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

Abstract

The invention provides a pectin methylesterase mutant designed based on a deep learning model as well as preparation and application of the pectin methylesterase mutant. The amino acid sequence of the pectin methylesterase mutant PME-DS3 is shown as SEQ ID NO: 2. According to the invention, a deep learning model proteinMPNN is utilized to redesign a pectin methylesterase sequence, and a mutant PME-DS3 of which the enzymatic activity is improved by more than 3 times compared with that of a wild type is obtained. Compared with a WT sequence, 61 amino acids of the PME-DS3 are replaced in total. The pectin methylesterase has wide application prospects in the fields of food industry, biomass material modification and the like.

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-DS3) designed by a deep learning model proteinMPNN, a preparation method thereof, and industrial applications thereof. Background Art

[0002] Pectin methylesterase (PME) is an important industrial enzyme that can catalyze the hydrolysis of methyl ester groups in pectin molecules and has important applications in the food industry. It can change the methylation degree of pectin, thereby affecting the gel properties of pectin and plays a key role in juice clarification, jam preparation and other processes. Improving the catalytic activity and stability of PME is of great significance for improving the efficiency of industrial applications.

[0003] Traditional enzyme engineering methods mainly rely on random mutation or structure-guided rational design, which often require a lot of experimental screening and have a low success rate. In recent years, artificial intelligence technology, especially deep learning, has made breakthrough progress in the field of protein design. ProteinMPNN, as a new protein sequence design model of graph neural network, can predict better amino acid sequence combinations based on known protein structure information. Summary of the invention

[0004] The purpose of the present invention is to provide a highly active pectin methylesterase PME-DS3, the enzyme activity of which is increased by more than 3 times compared with the wild-type pectin methylesterase (WT).

[0005] The objective of the present invention is achieved through the following technical solutions: The highly active pectin methylesterase mutant PME-DS3 of the present invention has an amino acid sequence as shown in SEQ ID NO: 2. The highly active pectin methylesterase mutant PME-DS3 was obtained by screening by using a deep learning model proteinMPNN, on the basis of multiple sequence alignment (MSA), keeping about 75% of the highly conserved residues unchanged, and performing sequence variation design on the remaining about 25% of the non-conserved residues.

[0006] The specific technical solutions are as follows: 1. Obtaining PME-DS3 mutants: Sequence design: The proteinMPNN model was used to redesign the PME sequence with the sequence and structure of wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) as input. Based on multiple sequence alignment (MSA), about 75% of the highly conserved residues were kept unchanged, and sequence variation design was performed on the remaining 25% of non-conserved residues.

[0007] Candidate sequence screening: Among the 30 variant sequences generated by the model, AlphaFold2 was used for structure prediction and quality assessment, and the mutant PME-DS3 with significantly improved activity was obtained through activity screening.

[0008] 2. The amino acid sites of PME-DS3 and WT sequences are different: WT pectin methylesterase sequence (SEQ ID NO:1):ATTYNAVVSKSSSDGKTFKTIADAIASAPAGSTPFVILIKNGVYNERLTITRNNLHLKGESRNGAVIAAATAAGTLKSDGSKWGTAGSSTITISAKDFSAQSLTIRNDFFDFPANQAKSDSDSSKIKDTQAVALYVTKSGDRAYFKDVSLVGYQDTLYVSGGRSFFSDC RISGTVDFIFFGDGTALFNNCDLVSRYRADVKSGNVSGYLTAPSTNINQKYGLVITNSRVIRESDSVPAKSYGLGRPWHPTTTFSDGRYADPNAIGQTVFLNTSMDNHIYGWDKMSGKDKNGNTIWFNPEDSRFFEYKSYGAGATVSKDRRQLTDAQAAEYTQSKVLGDWTPTLP PME-DS3 pectin methylesterase sequence (SEQ ID NO:2):GVVYNAVVSQSSTDGNTFATISAAIASAPAGDAPFVILIKNGTYNERLTITRNNLALKGESRNGAVISASTAAGTLKADGTKYGTIGSATITINAKDFAAESLTIRNDFDFPANQAKPASDSTKIKDTQAVALYVNANGDRAYFKDVALEGYQDTLYVSGGRSFFEDC VISGTVDFIFGDGTALFNNCDLVSRYRPDVPADEVLGYLTAPSTPISKKYGLVIRNSRVIRESDAVPANSYGLGRPWHPTTTFPDGRYADPNAIGQTVFLNTYMDNHIYGWDKMSGKDKNGNEIWFKPEDSRFFEYNSYGAGAPTGSDRRQLTDEEAKEYTEENVLGDYTPTLP Differential amino acid position information: By comparing the amino acid sequences of WT and PME-DS3, it was found that 61 amino acid sites in PME-DS3 were replaced. The main mutation sites are listed below (sequence positions refer to the WT sequence): Mutation site list: A1G,T2V,T3V,K10Q,S13T,K16N,K19A,A22S,D23A,S32D,T33A,V43T,H56A,A68S,A70S,S78A,S81T,W83 Y,A86I,S89A,S94N,S99A,Q101E,S118P,D119A,S123T,T136N,K137A,S138N,S148A,V150E,S166E,R16 9V,A196P,K199P,S200A,G201D,N202E,S204L,N213P,N215S,Q216K,T223R,S233A,K237N,S252P,S271 Y,T291E,N295K,K305N,T312P,V313T,S314G,K315S,A323E,Q324E,A326K,Q330E,S331E,K332N,W337Y 3. Preparation of PME-DS3: Gene synthesis: According to the amino acid sequence of PME-DS3, the corresponding gene sequence was synthesized by reverse translation and codon optimization. The codon usage preference of Pichia pastoris was taken into consideration during the optimization process to improve the expression efficiency of the gene.

