A pectin methylesterase mutant based on diffusion model design and preparation and application thereof

Abstract

The application provides a high-activity pectin methylesterase mutant designed based on a diffusion model, and preparation and application of the pectin methylesterase mutant, and the amino acid sequence of the pectin methylesterase mutant PME-DS12 is shown as SEQ ID NO:2. The application re-designs the structure of the pectin methylesterase by using RFdiffusion generated diffusion model, and a mutant PME-DS12 with significantly improved enzyme activity is screened. Compared with the wild-type pectin methylesterase, the enzyme activity of PME-DS12 is improved by about 6.3 times, and PME-DS12 has higher industrial application value. The mutant has wide application prospects in the fields of food processing, textile and the like. Especially in the aspect of flavor substance generation, the enzyme can provide the generation of aroma lipid substances by degrading macromolecular substances in food, so as to improve the aroma quality of products; and the enzyme can also be used in the papermaking industry, including biological bleaching of paper pulp, reduction of use of chemical reagents and reduction of environmental pollution.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and enzyme engineering, and in particular to a highly active pectin methylesterase (PME-DS12) designed by a generative diffusion model RFdiffusion, a preparation method thereof, and industrial applications thereof. Background Art

[0002] Pectin methylesterase is an enzyme that catalyzes the demethylation of pectin and has important applications in food processing, textiles, papermaking, bioenergy, and other industries. Improving the activity and stability of pectin methylesterase is crucial for improving industrial production efficiency and product quality.

[0003] While traditional enzyme engineering methods, such as directed evolution and rational design, have achieved some success in improving enzyme performance, they still suffer from limitations such as time-consuming, inefficient, and limited sequence space exploration. With the advancement of deep learning and diffusion modeling technologies, the use of deep learning-based diffusion models for protein sequence design and optimization offers unique advantages. The diffusion model RFdiffusion is a generative protein diffusion model based on deep learning that effectively generates novel protein structures based on structural information in protein sequences. Summary of the Invention

[0004] The purpose of the present invention is to provide a pectin methylesterase mutant PME-DS12 designed based on a diffusion model, and its preparation and application. The enzyme activity of the mutant PME-DS12 is increased by about 6.3 times compared with the wild-type pectin methylesterase (WT).

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A pectin methylesterase mutant, PME-DS12, was designed based on the diffusion model RFdiffusion, with its amino acid sequence shown in SEQ ID NO: 2. Using the RFdiffusion model, the amino acid sequence and structure of wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) were used as input. Combined with multiple sequence alignment (MSA) analysis, approximately 75% of highly conserved residues were retained, while sequence variations were designed for the remaining approximately 25% of non-conserved residues. This generated a new protein model and sequence, resulting in the screening of the highly active pectin methylesterase mutant PME-DS12.

[0007] The screening process involved using AlphaFold2 to perform structure prediction and quality assessment on multiple variant sequences generated by the model, and through activity screening, a mutant PME-DS12 with significantly improved activity was obtained.

[0008] The method for preparing the pectin methylesterase mutant PME-DS12 of the present invention comprises the following steps:

[0009] i. Synthesize the PME-DS12 gene sequence and clone it into an expression vector;

[0010] ii. transforming the recombinant vector into a host cell for expression;

[0011] iii. Obtain highly active PME-DS12 pectin methylesterase.

[0012] The specific technical solutions are as follows:

[0013] 1. Obtaining PME-DS12 Mutants

[0014] Sequence Design: Utilizing the RFdiffusion model, the amino acid sequence and structure of wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) were used as input, combined with multiple sequence alignment (MSA) analysis. Approximately 75% of highly conserved residues were retained, while sequence variations were designed for the remaining 25% of non-conserved residues. Multiple variant sequences were generated using the model, and structure prediction and quality assessment were performed using AlphaFold2. The mutant PME-DS12, which exhibited significantly enhanced enzyme activity, was identified.

[0015] Candidate sequence screening: AlphaFold2 was used to predict the structure of multiple variant sequences generated by the model to evaluate their stability and activity potential. Finally, enzyme activity screening was performed to identify the mutant PME-DS12 with significantly improved activity.

[0016] 2. Differences in amino acid sequences between PME-DS12 and WT

[0017] WT pectin methylesterase sequence (SEQ ID NO:1):ATTYNAVVSKSSSDGKTFKTIADAIASAPAGSTPFVILIKNGVYNERLTITRNNLHLKGESRNGAVIAAATAAGTLKSDGSKWGTAGSSTITISAKDFSAQSLTIRNDFFDFPANQAKSDSDSSKIKDTQAVALYVTKSGDRAYFKDVSLVGYQDTLYVSGGRSFFSDC RISGTVDFIFGDGTALFNNCDLVSRYRADVKSGNVSGYLTAPSTNINQKYGLVITNSRVIRESDSVPAKSYGLGRPWHPTTTFSDGRYADPNAIGQTVFLNTSMDNHIYGWDKMSGKDKNGNTIWFNPEDSRFFEYKSYGAGATVSKDRRQLTDAQAAEYTQSKVLGDWTPTLP

