Direct evolution of a regioselective halogenase for increased thermostability
AssigneeThe University of Chicago
InventorsJared C. Lewis, Catherine Poor, Mary Andorfer, James Payne
Life of this patent
Estimating the term…
Abstract
Compounds and methods are providing involving RebH variants with improved properties. directed evolution based on random mutagenesis was employed to generate a series of RebH variants. RebH variants with improved thermostability and increased activity at elevated temperatures were generated.
Claims23
An isolated RebH variant polypeptide of SEQ ID NO:
- 1 comprising at least one amino acid substitution, wherein the at least one amino acid substitution results in improved halogenating activity;
- and wherein the at least one amino acid substitution is selected from the group consisting of S2P;
- I52T;
- A58V;
- M71V, M71T, M71A, or M71C;
- N75K;
- E96V;
- D101G;
- S110P or S110L;
- F111S;
- G112S or G112D;
- L113D or L113N;
- L114P;
- S130L;
- K145M or K145R;
- N166S;
- F171I, K187R;
- D203A or D203G;
- T213A;
- V225I;
- K237E, V256I;
- T258A;
- D264G;
- T283A;
- L289P;
- F312L;
- T322I;
- T348A;
- L380F;
- T394M;
- F396Y or F396L;
- R400C;
- T413A;
- E423D;
- A442V;
- S448P;
- L453P;
- F458S or F458L;
- E461G;
- F465C or F465L;
- N467T;
- N470S;
- A476T or A476V;
- V481A;
- Q494R;
- T496R or T496A;
- G504S;
- and R509Q.
(canceled)
A RebH variant polypeptide comprising at least one amino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256, 258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 448, 453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or 509 in SEQ ID NO:1, wherein the RebH variant polypeptide is at least 60% identical to SEQ ID NO:1.
-53. (canceled)
A RebH variant comprising an amino acid sequence that is between 80% and 99% identical to SEQ ID NO:
- 1, wherein the RebH variant is capable of halogenating an aromatic substrate.
-75. (canceled)
Dependencies are inferred from the claim text.