Pectate lyase mutant based on diffusion model design as well as preparation and application thereof

Abstract

The invention provides a pectin lyase mutant designed based on a diffusion model as well as preparation and application of the pectin lyase mutant. The amino acid sequence of the pectin lyase mutant PL-DS11 is shown as SEQ ID NO: 2. The method comprises the following steps: redesigning a pectin lyase sequence by using a diffusion model RFdiffusion, and screening to obtain a mutant PL-DS11 of which the enzyme activity is remarkably improved in combination with multi-sequence alignment (MSA) analysis. Compared with wild pectate lyase, the enzyme activity of the PL-DS11 is improved by about 4.3 times, and the pectate lyase has higher industrial application value. The mutant has wide application prospects in the fields of food processing, feed processing, textile industry, papermaking and the like.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and enzyme engineering, and in particular to a high-activity pectin lyase (PL-DS11) designed by a generative diffusion model RFdiffusion, a preparation method thereof, and industrial applications thereof. Background Art

[0002] Pectin lyase (PL) is a type of pectinase that catalyzes the elimination and cleavage of pectin molecular chains. It is of great significance in the food processing industry to increase the yield of fruit juice. Pectin lyase (trans-eliminase) is a pectinase that cleaves pectin polymers by trans-elimination. The lyase breaks the glycosidic bond at the C-4 position and eliminates a H atom from the C-5 position to produce an unsaturated product. Pectin lyase is a type of enzyme that can catalyze the degradation of pectin substances. It has important application value in food processing, feed processing, textile, papermaking and other industrial fields. Improving the activity and stability of pectin lyase 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 diffusion generation 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 highly active pectin lyase PL-DS11, the enzyme activity of which is increased by about 4.3 times compared with the wild-type pectin lyase (WT).

[0005] To achieve the above object, the present invention provides the following technical solutions: A pectin lyase mutant PL-DS11 designed based on the diffusion model RFdiffusion, whose amino acid sequence is shown in SEQ ID NO: 2. The RFdiffusion model was used to take the sequence and structure of the wild-type pectin lyase (UniProtKB: D0VP31 / PDB ID: 3VMV) 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 about 25% of the non-conserved residues, and the highly active pectin lyase mutant PL-DS11 was screened and obtained.

[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 PL-DS11 with significantly improved activity was obtained.

[0007] The method for preparing the pectin lyase mutant PL-DS11 of the present invention comprises the following steps: i. Synthesize the PL-DS11 gene sequence and clone it into an expression vector; ii. transforming the recombinant vector into a host cell for expression; iii. Obtain highly active PL-DS11 pectin lyase.

[0008] The specific technical solutions are as follows: 1. Obtaining PL-DS11 mutants Sequence design: Using the RFdiffusion model, the sequence and structure of wild-type pectin lyase (UniProtKB: D0VP31 / PDB ID: 3VMV) 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 for a mutant PL-DS11 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, enzyme activity screening was performed to identify the mutant PL-DS11 with significantly improved activity.