[0018] PME-DS12 pectin methylesterase sequence (SEQ ID NO:2):GVVYNAVVSKSSSDGNTFATISDAIASAPAGDAPFVILIKNGTYNERLTITRNSLKGESRNGAVISAATAAGTLNADGTKYGTIGSATITINAKDFYAESLTIRNDFDFPANQAKPASDSTKIKDTQAVALYVNSNGDRAYFKDVALEGYQDTLYVSGGRSFFEDC VISGTVDFIFGDGTALFNNCDLVSRYRPDVAPDQVLGYLTAPSTHIDKKYGLVIKNSRVIRESDAVPAKSYGLGRPWHPTTTFPDGRYADPNAIGQTVFLNTYMDNHIYGWDKMSGKDKNGNEIWFKPEDSRFFEYNSYGAGAPTGSDRRQLTDEEAAEYTEENVLGDYTPSAP

[0019] Differential amino acid site information:

[0020] Comparison of the amino acid sequences of WT and PME-DS12 revealed that 58 amino acid substitutions occurred in PME-DS12. The main mutation sites are listed below (sequence positions refer to the WT sequence):

[0021] Mutation site list:

[0022] A1G, T2V, T3V, K16N, K19A, A22S, S32D, T33A, V43T, H56S, A68S, K77N, S78A, S81T, W83Y, A86I, S89A , S94N, S99Y, Q101E, S118P, D119A, S123T, T136N, K137S, S138N, S148A, V150E, S166E, R169V, A196P , K199A, S200P, G201D, N202Q, S204L, N213H, N215D, Q216K, T223K, S233A, S252P, S271Y, T291E, N2 95K, K305N, T312P, V313T, S314G, K315S, A323E, Q324E, Q330E, S331E, K332N, W337Y, T340S, L341A.

[0023] 3. Preparation of PME-DS12

[0024] Gene synthesis: Based on the amino acid sequence of PME-DS12, the corresponding gene sequence was synthesized using reverse translation and codon optimization. The optimization process took into account the codon usage preference of the host bacteria (such as E. coli) to improve gene expression efficiency.

[0025] Vector construction: The synthesized PME-DS12 gene sequence is cloned into a prokaryotic expression vector, such as pET-28a(+). Use specific restriction endonuclease sites (such as NcoI and XhoI) for digestion and ligation to construct the recombinant expression vector pET-28a(+)-PME-DS12.

[0026] Expression and Purification: The recombinant plasmid was transformed into Escherichia coli BL21(DE3). Expression was induced with IPTG under appropriate culture conditions. After harvesting the cells, the crude enzyme solution was obtained by ultrasonication and centrifugation. The PME-DS12 protein was purified using a Ni-NTA affinity chromatography column based on the 6×His tag.

[0027] Enzyme activity assay: The methanol oxidase method was used to determine the pectin methylesterase activity of PME-DS12 and WT. The results showed that the enzyme activity of PME-DS12 was more than 6.3 times that of WT.

[0028] 4. Applications of PME-DS12

[0029] As a highly active pectin methylesterase, PME-DS12 can be widely used in the following fields:

[0030] Flavor substance production: By degrading macromolecular substances in food, the production of aromatic lipids is provided, thereby improving the aroma quality of the product.

[0031] Food processing: used for juice clarification, jam production, etc. to improve product quality and production efficiency.

[0032] Textile industry: used in bio-enzyme desizing and bio-polishing to improve the quality of textiles.

[0033] Papermaking industry: used for biological bleaching of pulp, reducing the use of chemical reagents and reducing environmental pollution.

[0034] Compared to existing technologies, the present invention's greatest advantage lies in its redesign of the pectin methylesterase sequence using the diffusion model RFdiffusion, resulting in a mutant, PME-DS12, whose enzyme activity is approximately 6.3-fold higher than that of the wild-type. Compared to the wild-type sequence, PME-DS12 undergoes 58 amino acid substitutions. This pectin methylesterase has broad application prospects in various industries, including food processing (for juice clarification and jam production), textiles, and papermaking (for biobleaching of pulp, reducing the use of chemical reagents and minimizing environmental pollution). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : SDS-PAGE electrophoresis of WT and PME-DS12 purified proteins to evaluate the purity and molecular weight of the proteins.

[0036] Figure 2 : The results of enzyme activity assay showed that the catalytic activity of PME-DS12 was significantly higher than that of WT (wild type).

[0037] Figure 3 : Electrostatic surface potential maps of WT and PME-DS12 proteins (WT on the left, PME-DS12 on the right). The black dotted circle outlines the potential substrate binding pocket, showing that PME-DS12 has an increased positive potential in this area. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the embodiments (attached drawings):

[0039] Example 1: Construction of PME-DS12 gene

[0040] Based on the amino acid sequence of PME-DS12, the corresponding nucleotide sequence was obtained using reverse translation combined with codon optimization in Escherichia coli. The optimized PME-DS12 gene sequence was synthesized and cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant expression vector pET-28a(+)-PME-DS12.

