Bacterial cytochrome P450 protein variant and method of reducing concentration of fluorinated methane in sample using the same
Provided is a recombinant microorganism including an exogenous gene encoding a bacterial cytochrome P450 protein or a variant thereof, a composition including the recombinant P450 protein or the variant thereof, which is used for removing CH n F 4-n (n is an integer of 0 to 3) in a sample, and a method of reducing a concentration of CH n F 4-n in the sample.
1. A bacterial P450Cam variant polypeptide having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 selected from the group consisting of F351T, F351N, F351Q, and F351D and having an activity belonging to EC 1.14.15.1.
2. The variant polypeptide of claim 1 , wherein the variant polypeptide comprises a P450Cam variant comprising SEQ ID NO: 6 with a substitution at position F351 of SEQ ID NO: 6, wherein the substitution is F351T, F351N, F351Q, or F351D.
3. A composition comprising the P450Cam variant of claim 1 .
4. A polynucleotide encoding the bacterial P450Cam variant polypeptide of claim 1 .
5. A recombinant microorganism comprising an exogenous gene encoding the bacterial P450Cam variant polypeptide of claim 1 .
6. The recombinant microorganism of claim 5 , wherein the exogenous gene encodes a P450Cam variant polypeptide comprising SEQ ID NO: 6 with a substitution at position F351 of SEQ ID NO: 6, wherein the substitution is F351T, F351N, F351Q, or F351D.
7. The recombinant microorganism of claim 5 , further comprising an exogenous gene encoding a protein having an activity belonging to EC 1.1.1.49.
8. A method of reducing a concentration of fluorinated methane in a sample, the method comprising contacting a recombinant P450 protein, or a P450Cam variant polypeptide having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and having an activity belonging to EC 1.14.15.1, with the sample comprising fluorinated methane represented by CH n F4-n (n is an integer of 0 to 3) to reduce the concentration of fluorinated methane in the sample.
9. The method of claim 8 , wherein the contacting is performed in a sealed container.
10. A method of reducing a concentration of fluorinated methane in a sample, the method comprising contacting a recombinant microorganism with the sample comprising fluorinated methane represented by CH n F4-n (n is an integer of 0 to 3) to reduce the concentration of fluorinated methane in the sample, wherein the recombinant microorganism comprises an exogenous gene encoding a bacterial P450 protein or a variant thereof, wherein the variant is a P450Cam variant having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and having an activity belonging to EC 1.14.15.1.
11. The method of claim 10 , wherein the contacting is performed in a sealed container where the recombinant microorganism survives or is viable.
12. The method of claim 10 , wherein the contacting comprises culturing the recombinant microorganism in the presence of the sample.
13. The method of claim 10 , wherein the sample is industrial waste water or waste gas.
14. The method of claim 10 , wherein the recombinant microorganism further comprises an exogenous gene encoding a protein having an activity belonging to EC 1.1.1.49.
15. The method of claim 10 , wherein the recombinant microorganism comprises an exogenous gene encoding the P450Cam variant comprising a F351Y, F351T, F351N, F351Q, F351H, or F351D mutation.
16. A bacterial P450BM3 variant polypeptide having an amino acid alteration at an amino acid residue corresponding to position N320 of SEQ ID NO: 8 and having an activity belonging to EC 1.14.14.1.
17. The variant polypeptide of claim 16 , wherein the variant polypeptide comprises a P450BM3 variant comprising SEQ ID NO: 8 with a substitution at position N320.
18. The composition of claim 17 , wherein the substitution at amino acid position N320 is a N320W, N320F, N320G, N320P, N320S, or N320E mutation.
19. A composition comprising the P450BM3 variant polypeptide of claim 16 .
20. A polynucleotide encoding the bacterial P450BM3 variant polypeptide of claim 16 .
21. A recombinant microorganism comprising an exogenous gene encoding the bacterial P450BM3 variant polypeptide of claim 16 .
22. The recombinant microorganism of claim 21 , wherein the exogenous gene encodes a P450BM3 variant polypeptide comprising SEQ ID NO: 8 with a substitution at position N320.
23. The recombinant microorganism of claim 22 , wherein the substitution at position N320 is N320W, N320F, N320G, N320P, N320S, or N320E.
24. The recombinant microorganism of claim 21 , further comprising an exogenous gene encoding a protein having an activity belonging to EC 1.1.1.49.
25. A method of reducing a concentration of fluorinated methane in a sample, the method comprising contacting a P450BM3 variant polypeptide of claim 16 with the sample comprising fluorinated methane represented by CHnF4-n (n is an integer of 0 to 3) to reduce the concentration of fluorinated methane in the sample.
26. The method of claim 25 , wherein the contacting is performed in a sealed container.
27. A method of reducing a concentration of fluorinated methane in a sample, the method comprising contacting the recombinant microorganism of claim 21 with a sample comprising fluorinated methane represented by CHnF4-n (n is an integer of 0 to 3) to reduce the concentration of fluorinated methane in the sample.
28. The method of claim 27 , wherein the contacting is performed in a sealed container where the recombinant microorganism survives or is viable.
29. The method of claim 27 , wherein the contacting comprises culturing the recombinant microorganism in the presence of the sample.
30. The method of claim 27 , wherein the sample is industrial waste water or waste gas.
31. The method of claim 27 , wherein the recombinant microorganism further comprises an exogenous gene encoding a protein belonging to EC 1.1.1.49.