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A method for improving enzymes

A mutant and protein technology, applied in the fields of molecular biology and protein engineering, can solve problems such as limited effects, achieve the effects of improving efficiency, improving reliability and screening efficiency, improving protein stability and protein expression

Active Publication Date: 2021-12-24
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This optimization method has very limited effect

Method used

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  • A method for improving enzymes
  • A method for improving enzymes
  • A method for improving enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Carrying out structural simulation of proteins and obtaining high-precision protein structure models

[0064] Structural modeling of CPY87D20 (cucumber P450 enzyme) was performed using the homology modeling function of Rosetta. First, the fasta amino acid sequence file of CPY87D20 was used as input, and the homologous sequence of CPY87D20 was searched from the pfamA_32.0 or scope70_1_1.75 database using hhblits to obtain a multiple sequence alignment file in a3m format. Take this file as input, use hhsearch to retrieve the existing pdb structure from the pdb70 database, and obtain the pdb.hhr file. Use this file as input for homology modeling with RosettaCM. At the same time, the sdf structures of the ligands cucurbitenol (Cuol) and heme (heme) were obtained from Pubchem, and the parameter files were generated using the Rosetta program. Use Rosetta Ligand_Docking to dock heme and Cuol into the protein model to obtain the complex. Using the deep learning fr...

Embodiment 2

[0065] Embodiment 2, change the catalytic activity of enzyme

[0066] Using the psiblast program and using uniref90 as the database, the position-specific scoring matrix PSSM of CPY87D20 was obtained, which displayed the conservation information of each amino acid of CPY87D20. The PSSM scoring matrix is ​​analyzed to obtain potential replacements of catalytic centers or protein surface amino acids according to the purpose of the experiment (improving activity or stability). Using hhblits and uniclust30_2018_08 as the database, multiple sequence alignments were performed on CPY87D20, amino acid co-evolution analysis was performed using Gremlin, and potential replacement sites were obtained according to the purpose of the experiment. Selection of Substrate Cucurbitadienol Based on Protein Model Amino acids within the range exclude amino acid sites near the heme (because it may cause loss of activity). Replacement mutations with PSSM scores greater than 0 were screened against...

Embodiment 3

[0072] Embodiment 3, the effect of each mutant

[0073] The P450s mutant of the present invention is based on the amino acid sequence of CPY87D20 of cucumber (cucumis sativus), as shown in SEQ ID No.1, and multiple mutants including V4 have been obtained successively, for example: mutant V1, amino acid sequence As shown in SEQ ID No.2; mutant V2, the amino acid sequence is shown in SEQ ID No.3; mutant V3, the amino acid sequence is shown in SEQ ID No.4.

[0074]For another example, mutants further mutated on the basis of the above mutants: V2-I46L-A49L, V2-W119I-L125D, V2-R385Y, V2-W399K, V2-I439H, V2-E463P, V2-I46L-A49L- C343Y, V3-C343Y, V3-C343Y-S49L, V3-C343Y-I46L, V3-K73Y, V3-F89D, V3-Y432E, V3-L125D, V3-R383T, V3-W399D, etc., tested in batches under the same conditions . Several were found: both the yield of the target product 11H-Cuol, the substrate selection specificity of the enzyme, and the protein expression level were significantly higher than those of the wild ty...

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Abstract

The invention relates to a method for improving enzymes, which comprehensively utilizes technologies such as computational biology, big data analysis, and synthetic biology to simultaneously analyze the enzyme active region and the conservation of amino acids on its protein surface on the basis of high-precision protein structure simulation And co-evolutionary information, combined with protein energy calculation and other methods to rationally design mutants, provides a new set of ideas for improving proteins and efficiently screening high-quality mutants.

Description

technical field [0001] The invention relates to an enzyme improvement method, in particular to designing the enzyme by combining protein surface amino acids and catalytic center amino acids, and belongs to the fields of molecular biology and protein engineering. Background technique [0002] Enzymes are macromolecular substances with catalytic functions, which can catalyze almost all chemical reactions in the life process. Their precise spatial structure makes them have the advantages of high catalytic efficiency and strong specificity. How to design and modify rationally to obtain new enzymes with higher catalytic efficiency, stronger selectivity, stability and higher expression levels is becoming a research hotspot. [0003] At present, the engineering improvement strategies of enzymes are mainly directed evolution and rational design. Directed evolution is to carry out amino acid mutations through specific methods, generate sequence diversity, and repeatedly screen mutan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B40/00G16B20/50C12N9/02
CPCG16B40/00G16B20/50C12N9/0081C12Y114/15006
Inventor 黄三文尚轶张晓鹏姚垠颖罗威
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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