Methods and systems to produce lignin-modifying enzymes and uses thereof
Embodiments of the invention are generally directed to lignin-modifying enzymes and systems and methods of their manufacture. In many embodiments, the lignin-modifying enzymes are lignin-degrading enzymes capable of breaking down lignin into component parts that are usable for other purposes. Several embodiments are directed to systems for producing lignin-modifying enzymes in vivo, including in yeast and/or plant species, and certain embodiments are directed to methods of creating these systems, including transfecting the species to produce lignin-modifying enzymes.
1. A method to produce lignin-degrading enzymes comprising:
obtaining an expression vector comprised of DNA and encoding a lignin-degrading peroxidase, a signal peptide to export the lignin-degrading peroxidase to an apoplast, and a constitutive promoter to drive transcription of the lignin-degrading peroxidase and the signal peptide, wherein the lignin-degrading peroxidase is encoded by a sequence selected from SEQ ID NO: 1, SEQ ID NO: 49, SEQ ID NO: 69, and SEQ ID NO: 74;
transforming a plant with the expression vector;
allowing the plant to express the lignin-degrading peroxidase contained within the expression vector by allowing the plant to grow for up to 15 days; and
extracting the lignin-degrading peroxidase.
2. The method of claim 1 , wherein the extracting step utilizes vacuum infiltration and centrifugation to extract the lignin-degrading peroxidase.
3. The method of claim 1 , further comprising quantifying the lignin-degrading peroxidase.
4. The method of claim 3 , wherein the lignin-degrading peroxidase is selected from the group consisting of lignin peroxidases, versatile peroxidases, and manganese peroxidases.
5. The method of claim 1 , the plant is N. benthamiana.
6. The method of claim 1 , wherein the transforming step utilizes Agrobacterium transformation, particle bombardment, or electroporation.
7. The method of claim 1 , wherein the extracting step utilizes ion-exchange chromatography, size-exclusion chromatography, or immunoaffinity chromatography.
8. The method of claim 3 , wherein quantifying is accomplished via western blotting, ELISA, fluorescence spectroscopy, or UV-Vis spectroscopy.
9. The method of claim 1 , further comprising testing the activity of the extracted lignin-degrading peroxidase.
10. The method of claim 9 , wherein the testing step is accomplished by testing the activity of the extracted lignin-degrading peroxidase against a colorimetric dye.
11. The method of claim 10 , wherein the colorimetric dye is 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 2,6-Dichlorophenolindophenol (DCIP).
12. The method of claim 9 , wherein the testing step is accomplished by testing the activity of the extracted lignin-degrading peroxidase against a model lignin dimer.
13. The method of claim 12 , wherein the model lignin dimer is β-O-4.
14. The method of claim 1 , wherein the signal peptide is encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 78-80.
15. The method of claim 1 , wherein the lignin-degrading peroxidase is encoded by SEQ ID NO: 69.
16. The method of claim 1 , wherein the lignin-degrading peroxidase is a versatile peroxidase.
17. The method of claim 1 , wherein the constitutive promoter is a CAMV 35S promoter.
18. The method of claim 1 , wherein the wherein the lignin-degrading peroxidase is encoded by SEQ ID NO: 74.
19. The method of claim 1 , wherein the wherein the lignin-degrading peroxidase is encoded by SEQ ID NO: 1.
20. The method of claim 1 , wherein the wherein the lignin-degrading peroxidase is encoded by SEQ ID NO: 49.