[0010] 2. Differences in amino acid sites between PL-DS11 and WT sequences WT pectin lyase sequence (SEQ ID NO:1):GPQGYASMNGGTTGGAGGRVEYASTGAQIQQLIDNRSRSNNPDEPLTIYVNGTITQGNSPQSLIDVKNHRGKAHEIKNISIIGVGTNGEFDGIGIRLSNAHNIIIQNVSIHHVREGEGTAIEVTDDSKNVWIDHNEFYSEFPGNGDSDYYDGLVDMKRN AEYITVSWNKFENHWKTMLVGHTDNASLAPDKITYHHNYFNNLNSRVPLIRYADVHMFNNYFKDINDTAINSRVGARVFVENNYFDNVGSGQADPTTGFIKGPVGWFYGSPSTGYWNLRGNVFVNTPNSHLNSTTNFTPPYSYQVQSATQAKSSVEQHSGVGVIN PL-DS11 pectin lyase sequence (SEQ ID NO:2):TVTGYASLNGGTTGGSGGKTVTATTGKEIQQLIKDRCLSGKTDEPLTIYVNGTITPENSDSGLIDVKNHRTKTAPIKNISIIGVGTKGEFDGIGIRLTSAHNIIIQNVKIHHVKEESGNGIEVTDGSKNVWIDHNEFYSGETLPDDSDYYDGLVDFKRN AEYITVSWNKFENHWKAMLVGHTDNLELRSDNITYHHNLFSNLNSRVPLIRYANVHMFNNYFKDITDTAINSRVGARVYVENNYFDNVGSKEKDKETGKVGTPVGNFYESSENGYWNLSGNIFNNTNTENLGSTTNFTPPYSYDVLDAETAKEKVLKESGVGNNL Differential amino acid position information: By comparing the amino acid sequences of WT and PL-DS11, it was found that 91 amino acid sites of PL-DS11 were replaced. The main mutation sites are listed below (sequence positions refer to the WT sequence): Mutation site list: G1T,P2V,Q3T,M8L,A16S,R19K,V20T,E21V,Y22T,S24T,A27K,Q28E,D34K,N3 5D,S37C,R38L,N40G,N41K,P42T,Q56P,G57E,P60D,Q61S,S62G,G71T,A73T,H 74A,E75P,N87K,S98T,N99S,S109K,R114K,G116E,E117S,T119N,A120G,D126 G,E140G,F141E,P142T,G143L,N144P,G145D,M156F,T176A,A185L,S186E,A1 88R,P189S,K191N,Y198L,N200S,D213N,N225T,F238Y,G250K,Q251E,A252K, P254K,T255E,F258K,I259V,K260G,G261T,W265N,G268E,P270S,S271E,T272 N,R278S,V281I,V283N,P286N,N287T,S288E,H289N,N291G,Q303D,Q305L,S3 06D, T308E, Q309T, S312E, S313K, E315L, Q316K, H317E, V322N, I323N, N324L.

[0011] 3. Preparation of PL-DS11 Gene synthesis: Based on the amino acid sequence of PL-DS11, 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 PL-DS11 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(+)-PL-DS11.

[0013] Expression and purification: The recombinant plasmid was transformed into Escherichia 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 PL-DS11 protein was purified using a Ni-NTA affinity chromatography column based on the 6×His tag.

[0014] Enzyme activity determination: The DNS method was used to determine the enzyme activity of PL-DS11 and WT pectin lyase. The results showed that the enzyme activity of PL-DS11 was more than 4.3 times that of WT.

[0015] 4. Application of PL-DS11 As a highly active pectin lyase, PL-DS11 can be widely used in the following fields: Food processing: used for juice clarification, jam production, etc. to improve product quality and production efficiency.

[0016] Feed processing: Since plant raw materials contain pectin, it is an anti-nutritional factor that affects the utilization rate of feed. Adding pectinase to feed can effectively degrade pectin and effectively promote the absorption of nutrients, thereby improving the utilization rate of feed.

[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] The biggest feature of the present invention is that the pectin lyase sequence is redesigned by using the diffusion model RFdiffusion, and the mutant PL-DS11 is obtained, whose enzyme activity is about 4.3 times higher than that of the wild type. Compared with the WT sequence, PL-DS11 has a total of 91 amino acids replaced. The pectin lyase has broad application prospects in industrial fields such as food processing, feed processing, textiles and papermaking. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : SDS-PAGE electrophoresis of WT and PL-DS11 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 PL-DS11 was significantly higher than that of WT (wild type).

[0022] Figure 3 : Electrostatic surface potential maps of WT and PL-DS11 proteins (WT on the left, PL-DS11 on the right). The black dotted circle outlines the potential substrate binding pocket, showing that PL-DS11 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 PL-DS11 gene According to the amino acid sequence of PL-DS11, the corresponding nucleotide sequence was obtained by reverse translation combined with codon optimization of Escherichia coli. The optimized PL-DS11 gene sequence was synthesized and cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant expression vector pET-28a(+)-PL-DS11.