[0009] Vector construction: The synthesized PME-DS3 gene sequence was cloned into the Pichia pastoris expression vector pPICZαA. EcoRI and XbaI restriction endonuclease sites were used for restriction digestion and ligation to construct the recombinant expression vector pPICZαA-PME-DS3.

[0010] Expression and purification: The recombinant plasmid was transformed into Pichia pastoris GS115. Expression was performed under methanol induction conditions. After the fermentation supernatant was collected, it was initially purified by centrifugation, ultrafiltration, etc., and then further purified by cation exchange chromatography to obtain high-purity PME-DS3 protein.

[0011] Enzyme activity determination: The enzyme activity of PME-DS3 and WT pectin methylesterase was determined by methanol oxidase method. The results showed that the enzyme activity of PME-DS3 was more than 3 times that of WT.

[0012] Applications of PME-DS3: As a highly active pectin methylesterase, PME-DS3 can be widely used in the following fields: Food Industry: 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.

[0013] Juice clarification: Improve the clarity and stability of juice.

[0014] Jam preparation: Improves the texture and gelling properties of jam.

[0015] Fruit and vegetable processing: improving processing efficiency and product quality.

[0016] Modification of pectin-based materials: Regulating the methylation degree of pectin to prepare biomaterials with specific properties.

[0017] Modification of plant cellulose materials: improving the performance and application range of materials.

[0018] The innovation of the present invention is to use the deep learning model proteinMPNN to redesign the pectin methylesterase sequence and obtain a mutant PME-DS3 whose enzyme activity is more than 3 times higher than that of the wild type. Compared with the WT sequence, a total of 61 amino acids in PME-DS3 have been replaced. The pectin methylesterase has broad application prospects in the fields of food industry and biomaterial modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : SDS-PAGE electrophoresis of WT and PME-DS3 purified proteins to evaluate the purity and molecular weight of the proteins.

[0020] Figure 2 : Enzyme activity comparison diagram, showing that the catalytic activity of PME-DS3 is significantly higher than that of WT (wild type).

[0021] Figure 3 : Comparison of the structural models of WT and PME-DS3 (A: WT structure; B: PME-DS3 structure; C: structural overlay), showing the impact of key mutation sites on the enzyme structure. DETAILED DESCRIPTION

[0022] The following examples are used to further illustrate the present invention but are not intended to limit the scope of the present invention.

[0023] Example 1: Synthesis and cloning of PME-DS3 gene 1. Gene synthesis: Based on the amino acid sequence of PME-DS3 (SEQ ID NO: 2), the PME-DS3 gene sequence was designed and synthesized using reverse translation software and combined with the codon usage preference of Pichia pastoris.

[0024] 2. Vector construction: The synthesized PME-DS3 gene sequence and the pPICZαA vector were double-digested with EcoRI and XbaI, respectively. The digested products were connected with T4 DNA ligase to construct the recombinant expression vector pPICZαA-PME-DS3.

[0025] 3. Screening of positive clones: Transform the ligation product into E. coli DH5α competent cells and spread them on LB plates containing Zeocin for screening. Pick single clones, extract plasmids, identify by PCR and double enzyme digestion, and screen positive clones.

[0026] 4. Sequencing verification: Send the recombinant plasmid of the positive clone to a sequencing company for sequencing to verify the correctness of the PME-DS3 gene sequence.

[0027] Example 2: Expression and purification of PME-DS3 1. Transformation of Pichia pastoris: Linearize the recombinant plasmid pPICZαA-PME-DS3 verified by sequencing, and then electrotransform Pichia pastoris GS115 competent cells. Spread the transformed cells on a YPDS plate containing Zeocin for screening.

