Determination of ligninases activities by nano-structure initiator mass spectrometry
A method for rapid, high throughput screening of the activities of enzymes, especially ligninases and its enzyme cocktails, using nanostructure initiator mass spectrometry (NIMS) surfaces, substrates and methodology.
1. A method for analysis of enzyme activity in a sample, comprising:
providing NIMS substrates incorporating one or more of, or simulating H, G, or S lignin units linked by one or more, beta-O-4, 5-5′ (biphenyl), β-5 (phenylcoumaran), β-β, and 4-O-5 (diaryl ether) linkages, and tagging each substrate of the NIMS substrates designed with a unique mass fluorous tag or perfluoronated affinity moiety;
performing ligninase oxidation reactions on the tagged NIMS substrates designed to generate tagged reaction products using one or more enzymes in the sample; and
identifying and analyzing the tagged reaction products of the ligninase oxidation reactions by nanostructure-initiator mass spectrometry (NIMS).
2. The method of claim 1 , wherein identifying and analyzing the reaction product of the ligninase oxidation reactions by NIMS comprises applying the reaction products to a hydrophobic NIMS chip surface, wherein the tagged reaction products interact with the NIMS chip surface via fluorous-phase-interactions.
3. The method of claim 1 , wherein the method further comprising: performing kinetics study and quantification of ratios of products to substrates of the ligninase oxidation reactions to determine the enzyme activity in the sample.
4. The method of claim 1 , wherein the providing step comprises designing the NIMS substrates.
5. The method of claim 1 , the providing step comprises providing a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a beta-O-4 linkage.
6. The method of claim 5 , wherein the NIMS substrate is Compound A.
7. The method of claim 6 , wherein the NIMS substrate is Compound A-1a or A-1b.
8. The method of claim 1 , the providing step comprises providing a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a 5-5′ (biphenyl) linkage.
9. The method of claim 8 , wherein the NIMS substrate is Compound B.
10. The method of claim 9 , wherein the NIMS substrate is Compound B-1a or B-1b.
11. The method of claim 1 , the providing step comprises providing a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a β-5 (phenylcoumaran) linkage.
12. The method of claim 11 , wherein the NIMS substrate is Compound C.
13. The method of claim 12 , wherein the NIMS substrate is Compound C-1a or C-1b.
14. The method of claim 1 , the providing step comprises providing a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a β-β linkage.
15. The method of claim 14 , wherein the NIMS substrate is Compound D.
16. The method of claim 15 , wherein the NIMS substrate is Compound D-1a or D-1b.
17. The method of claim 1 , the providing step comprises providing a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a 4-O-5 (diaryl ether) linkage.
18. The method of claim 17 , wherein the NIMS substrate is Compound E.
19. The method of claim 18 , wherein the NIMS substrate is Compound E-1a or E-1b.
20. The method of claim 1 , wherein the mass fluorous tag or perfluoronated affinity moiety is Compound 1a or 1b.
21. The method of claim 1 , wherein the providing step comprises providing a mixture of NIM substrates comprising (1) a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a beta-O-4 linkage; (2) a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a 5-5′ (biphenyl) linkage; (3) a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a β-5 (phenylcoumaran) linkage; (4) a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a β-β linkage; and, (5) a NIMS substrate incorporating, or simulating H, G, or S lignin units linked by, a 4-O-5 (diaryl ether) linkage.
22. The method of claim 21 , wherein the mixture of NIMS substrates comprises Compound A, Compound B, Compound C, Compound D, and Compound E.
23. The method of claim 22 , wherein the mixture of NIMS substrates comprises Compound A-1a or A-1b, Compound B-1a or B-1b, Compound C-1a or C-1b, Compound D-1a or D-1b, and Compound E-1a or E-1b.