[0024] 1. Enzyme digestion and ligation: Use NcoI and XhoI to digest the pET-28a(+) vector and PL-DS11 gene fragment.

[0025] 2. Ligation reaction: The PL-DS11 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 PL-DS11 gene sequence.

[0029] Example 2: Expression and purification of PL-DS11 1. Strain transformation and culture: Transform the pET-28a(+)-PL-DS11 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: PL-DS11 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: Use DNS method to determine the reducing sugar content produced by enzyme reaction. Measure the absorbance at 540 nm and calculate the enzyme activity.

[0035] 3. Result analysis: The enzyme activity of PL-DS11 is 4.3 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 PL-DS11 and WT pectin lyase to determine the site of mutation.

[0037] 2. Structural prediction: The three-dimensional structure of PL-DS11 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 lyase was redesigned and a highly active mutant PL-DS11 was successfully obtained. Compared with the wild type, the enzyme activity of PL-DS11 increased by about 4.3 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 lyase mutant designed based on diffusion model and its preparation and application <210> 1 <211> 324 <212> PRT <213> Pectin lyase mutant PL-DS11 <400> 1 TVTGYASLNGGTTGGSGGKTVTATTGKEIQQLIKDRCLSGKTDEPLTIYVNGTITPENSDSGLIDVKNHRTKTAPIKNISIIGVGTKGEFDGIGIRLTSAHNIIIQNVKIHHVKEESGNGIEVTDGSKNVWIDHNEFYSGETLPDDSDYYDGLVDFKRNAEYITVSWNKFENHWKAMLVGHTDNLELRSDNITYHHNLFSNLNSRVPLIRYANVHMFNNYFKDITDTAINSRVGARVYVENNYFDNVGSKEKDKETGKVGTPVGNFYESSENGYWNLSGNIFNNTNTENLGSTTNFTPPYSYDVLDAETAKEKVLKESGVGNNL

Claims

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

2.

2. The pectin lyase mutant according to claim 1, characterized in that: The RFdiffusion generative diffusion model was used with the sequence and structure of the wild-type pectin lyase (UniProtKB: D0VP31 / PDB ID: 3VMV) 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 non-conserved residues, and a highly active pectin lyase mutant (PL-DS11) was screened and obtained.

3. The pectin lyase 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 PL-DS11 with significantly improved activity was obtained.

4. The pectin lyase mutant according to claim 1, characterized in that: Compared with the wild-type pectin lyase (SEQ ID NO: 1), 91 amino acid sites are replaced, and the main mutation sites are as follows (sequence position refers to the WT sequence): G1T,P2V,Q3T,M8L,A16S,R19K,V20T,E21V,Y22T,S24T,A27K,Q28E,D34K,N3 5D,S37C,R38L,N40G,N41K,P42T,Q56P,G57E,P60D,Q61S,S62G,G71T,A73T,H 74A,E75P,N87K,S98T,N99S,S109K,R114K,G116E,E117S,T119N,A120G,D126 G,E140G,F141E,P142T,G143L,N144P,G145D,M156F,T176A,A185L,S186E,A1 88R,P189S,K191N,Y198L,N200S,D213N,N225T,F238Y,G250K,Q251E,A252K, P254K,T255E,F258K,I259V,K260G,G261T,W265N,G268E,P270S,S271E,T272 N,R278S,V281I,V283N,P286N,N287T,S288E,H289N,N291G,Q303D,Q305L,S3 06D, T308E, Q309T, S312E, S313K, E315L, Q316K, H317E, V322N, I323N, N324L.

5. A method for preparing the pectin lyase mutant according to claim 1, characterized in that: The following steps are involved: The PL-DS11 gene sequence was synthesized and cloned into an expression vector; Transforming the recombinant vector into host cells for expression; Obtain high-activity PL-DS11 pectin lyase.

6. Use of the pectin lyase mutant according to claim 1 in pectin degradation, juice clarification, jam preparation, feed processing, textile processing and papermaking industry.

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