Pectin methylesterase mutant designed based on diffusion model as well as preparation and application of pectin methylesterase mutant

Abstract

The invention provides a high-activity pectin methylesterase mutant designed based on a diffusion model as well as preparation and application of the high-activity pectin methylesterase mutant. The amino acid sequence of the pectin methylesterase mutant PME-DS12 is shown as SEQ ID NO: 2. According to the invention, the structure of the pectin methylesterase is redesigned by utilizing an RFdiffusion generative diffusion model, and the mutant PME-DS12 of which the enzyme activity is remarkably improved is obtained by screening. Compared with wild pectin methylesterase, the enzymatic activity of the PME-DS12 is improved by about 6.3 times, and the PME-DS12 has higher industrial application value. The mutant has wide application prospects in the fields of food processing, spinning and the like. In particular, in the aspect of flavor substance generation, macromolecular substances in food are degraded by the enzyme, so that the generation of aroma lipid substances is provided, and the aroma quality of a product is improved; the method can also be used in the papermaking industry, including biological bleaching of paper pulp, reduces the use of chemical reagents, and reduces 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 a type of enzyme that can catalyze the demethylation of pectin substances and has important application value in food processing, textile, papermaking, bioenergy and other industrial fields. Improving the activity and stability of pectin methylesterase is of great significance for improving industrial production efficiency and product quality.

[0003] Traditional enzyme engineering methods, such as directed evolution and rational design, have achieved certain results in improving enzyme performance, but they still have limitations such as long time consumption, low efficiency, and limited exploration of sequence space. With the development of deep learning and diffusion model technology, the use of deep learning diffusion models for protein sequence design and optimization has unique advantages. The diffusion model RFdiffusion is a protein generative diffusion model based on deep learning, which can effectively generate new protein structures based on the structural information in the protein sequence. 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: A pectin methylesterase mutant PME-DS12 designed based on the diffusion model RFdiffusion, whose amino acid sequence is shown in SEQ ID NO: 2. The RFdiffusion model is used to take the amino acid sequence and structure of wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) as input, combined with multiple sequence alignment (MSA) analysis, keeping about 75% of highly conserved residues unchanged, and performing sequence variation design on the remaining about 25% of non-conserved residues, thereby generating a new protein model and sequence, and screening the highly active pectin methylesterase mutant PME-DS12.

[0006] The screening process was to use 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.

[0007] The method for preparing the pectin methylesterase mutant PME-DS12 of the present invention comprises the following steps: i. Synthesize the PME-DS12 gene sequence and clone it into an expression vector; ii. transforming the recombinant vector into a host cell for expression; iii. Obtain highly active PME-DS12 pectin methylesterase.

[0008] The specific technical solutions are as follows: 1. Obtaining PME-DS12 mutants Sequence design: 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, keeping about 75% of highly conserved residues unchanged, and performing sequence variation design on the remaining 25% of non-conserved residues. Multiple variant sequences were generated through the model, and AlphaFold2 was used for structure prediction and quality assessment to screen out the mutant PME-DS12 with significantly improved enzyme activity.

[0009] Candidate sequence screening: Among the multiple variant sequences generated by the model, AlphaFold2 was used for structural prediction to evaluate their stability and activity potential. Finally, through enzyme activity screening, the mutant PME-DS12 with significantly improved activity was identified.

