24.
6. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the encoded polypeptide having laccase activity comprises catalyzing the oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).
7. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the encoded polypeptide having laccase activity comprises a peroxidase activity.
8. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the encoded polypeptide having laccase activity comprises oxidation of valencene.
9. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the encoded polypeptide having laccase activity comprises oxidation of an aromatic amine.
10. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the encoded polypeptide having laccase activity retains the laccase activity after exposure to a temperature range of 55° C. to 75° C.
11. A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a laccase activity, wherein the probe comprises at least 60 to 150 consecutive bases of the nucleic acid of claim 1 , wherein the probe identifies the nucleic acid by hybridization under high stringency conditions, wherein the high stringency conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes, and the identified nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:23, and the identified nucleic acid encodes a polypeptide having laccase activity.
12. The nucleic acid of claim 1 , wherein the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:24.
13. An expression cassette comprising the nucleic acid of claim 1 .
14. A vector comprising the nucleic acid of claim 1 .
15. A cloning vehicle comprising the nucleic acid of claim 1 .
16. An isolated transformed cell comprising the nucleic acid of claim 1 .
17. The isolated transformed cell of claim 16 , wherein the cell is a yeast cell.
18. A method for producing a recombinant polypeptide having laccase activity, comprising:
(a) transforming an isolated host cell with a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises the sequence of claim 1 ; and
(b) expressing the nucleic acid of (a) under conditions that allow expression of the polypeptide, thereby producing the recombinant polypeptide.
19. The method of claim 18 , wherein the host cell is a yeast cell.
20. The method of claim 19 , wherein the host is selected from the group consisting of: a Schizosaccharomyces sp., a Saccharomyces sp., and a Pichia sp.
21. The method of claim 20 , wherein the host is Schizosaccharomyces pombe.
22. The method of claim 20 , wherein the host is Saccharomyces cerevisiae.
23. The method of claim 20 , wherein the host is Pichia pastoris.
24. The method of claim 18 , wherein the host cell is E. coli.
25. The method of claim 18 , wherein the host cell is Bacillus cereus.
26. A method for isolating or recovering a nucleic acid encoding a polypeptide with laccase activity from an environmental sample comprising:
(a) providing the probe of claim 11 ;
(b) isolating a nucleic acid from the environmental sample or treating the environmental sample so that the nucleic acid is accessible for hybridization to the probe;
(c) combining the isolated nucleic acid or the treated environmental sample of (b) with the probe; and
(d) isolating a nucleic acid that specifically hybridizes under high stringency conditions, with the probe,
wherein the high stringency conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes;
thereby isolating or recovering a nucleic acid having at least 95% identity to SEQ ID NO:23 encoding a polypeptide with laccase activity from an environmental sample.
27. A method for oxidizing an aromatic amine, comprising:
(a) providing a polypeptide encoded by the nucleic acid of claim 1 ;
(b) providing an aromatic amine; and
(c) reacting the polypeptide of (a) with the aromatic amine of (b) under conditions that facilitate oxidizing the aromatic amine by the laccase enzymatic reaction.
28. The method of claim 27 , wherein the aromatic amine is 2,2′-azino-bis(3 -ethylbenzthiazoline-6-sulfonic acid) (ABTS).
29. A method for oxidizing valencene, comprising the following steps:
(a) providing a polypeptide encoded by the nucleic acid of claim 1 ;
(b) providing valencene; and
(c) reacting the polypeptide of step (a) with the valencene under conditions that facilitate the laccase activity of the polypeptide; thereby oxidizing the valencene.