[0028] 2. Induce expression: Pick a single clone, inoculate it in BMGY medium, and culture it at 30°C, 250 rpm until OD600 reaches 2-6. Collect the bacteria by centrifugation, resuspend them in BMMY medium, add 0.5% methanol to induce expression, add methanol every 24 hours, and continue induction for 72 hours.

[0029] 3. Protein purification: Collect the fermentation supernatant and centrifuge at 4°C, 12,000 rpm for 20 minutes to remove cell debris. Concentrate the supernatant by ultrafiltration and then purify it using a cation exchange column. Collect the target protein peak and perform SDS-PAGE analysis to verify the protein purity and molecular weight (see Figure 1 ).

[0030] Example 3: Enzyme activity assay 1. Enzyme activity determination method: The methanol oxidase method was used to determine the activity of PME-DS3 and WT pectin methylesterase. 0.5% citrus pectin was used as substrate and the reaction was carried out at 37°C and pH 4.8.

[0031] 2. Result analysis: The results showed that the enzyme activity of PME-DS3 was more than 3 times that of WT (see Figure 2 ), indicating that the mutants designed using proteinMPNN significantly improved the catalytic efficiency of the enzyme.

[0032] Example 4: Functional analysis of mutation sites 1. Sequence alignment: Align the amino acid sequences of PME-DS3 and WT pectinase to determine the site of mutation.

[0033] 2. Structural prediction: AlphaFold2 was used to predict the three-dimensional structure of PME-DS3, and it was found that the mutation sites were mainly concentrated in the region related to substrate binding (see Figure 3 ).

[0034] 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. Conclusion: This paper successfully obtained a highly active pectin methylesterase mutant PME-DS3 through deep learning-assisted design. The enzyme has higher catalytic activity and better stability, and has important application value in the food industry and biomaterials. The experimental results show that PME-DS3 significantly improves the catalytic efficiency while maintaining the original substrate specificity, providing a better choice for industrial applications.

[0036] Sequence Listing <110> China National Tobacco Corporation Zhengzhou Tobacco Research Institute <120> A pectin methylesterase mutant designed based on deep learning model and its preparation and application <210> 1 <211> 342 <212> PRT <213> Pectin methylesterase mutant PME-DS3 <400> 1 GVVYNAVVSQSSTDGNTFATISAAIASAPAGDAPFVILIKNGTYNERLTITRNNLALKGESRNGAVISASTAAGTLKADGTKYGTIGSATITINAKDFAAESLTIRNDFDFPANQAKPASDSTKIKDTQAVALYVNANGDRAYFKDVALEGYQDTLYVSGGRSFFEDCVIS GTVDFIFFGDGTALFNNCDLVSRYRPDVPADEVLGYLTAPSTPISKKYGLVIRNSRVIRESDAVPANSYGLGRPWHPTTTFPDGRYADPNAIGQTVFLNTYMDNHIYGWDKMSGKDKNGNEIWFKPEDSRFFEYNSYGAGAPTGSDRRQLTDEEAKEYTEENVLGDYTPTLP.

Claims

1. A pectin methylesterase mutant designed based on a deep learning 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 proteinMPNN model was used to redesign the sequence and structure of wild-type pectin methylesterase (UniProtKB: P0C1A8 / PDB ID: 1QJV) as input. Based on multiple sequence alignment (MSA), about 75% of the highly conserved residues were kept unchanged, and sequence variations were designed for the remaining 25% of non-conserved residues. The highly active pectin methylesterase mutant PME-DS3 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-DS3 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), 61 amino acid sites are replaced, and the main mutation sites are listed below (sequence position refers to the WT sequence): A1G,T2V,T3V,K10Q,S13T,K16N,K19A,A22S,D23A,S32D,T33A,V43T,H56A,A68S,A70S,S78A,S81T,W83 Y,A86I,S89A,S94N,S99A,Q101E,S118P,D119A,S123T,T136N,K137A,S138N,S148A,V150E,S166E,R16 9V,A196P,K199P,S200A,G201D,N202E,S204L,N213P,N215S,Q216K,T223R,S233A,K237N,S252P,S271 Y, T291E, N295K, K305N, T312P, V313T, S314G, K315S, A323E, Q324E, A326K, Q330E, S331E, K332N, W337Y.

5. A method for preparing the pectin methylesterase mutant according to claim 1, characterized in that: The following steps are involved: 1) Synthesize the PME-DS3 gene sequence and clone it into the expression vector; 2) Transform the recombinant vector into host cells for expression; 3) Obtain highly active PME-DS3 pectin methylesterase.

6. Use of the pectin methylesterase mutant according to claim 1 in pectin demethylation, juice clarification, jam preparation or biomass material modification.

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