[0010] 2. Differences in amino acid sites between PME-DS12 and WT WT pectin methylesterase sequence (SEQ ID NO:1):ATTYNAVVSKSSSDGKTFKTIADAIASAPAGSTPFVILIKNGVYNERLTITRNNLHLKGESRNGAVIAAATAAGTLKSDGSKWGTAGSSTITISAKDFSAQSLTIRNDFFDFPANQAKSDSDSSKIKDTQAVALYVTKSGDRAYFKDVSLVGYQDTLYVSGGRSFFSDC RISGTVDFIFFGDGTALFNNCDLVSRYRADVKSGNVSGYLTAPSTNINQKYGLVITNSRVIRESDSVPAKSYGLGRPWHPTTTFSDGRYADPNAIGQTVFLNTSMDNHIYGWDKMSGKDKNGNTIWFNPEDSRFFEYKSYGAGATVSKDRRQLTDAQAAEYTQSKVLGDWTPTLP PME-DS12 pectin methylesterase sequence (SEQ ID NO:2):GVVYNAVVSKSSSDGNTFATISDAIASAPAGDAPFVILIKNGTYNERLTITRNSLKGESRNGAVISAATAAGTLNADGTKYGTIGSATITINAKDFYAESLTIRNDFDFPANQAKPASDSTKIKDTQAVALYVNSNGDRAYFKDVALEGYQDTLYVSGGRSFFEDC VISGTVDFIFGDGTALFNNCDLVSRYRPDVAPDQVLGYLTAPSTHIDKKYGLVIKNSRVIRESDAVPAKSYGLGRPWHPTTTFPDGRYADPNAIGQTVFLNTYMDNHIYGWDKMSGKDKNGNEIWFKPEDSRFFEYNSYGAGAPTGSDRRQLTDEEAAEYTEENVLGDYTPSAP Differential amino acid position information: By comparing the amino acid sequences of WT and PME-DS12, it was found that 58 amino acid sites in PME-DS12 were replaced. The main mutation sites are listed below (sequence positions refer to the WT sequence): Mutation site list: 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.

[0011] 3. Preparation of PME-DS12 Gene synthesis: Based on the amino acid sequence of PME-DS12, the corresponding gene sequence was synthesized by reverse translation and codon optimization. The codon usage preference of the host bacteria (such as Escherichia coli) was taken into account during the optimization process to improve the expression efficiency of the gene.

[0012] Vector construction: The synthesized PME-DS12 gene sequence is cloned into a prokaryotic expression vector, such as pET-28a(+). Specific restriction endonuclease sites (such as NcoI and XhoI) are used for digestion and ligation to construct the recombinant expression vector pET-28a(+)-PME-DS12.

[0013] Expression and purification: The recombinant plasmid was transformed into E. coli BL21 (DE3). Under appropriate culture conditions, IPTG was used to induce expression. After the bacteria were collected, the crude enzyme solution was obtained by ultrasonic disruption, centrifugation, etc. The PME-DS12 protein was purified using a Ni-NTA affinity chromatography column based on the 6×His tag.

[0014] Enzyme activity determination: The methanol oxidase method was used to determine the enzyme activity of PME-DS12 and WT pectin methylesterase. The results showed that the enzyme activity of PME-DS12 was more than 6.3 times that of WT.

[0015] 4. Applications of PME-DS12 As a highly active pectin methylesterase, PME-DS12 can be widely used in the following fields: Generation of flavor substances: By degrading macromolecular substances in food, the generation of aromatic lipids is provided, thereby improving the aroma quality of the product.

[0016] Food processing: used for juice clarification, jam production, etc. to improve product quality and production efficiency.

[0017] Textile industry: used in bio-enzyme desizing and bio-polishing to improve the quality of textiles.

[0018] Papermaking industry: used for biological bleaching of pulp, reducing the use of chemical reagents and reducing environmental pollution.

[0019] Compared with the prior art, the biggest feature of the present invention is that the pectin methylesterase sequence is redesigned by using the diffusion model RFdiffusion, and the mutant PME-DS12 is obtained, whose enzyme activity is about 6.3 times higher than that of the wild type. Compared with the WT sequence, a total of 58 amino acids in PME-DS12 are replaced. The pectin methylesterase has broad application prospects in industrial fields such as food processing (juice clarification, jam production), textiles, and papermaking (for biological bleaching of pulp, reducing the use of chemical reagents, and reducing environmental pollution). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : SDS-PAGE electrophoresis of WT and PME-DS12 purified proteins to evaluate the purity and molecular weight of the proteins.

[0021] 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).

