Mutations for improving activity and thermostability of PETASE enzymes
This invention relates to engineered plastic-degrading enzymes with improved functional features. In particular, modified PETases capable of hydrolysis of PET or polyethelene terephthalate plastic polymer are disclosed herein. These engineered PETases contain one or more amino acid modifications at specified residue or residues, for example, N233, and exhibit improved enzymatic activity as well as thermostability.
1 . An engineered PET (poly(ethylene terephthalate)) hydrolase comprising an amino acid sequence at least 90% identical to SEQ ID NO:1 and having at least one mutation corresponding to N233 relative to SEQ ID NO:1.
2 . The engineered PET hydrolase of claim 1 , wherein the at least one mutation is N233K.
3 . The engineered PET hydrolase of claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO: 1, 2 or 3.
4 . The engineered PET hydrolase of claim 1 , wherein the amino acid sequence is identical to SEQ ID NO: 1, 2 or 3 except for the at least one mutation.
5 . The engineered PET hydrolase of claim 1 , wherein the amino acid sequence has at least two mutations corresponding to a position relative to SEQ ID NO: 1 selected from the group consisting of N233K, S58E, S58A, N114T, S121E, N225C, M262L, T270V, T140D, S61T, I208V, and R224Q.
6 . The engineered PET hydrolase of claim 1 , wherein the amino acid sequence has two or three mutations corresponding to a position relative to SEQ ID NO: 1 selected from the group consisting of N233K, S58E, S58A, N114T, S121E, N225C, M262L, T270V, T140D, S61T, I208V, R224Q, S58Y, S58M, S58L, S58V, S58P, S61D, S61E, S61Y, S61F, N114H, N114L, N114R, N114S, N114T, T140Y, T140L, T140I, T140V, T140S, S121E, I208M, I208H, I208F, I208Y, I208P, I208A, R224D, R224E, R224S, R224T, R224N, R224Q, N225N, N225I, N225V, N225M, N225A, N225L, N225S, N225T, N233R, N233Y, N233H, N233P, M262L, M262I, M262A, M262V, M262F, M262W, T270Y, T270R, and T270H.
7 . A polynucleotide encoding the engineered PET hydrolase of claim 1 .
8 . The polynucleotide of claim 7 , wherein the engineered PET hydrolase further comprises a signal peptide.
9 . A vector comprising a promoter operably linked to the polynucleotide of claim 7 such that the promoter controls expression of the engineered PET hydrolase.
10 . A host cell comprising the polynucleotide of claim 7 .
11 . The host cell of claim 10 , wherein the host cell is a microbial cell.
12 . The host cell of claim 10 , wherein the host cell is a bacterial cell.
13 . The host cell of claim 12 , wherein the bacterial cell is Pseudomonas putida.
14 . The host cell of claim 10 , wherein the host cell is a fungal cell.
15 . A method of degrading poly(ethylene terephthalate) (PET) comprising contacting PET with the engineered PET hydrolase of claim 1 under conditions to degrade the PET.
16 . The method of claim 15 , comprising contacting the PET with a host cell that expresses and secretes the engineered PET hydrolase.
17 . The method of claim 15 , wherein the engineered PET hydrolase is purified.
18 . The method of claim 15 , wherein the conditions include an incubation at a temperature of between 25-70° C.