- Document Number:
20240194289
- Appl. No:
18/553096
- Application Filed:
April 20, 2022
- نبذة مختصرة :
The present invention provides a computational method for designing artificial variants which have activity towards non-natural substrates. The present invention provides a special method to process stability evaluation results and creatively combines a process of calculating free energy barrier, which can improve the accuracy of the virtual screening of enzyme variants. The computational method disclosed by this invention greatly reduce the number of variants to be constructed and tested in the wet lab. In some cases, this method achieved the effect of enzyme engineering that cannot be achieved by traditional directed enzyme evolution methods.
- Assignees:
ENZYMASTER (NINGBO) BIO-ENGINEERING CO., LTD. (Ningbo, Zhejiang, CN)
- Claim:
1-10. (canceled)
- Claim:
11. A method, implemented using a computer system comprising one or more processors and system memory, for designing artificial variants of a given enzyme having catalytic activity for non-natural substrate(s), said method comprising the steps of: (1) introducing into said computer system a structural model for said given enzyme, wherein said structural model comprises a three dimensional computational representation of said enzyme in a catalytic conformation; (2) using said one or more processors to computationally model enzyme-substrate binding, determine substrate binding site(s) and binding conformation(s) for said given enzyme, and generate a three-dimensional computational representation of an enzyme-substrate complex; (3) using said one or more processors to perform systematic molecular docking analyses on the enzyme-substrate complex generated in step (2) to identify candidate positions for enzyme mutagenesis and specify amino acid substitutions; (4) using said one or more processors to virtually screen on the basis of protein structure stability evaluation methods all possible combinations of specified amino acid substitutions of all candidate positions to predict beneficial substitutions for each candidate position; and (5) using said one or more processors to virtually screen on the basis of free energy barrier calculations all possible combinations of predicted beneficial substitutions of all candidate positions identified in step (4) to identify catalytically active variants for a given enzyme.
- Claim:
12. The method according to claim 1, wherein the structural model for said given enzyme introduced in step (1) is obtained from a protein data bank database or predicted by modeling software.
- Claim:
13. The method according to claim 1, wherein the three-dimensional computational representation of an enzyme-substrate complex of step (2) is generated using software selected from the group consisting of Yasara, Discovery studio and Rosetta.
- Claim:
14. The method according to claim 1, wherein the evaluation methods for protein structure stability utilized in step (4) are selected from the group consisting of ddg_monomer, Cartesian_ddg, FoldX, Provean, ELASPIC and Amber TI.
- Claim:
15. The method according to claim 1, wherein results of the evaluation methods for protein structure stability utilized as a virtual screen step (4) are processed by a statistical method comprising the following steps: (i) determine the free energy difference (ΔΔG) of all calculated variants and sort from low to high in terms of numeric values, wherein a high numeric value corresponds to a stable cluster and a low numeric value corresponds to an unstable cluster; (ii) select a number of top-ranked stable clusters and bottom-ranked unstable clusters for frequency analysis wherein, fora specific amino acid position, an amino acid substitution with a higher frequency in an unstable cluster is subtracted from the amino acid substitutions having a higher frequency in a stable cluster to obtain the theoretically stable substitutions at the specific position; and (iii) combine the substitutions at each position determined to be stable in step (ii) to obtain a set of stable variants that correspond to predicted beneficial substitutions as predicted by computer virtual screening.
- Claim:
16. The method according to claim 1, wherein the “free energy barrier” utilized as a virtual screen step (5) is defined as the energy difference between the lowest energy point, which corresponds to the optimal conformation of an enzyme and substrate in a free state, and the highest energy point, which corresponds to the optimal conformation an enzyme-substrate complex in an activated state.
- Claim:
17. An artificial aminolyase variant designed using the method of claim 1, wherein said variant catalyzes the synthesis of β-alanine from acrylic acid which is a non-natural substrate for SEQ ID NO: 24, and the said variant has amino acid substitutions X326T or X326V as compared to SEQ ID NO: 24.
- Claim:
18. The artificial aminolyase variant according to claim 7, wherein the amino acid sequence of the artificial aminolyase variant further comprises amino acid substitutions (i) X187I or X187C, (ii) X321C, or (iii) X324L or X324V as compared to SEQ ID NO: 24.
- Claim:
19. The artificial aminolyase variant according to claim 7, wherein the amino acid sequence of said variant is set forth in SEQ ID NO: 26 and SEQ ID NO: 28.
- Claim:
20. The artificial aminolyase variant according to claim 7, wherein said variant catalyzes the synthesis of β-alanine from acrylic acid in conditions with temperature range of 30-60° C. and/or pH range of 7-11.
- Current International Class:
16; 12; 16
- الرقم المعرف:
edspap.20240194289
Processing Request
No Comments.