Directed evolution of a regioselective halogenase for increased thermostability
AssigneeThe University of Chicago
InventorsJared C. Lewis, Catherine Poor, Mary Andorfer, James Payne
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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.
Claims16
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, M71C, N75K, E96V, D101G, S110P, S110L, F111S, G112S, G112D, L113D, L113N, L114P, S130L, K145M, K145R, N166S, F171I, K187R, D203A, D203G, T213A, V225I, K237E, V256I, T258A, D264G, T283A, L289P, F312L, T322I, T348A, L380F, T394M, F396Y, F396L, R400C, T413A, E423D, A442V, S448P, L453P, F458S, F458L, E461G, F465C, F465L, N467T, N470S, A476T, A476V, V481A, Q494R, T496R, T496A, G504S, and R509Q, wherein the RebH variant polypeptide is at least 85% identical to SEQ ID NO:1.
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 85% identical to SEQ ID NO:1.
Dependencies are inferred from the claim text.