High-activity pectinase mutant DS-4 designed based on deep learning model and its preparation and application

Abstract

The present invention provides a highly active pectinase mutant DS-4 designed based on a deep learning model, as well as its preparation and application. The amino acid sequence of the pectinase mutant DS-4 is shown in SEQ ID NO: 2. Using the deep learning model ProteinMPNN, the pectinase sequence was redesigned to obtain a mutant DS-4 with an enzyme activity more than 2 times higher than that of the wild type. Compared with the WT sequence, DS-4 has a total of 70 amino acid substitutions. This pectinase has broad application prospects in industrial fields such as food processing, textiles, papermaking, and bioenergy.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and enzyme engineering, and in particular to a highly active pectinase DS-4 designed by a deep learning model ProteinMPNN, a preparation method thereof, and industrial applications thereof. Background Art

[0002] Pectinase is a type of enzyme that hydrolyzes pectin and has important applications in food processing, textiles, papermaking, bioenergy, and other industries. Improving the activity and stability of pectinase is of great significance for industrial production.

[0003] Traditional enzyme engineering methods, such as directed evolution and rational design, have achieved some success in improving enzyme performance. However, they suffer from limitations such as time-consuming design, low efficiency, and limited sequence space exploration. With the development of deep learning technology, the use of deep learning models for protein design and reconstruction has become an emerging trend. ProteinMPNN is a deep learning-based protein sequence design model that can generate sequences with optimized functionality while maintaining the protein's tertiary structure. Summary of the Invention

[0004] The purpose of the present invention is to provide a high-activity pectinase DS-4, the enzyme activity of which is 2 times higher than that of wild-type pectinase (WT).

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] The highly active pectinase mutant DS-4 of the present invention has an amino acid sequence as shown in SEQ ID NO: 2. The highly active pectinase mutant DS-4 was obtained by screening using the deep learning model ProteinMPNN based on multiple sequence alignment (MSA), keeping approximately 70% of the highly conserved residues unchanged and performing sequence variation design on the remaining approximately 30% of non-conserved residues.

[0007] The specific technical solutions are as follows:

[0008] 1. Acquisition of DS-4 mutants:

[0009] Sequence Design: The protein MPNN model was used to redesign the pectinase sequence using the crystal structure of the wild-type pectinase (PDB ID: 3VMW). Based on a multiple sequence alignment (MSA), approximately 70% of the highly conserved residues were kept unchanged, while sequence variations were designed for the remaining approximately 30% of non-conserved residues.

[0010] Candidate sequence screening: Among the 20 variant sequences generated by the model, AlphaFold2 was used for structure prediction and quality assessment, and the mutant DS-4 with significantly improved activity was obtained through activity screening.

[0011] 2. Differences in amino acid positions between DS-4 and WT sequences:

[0012] WT pectinase sequence (SEQ ID NO: 1):

[0013] SNGPQGYASMNGGTTGGAGGRVEYASTGAQIQQLIDNRSRSNNPDEPLTIYVNGTITQGNSPQSLIDVKNHRGKAHEIKNISIIGVGTNGEFDGIGIRLSNAHNIIIQNVSIHHVREGEGTAIEVTDDSKNVWIDHNEFYSEFPGNGDSDYYDGLVDMKRNAE YITVSWNKFENHWKTMLVGHTDNASLAPDKITYHHNYFNNLNSRVPLIRYADVHMFNNYFKDINDTAINSRVGARVFVENNYFDNVGSGQADPTTGFIKGPVGWFYGSPSTGYWNLRGNVFVNTPNSHLNSTTNFTPPYSYQVQSATQAKSSVEQHSGVGVIN

[0014] DS-4 pectinase sequence (SEQ ID NO: 2):

