Laccases for bio-bleaching
Provided herein are isolated laccase enzymes and nucleic acids encoding them. Also provided are mediators for laccase reactions. Also provided herein are methods for using laccases to oxidize lignins and other phenolic and aromatic compounds, such as for bio-bleaching and decolorization of wood pulp under high temperature and pH conditions to facilitate a substantial reduction in use of bleaching chemicals, as well as for treatment of fibers.
1. An isolated polypeptide, comprising:
an amino acid sequence having 90-99% sequence identity to the amino acid sequence of the polypeptide of SEQ ID NO: 2, wherein said isolated polypeptide comprises laccase activity, oxidizes lignin under conditions of pH greater than or equal to pH 8, and retains laccase activity for at least about 5 minutes at greater than or equal to 60° C.
2. The isolated polypeptide of claim 1 , lacking its associated signal peptide.
3. A method of delignifying a composition comprising lignin, comprising:
contacting a phenolic substrate with the polypeptide of claim 1 .
4. The method of claim 3 , further comprising contacting the composition with a mediator.
5. The method of claim 4 , wherein said mediator is a compound represented by Formula I or Formula II, wherein Formula I is represented by
wherein R 1 , R 2 R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of H, CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH 2 (CH 3 ) 2 , C(CH 3 ) 3 , CH 2 CH 2 CH 2 , CH 3 , alkyl, aryl, phenyl, CF 3 , OC(CH 3 ) 3 , OCH(CH 3 ) 2 , CH 2 CH 3 , C(O)OCH 3 , COOH, OCH 3 , OH, OCF 3 , NH 2 , NH(CH 3 ), N(CH 3 ) 2 , CH 2 NH 2 , CH 2 CH 2 NH 2 , Br, and Cl, and wherein Formula II is represented by
wherein R 1 , R 2 R 3 , R 4 , R 5 R 6 , R 7 , R 8 , R 9 and R 10 are independently selected from the group consisting of H, CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH 2 (CH 3 ) 2 , C(CH 3 ) 3 , CH 2 CH 2 CH 2 , CH 3 , alkyl, aryl, phenyl, CF 3 , OC(CH 3 ) 3 , OCH(CH 3 ) 2 , CH 2 CH 3 , C(O)OCH 3 , COOH, OCH 3 , OH, OCF 3 , NH 2 , NH(CH 3 ), N(CH 3 ) 2 , CH 2 NH 2 , CH 2 CH 2 NH 2 , Br, and Cl.
6. The method of claim 4 , wherein the mediator is
7. The method of claim 4 , wherein the mediator is selected from the group consisting of violuric acid; 2,6,6-tet-rarmethylpiperidein-1-yloxy (TEMPO); 1-hydroxybenzotriazole (HBT); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); syringaldazine; N-benzoyl-N-phenyl hydroxylamine (BPHA); N-hydroxyphthalimide, 3-Hydroxy-1,2,3-benzotriazin-4-one; promazine; 1, 8-Dihydroxy-4,5-dinitroanthraquinone; phenoxazine; anthraquinone; 2-hydroxy-1,4-naphthoquinone; phenothiazine; anthrone; anthracene, anthrarufin, anthrarobin; dimethoxyphenol (DMP); ferulic acid; catechin; epicatechin; homovanillic acid (HMV); and 2,3-dihydroxybenzoic acid (2,3-DHB); or any combination thereof.
8. The method of claim 3 , wherein said delignification reaction proceeds under conditions of between pH 8 and pH 11.
9. The method of claim 3 , wherein said delignification reaction proceeds at a temperature of at least 50° C.
10. A method of oxidizing a composition comprising a fiber, comprising:
contacting said composition with the polypeptide of claim 1 .
11. The method of claim 10 , further comprising contacting the composition with a mediator.
12. The method of claim 11 , wherein said mediator is a compound represented by Formula I or Formula II.
13. The method of claim 11 , wherein the mediator is
14. The method of claim 11 , wherein the mediator is selected from the group consisting of violuric acid; 2,6,6-tet-rarmethylpiperidein-1-yloxy (TEMPO); 1-hydroxybenzotriazole (HBT); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); syringaldazine; N-benzoyl-N-phenyl hydroxylamine (BPHA); N-hydroxyphthalimide, 3-Hydroxy-1,2,3-benzotriazin-4-one; promazine; 1, 8-Dihydroxy-4, 5-dinitroanthraquinone; phenoxazine; anthraquinone; 2-hydroxy-1,4-naphthoquinone; phenothiazine; anthrone; anthracene, anthrarufin, anthrarobin; dimethoxyphenol (DMP); ferulic acid; catechin; epicatechin; homovanillic acid (HMV); and 2,3-dihydroxybenzoic acid (2,3-DHB); or any combination thereof.
15. The method of claim 10 , wherein said oxidation reaction proceeds under conditions of between pH 8 and pH 11.
16. The method of claim 10 wherein said oxidation reaction proceeds at a temperature of at least 50° C.
17. A method of bleaching a composition comprising pulp or paper, comprising:
contacting said composition with the polypeptide of claim 1 .
18. The method of claim 17 , further comprising contacting the composition with a mediator.
19. The method of claim 18 , wherein said mediator is a compound represented by Formula I or Formula II.
20. The method of claim 19 , wherein the mediator is
21. The method of claim 19 , wherein the mediator is selected from the group consisting of violuric acid; 2,6,6-tet-rarmethylpiperidein-1-yloxy (TEMPO); 1-hydroxybenzotriazole (HBT); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); syringaldazine; N-benzoyl-N-phenyl hydroxylamine (BPHA); N-hydroxyphthalimide, 3-Hydroxy-1,2,3-benzotriazin-4-one; promazine; 1,8-Dihydroxy-4,5-dinitroanthraquinone; phenoxazine; anthraquinone; 2-hydroxy-1,4-naphthoquinone; phenothiazine; anthrone; anthracene; anthrarufin; anthrarobin; dimethoxyphenol (DMP); ferulic acid; catechin; epicatechin; homovanillic acid (HMV); and 2,3-dihydroxybenzoic acid (2,3-DHB); or any combination thereof.
22. The method of claim 19 , wherein said bleaching reaction proceeds under conditions of between pH 8 and pH 11.
23. The method of claim 19 , wherein said bleaching reaction proceeds at a temperature of at least 50° C.