[0041] 1. Enzyme digestion and ligation: Use NcoI and XhoI to digest the pET-28a(+) vector and the PME-DS12 gene fragment.

[0042] 2. Ligation reaction: The PME-DS12 gene fragment after enzyme digestion is connected to the linearized vector under the action of T4 DNA ligase.

[0043] 3. Transformation and screening: The ligation product was transformed into E. coli DH5α competent cells, spread on LB plates containing kanamycin, and cultured at 37°C overnight.

[0044] 4. Identification of positive clones: Select single colonies for bacterial liquid PCR and enzyme digestion identification to screen out the correct recombinant plasmid.

[0045] 5. Sequencing verification: Send the recombinant plasmid to a sequencing company for sequence determination to ensure the accuracy of the PME-DS12 gene sequence.

[0046] Example 2: Expression and purification of PME-DS12

[0047] 1. Transformation and culture of bacterial strains: Transform the pET-28a(+)-PME-DS12 recombinant plasmid into competent Escherichia coli BL21 (DE3) cells.

[0048] 2. Induce expression: Culture the strain in LB medium and shake culture at 37℃ until the OD600 is about 0.6. Add IPTG to a final concentration of 0.5 mM, lower the temperature to 16℃, and continue to culture for 12-16 hours to induce protein expression.

[0049] 3. Protein extraction: Collect the cultured bacteria, resuspend them in lysis buffer, and use ultrasonication to disrupt the cells to obtain crude enzyme solution.

[0050] 4. Protein purification: PME-DS12 protein was purified by Ni-NTA affinity chromatography based on the His tag. The purified protein was analyzed by SDS-PAGE, showing a clear band at approximately 40 kDa (see Figure 1 ), with higher purity.

[0051] Example 3: Determination of enzyme activity

[0052] 1. Reaction system: 0.5% (w / v) citrus pectin was used as the substrate, and the enzymatic reaction was carried out at pH 4.8 and 37°C.

[0053] 2. Assay: Use the methanol oxidase method to determine the reducing sugar content produced by the enzyme reaction. Measure the absorbance at 540 nm and calculate the enzyme activity.

[0054] 3. Result analysis: The enzyme activity of PME-DS12 is 6.3 times higher than that of WT (see Figure 2 ), indicating that the mutants designed by RFdiffusion significantly improved the catalytic efficiency of the enzyme.

[0055] Example 4: Functional analysis of mutation sites

[0056] 1. Sequence alignment: Align the amino acid sequences of PME-DS12 and WT pectin methylesterase to determine the mutation site.

[0057] 2. Structure prediction: The three-dimensional structure of PME-DS12 was predicted using AlphaFold2, and it was found that the mutation sites were mainly concentrated in the region related to substrate binding (see Figure 3 ).

[0058] 3. Reasons for increased activity: Mutations cause conformational changes in the protein's active site, enhancing the binding ability of the enzyme to the substrate and improving catalytic efficiency.

[0059] in conclusion

[0060] By utilizing the diffusion model RFdiffusion, a highly active pectin methylesterase mutant, PME-DS12, was successfully generated. Compared to the wild-type, PME-DS12 exhibits approximately 6.3-fold increased enzyme activity. This enzyme has important applications in various industrial fields.

[0061] Sequence Listing

[0062] <110> China National Tobacco Corporation Zhengzhou Tobacco Research Institute

[0063] <120> A pectin methylesterase mutant designed based on a diffusion model and its preparation and application

[0064] <210> 1

[0065] <211> 342

[0066] <212> PRT

[0067] <213> Pectin methylesterase mutant PME-DS12

[0068] <400> 1

[0069] GVVYNAVVSKSSSDGNTFATISDAIASAPAGDAPFVILIKNGTYNERLTITRNNLSLKGESRNGAVISAATAAGTLNADGTKYGTIGSATITINAKDFYAESLTIRNDFDFPANQAKPASDSTKIKDTQAVALYVNSNGDRAYFKDVALEGYQDTLYVSGGRSFFEDCVISGTVDFIFGDGTALFNNCDLVSRYRPDVAPDQVLGYLTAPSTHIDKKYGLVIKNSRVIRESDAVPAKSYGLGRPWHPTTTFPDGRYADPNAIGQTVFLNTYMDNHIYGWDKMSGKDKNGNEIWFKPEDSRFFEYNSYGAGAPTGSDRRQLTDEEAAEYTEENVLGDYTPSAP

Claims

1. A pectin methylesterase mutant designed based on a diffusion model, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

2. A method for preparing the pectin methylesterase mutant according to claim 1, characterized in that: The following steps are involved: 1) Synthesize the sequence as shown in SEQ ID NO: 2 and clone it into an expression vector; 2) Transform the recombinant vector into host cells for expression; 3) Obtain highly active PME-DS12 pectin methylesterase.

3. Use of the pectin methylesterase mutant according to claim 1 in pectin demethylation, juice clarification, jam preparation, textile processing or papermaking.

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