[0015] GPQGYASLNGGTTGGSGGETATASTGKEIQDLIDTRSKSSNPNTPLTIYVNGTITPANSDTPLIDVKNHRGSEYPITNISIIGVGTNGEFDGIGIRLSNAHNIIIQNVTIHHVKEGTGTAIEVTDNSKNVWIDHNEFYSDYPGNGDSDYYDGLVDFKRNAEY ITVSWNKFENHWKAMLVGHTDNEALAPDNITYHHNLFKNLNSRVPLIRYANVHMFNNYFEDIKDTAINSRVGARVWVENNYFDNVGSGKKDPVTGTVRGPVGHYYGSSSPGYWNLEGNVFNNTPHDGLKSTTDFKPPYSYTVLSAEEAKTYVLKYSGVGVVT

[0016] Differential amino acid site information:

[0017] Comparison of the amino acid sequences of WT and DS-4 revealed that 70 amino acid substitutions occurred in DS-4. The main mutation sites are listed below (sequence positions refer to the WT sequence):

[0018] Mutation site list:

[0019] M8L, A16S, R19E, V20T, E21A, Y22T, A27K, Q28E, Q31D, N35T, R38K, N40S, D43N, E44T, Q56P, G57A, P60D, Q61T, S62P, K72S , A73E, H74Y, E75P, K77T, S109T, R114K, E117T, D126N, E140D, F141Y, M156F, T176A, A185E, S186A, K191N, Y198L, N200K, D213N, K222E, N225K, F238W, Q251K, A252K, T255V, F258T, I259V, K260R, W265H, F266Y, P270S, T272P, R278E, V283N, N2 87H, S288D, H289G, N291K, N295D, T297K, Q303T, Q305L, T308E, Q309E, S312T, S313Y, E315L, Q316K, H317Y, I323V, N324T

[0020] 3. Preparation method of DS-4:

[0021] Gene synthesis: Based on the amino acid sequence of DS-4, 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.

[0022] Vector construction: The synthesized DS-4 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(+)-DS-4.

[0023] Expression and Purification: The recombinant plasmid was transformed into Escherichia coli BL21(DE3). Expression was induced using IPTG under appropriate culture conditions. After harvesting the cells, the crude enzyme solution was obtained by ultrasonication and centrifugation. The DS-4 protein was purified using a Ni-NTA affinity chromatography column based on the 6×His tag.

[0024] Enzyme activity assay: The DNS method was used to determine the enzymatic activity of DS-4 and WT pectinase. The results showed that the enzymatic activity of DS-4 was more than twice that of WT.

[0025] Applications of DS-4:

[0026] As a highly active pectinase, DS-4 can be widely used in the following fields:

[0027] Food processing: used for juice clarification, jam production, etc. to improve product quality and production efficiency.

[0028] Textile industry: used in bio-enzyme desizing and bio-polishing to improve the quality of textiles.

[0029] Papermaking industry: used for biological bleaching of pulp, reducing the use of chemical reagents and reducing environmental pollution.

[0030] Bioenergy: Participate in biomass degradation, improve raw material conversion efficiency, and promote bioenergy production.

[0031] A key feature of this invention is the redesign of the pectinase sequence using the deep learning model ProteinMPNN, resulting in the generation of a mutant, DS-4, whose enzyme activity is more than two-fold higher than that of the wild-type. Compared to the wild-type sequence, DS-4 undergoes 70 amino acid substitutions. This pectinase has broad application prospects in industries such as food processing, textiles, papermaking, and bioenergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : The results of enzyme activity assay showed that the catalytic activity of DS-4 was significantly higher than that of WT (wild type).

[0033] Figure 2 : SDS-PAGE electrophoresis of WT and DS-4 purified proteins to evaluate the purity and molecular weight of the proteins.

[0034] Figure 3 : Electrostatic surface potential maps of WT and DS-4 proteins (WT on the left, DS-4 on the right). The black dotted circle outlines the potential substrate binding pocket, showing that DS-4 has an increased positive potential in this area. DETAILED DESCRIPTION

[0035] The present invention is described in further detail below in conjunction with the embodiments (attached drawings):

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials used are commercially available.