[0022] Figure 3 : Electrostatic surface potential diagram 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

[0023] The present invention is further described in detail below in conjunction with the embodiments (attached drawings): Example 1: Construction of PME-DS12 gene According to the amino acid sequence of PME-DS12, the corresponding nucleotide sequence was obtained by reverse translation combined with codon optimization of 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.

[0024] 1. Enzyme digestion and ligation: Use NcoI and XhoI to digest the pET-28a(+) vector and PME-DS12 gene fragment.

[0025] 2. Ligation reaction: The PME-DS12 gene fragment after restriction digestion and the linearized vector were connected under the action of T4 DNA ligase.

[0026] 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.

[0027] 4. Identification of positive clones: Select single colonies for bacterial liquid PCR and enzyme digestion identification to screen out the correct recombinant plasmid.

[0028] 5. Sequencing verification: Send the recombinant plasmid to a sequencing company for sequence determination to ensure the accuracy of the PME-DS12 gene sequence.

[0029] Example 2: Expression and purification of PME-DS12 1. Strain transformation and culture: Transform the pET-28a(+)-PME-DS12 recombinant plasmid into Escherichia coli BL21 (DE3) competent cells.

[0030] 2. Induce expression: Culture the strain in LB medium, shake and culture at 37℃ until OD600 is about 0.6, add IPTG with a final concentration of 0.5 mM, lower the temperature to 16℃, and continue to culture for 12-16 hours to induce protein expression.

[0031] 3. Protein extraction: Collect the cultured bacteria, resuspend them in lysis buffer, and use ultrasound to disrupt the cells to obtain crude enzyme solution.

[0032] 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 about 40 kDa (see Figure 1 ), with higher purity.

[0033] Example 3: Determination of enzyme activity 1. Reaction system: 0.5% (w / v) citrus pectin was used as substrate, and the enzymatic reaction was carried out at pH 4.8 and 37°C.

[0034] 2. Determination method: The reducing sugar content produced by the enzyme reaction was determined using the methanol oxidase method. The absorbance was measured at 540 nm and the enzyme activity was calculated.

[0035] 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.

[0036] Example 4: Functional analysis of mutation sites 1. Sequence alignment: Align the amino acid sequences of PME-DS12 and WT pectin methylesterase to determine the mutation site.

[0037] 2. Structural 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 ).

[0038] 3. Reasons for increased activity: Mutations cause conformational changes in the active site of the protein, which enhances the binding ability of the enzyme to the substrate and improves catalytic efficiency.

[0039] in conclusion By using the diffusion model RFdiffusion, the sequence of pectin methylesterase was redesigned and a highly active mutant PME-DS12 was successfully obtained. Compared with the wild type, the enzyme activity of PME-DS12 increased by about 6.3 times. This enzyme has important application value in various industrial fields.

[0040] Sequence Listing <110> China National Tobacco Corporation Zhengzhou Tobacco Research Institute <120> A pectin methylesterase mutant designed based on diffusion model and its preparation and application <210> 1 <211> 342 <212> PRT <213> PME-DS12 <400> 1 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. The pectin methylesterase mutant according to claim 1, characterized in that: The RFdiffusion generative diffusion model was used with the amino acid sequence and structure of the wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) as input, combined with multiple sequence alignment (MSA) analysis, keeping about 75% of the highly conserved residues unchanged, and performing sequence variation design on the remaining approximately 25% of the non-conserved residues. A highly active pectin methylesterase mutant (PME-DS12) was screened and obtained.

3. The pectin methylesterase mutant according to claim 2, characterized in that: The screening process was to use 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.

4. The pectin methylesterase mutant according to claim 1, characterized in that: Compared with the wild-type pectin methylesterase (SEQ ID NO: 1), 58 amino acid sites were replaced, and the main mutation sites are as follows (sequence position refers to the WT sequence): 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.

5. A method for preparing the pectin methylesterase mutant according to claim 1, characterized in that: The following steps are involved: The PME-DS12 gene sequence was synthesized and cloned into an expression vector; Transforming the recombinant vector into host cells for expression; Highly active PME-DS12 pectin methylesterase was obtained.

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

Images