[0037] Example 1: Construction of DS-4 gene

[0038] Based on the amino acid sequence of DS-4, the corresponding nucleotide sequence was obtained using reverse translation combined with codon optimization in E. coli. The optimized DS-4 gene sequence was synthesized and cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant expression vector pET-28a(+)-DS-4.

[0039] 1. Enzyme digestion and ligation: Use NcoI and XhoI to digest the pET-28a(+) vector and DS-4 gene fragment.

[0040] 2. Ligation reaction: The DS-4 gene fragment after enzyme digestion is connected to the linearized vector under the action of T4 DNA ligase.

[0041] 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.

[0042] 4. Identification of positive clones: Select single colonies for bacterial liquid PCR and enzyme digestion identification to screen out the correct recombinant plasmid.

[0043] 5. Sequencing verification: Send the recombinant plasmid to a sequencing company for sequence determination to ensure the accuracy of the DS-4 gene sequence.

[0044] Example 2: Expression and purification of DS-4

[0045] 1. Strain transformation and culture: The pET-28a(+)-DS-4 recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells.

[0046] 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 0.5mM, lower the temperature to 16℃, and continue to culture for 12-16 hours to induce protein expression.

[0047] 3. Protein extraction: Collect the cultured bacteria, resuspend them in lysis buffer, and use ultrasonication to disrupt the cells to obtain crude enzyme solution.

[0048] 4. Protein purification: DS-4 protein was purified by Ni-NTA affinity chromatography based on the His tag. The purified protein was analyzed by SDS-PAGE and showed a clear band at about 40 kDa (see Figure 2 ), with higher purity.

[0049] Example 3: Determination of enzyme activity

[0050] 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.

[0051] 2. Determination method: Use the DNS method to determine the reducing sugar content produced by the enzyme reaction. Measure the absorbance at 540 nm and calculate the enzyme activity.

[0052] 3. Result analysis: The enzyme activity of DS-4 is more than 2 times that of WT (see Figure 1 ), indicating that the mutants designed by ProteinMPNN significantly improved the catalytic efficiency of the enzyme.

[0053] Example 4: Functional analysis of mutation sites

[0054] 1. Sequence alignment: Align the amino acid sequences of DS-4 and WT pectinase to determine the site of mutation.

[0055] 2. Structure prediction: AlphaFold2 was used to predict the three-dimensional structure of DS-4, and it was found that the mutation sites were mainly concentrated in the region related to substrate binding (see Figure 3 ).

[0056] 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.

[0057] in conclusion

[0058] By utilizing the deep learning model ProteinMPNN, we successfully redesigned the pectinase sequence and generated a highly active mutant, DS-4. Compared to the wild-type, DS-4 exhibits over 2-fold increased enzymatic activity. This enzyme has important applications in a variety of industrial fields.

Claims

1. A highly active pectinase mutant DS-4 designed based on a deep learning model, characterized by: Its amino acid sequence is shown in SEQ ID NO:

2.

2. The highly active pectinase mutant DS-4 according to claim 1, characterized in that: The protein MPNN model was used to redesign the pectinase sequence using the crystal structure of the wild-type pectinase (PDB ID: 3VMW) as input. Based on multiple sequence alignment, approximately 70% of the highly conserved residues were kept unchanged, and sequence variations were designed for the remaining approximately 30% of non-conserved residues. This resulted in the screening of the highly active pectinase mutant DS-4.

3. The highly active pectinase mutant DS-4 according to claim 2, characterized in that: The screening process involved using AlphaFold2 to perform structure prediction and quality assessment on multiple variant sequences generated by the model, and obtaining the mutant DS-4 with significantly improved activity through activity screening.

4. A method for preparing the pectinase mutant DS-4 according to claim 1, characterized in that: The following steps are involved: 1) Synthesize the DS-4 gene sequence and clone it into an expression vector; 2) Transforming the recombinant vector into host cells for expression; 3) Highly active DS-4 pectinase was obtained.

5. Use of the pectinase mutant DS-4 according to claim 1 in the preparation of juice and jam.

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