Improved Penicillium pectate lyase

Abstract

The improved Penicillium pectin lyase was sequenced and optimized through molecular design and simulation, allowing for the screening of a thermostable pectin lyase sequence. This optimization expands the pectin lyase's operating conditions, increasing its optimal pH by 1 and its optimal reaction temperature by 15°C compared to wild Penicillium pectin lyase.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to sequence optimization, expression and application of Penicillium pectin lyase. Background Art

[0002] Pectin is a high-molecular-weight polymer widely found in higher plants, bonding the intercellular matrix and primary cell walls and supporting plant structure. Galacturonic acid, linked by α-1,4-glycosidic bonds, forms the pectin backbone. Pectin is partially methylated, and xylan and arabinogalactan are modified with side chains. Pectin is typically degraded using pectinases, collectively known as pectinases. These enzymes include pectate lyase (PEL, EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), pectate lyase (EC 4.2.2.2), exopolygalacturonase (EC 3.2.1.67), exopolygalacturonate lyase (EC 4.2.2.9), and exopoly-α-galacturonidase (EC 3.2.1.82) (Kanchan Yadav et al., 2023). Pectin lyase is an important component of pectinase. According to the optimal pH of the enzyme reaction, it can be divided into acidic pectin lyase and alkaline pectin lyase. Pectin lyase plays an important role in the degradation of pectin. Pectin lyase removes 6-methyl-D-4-5-galacturonic acid residues from pectin. It catalyzes the elimination and cleavage of pectin molecular chains, producing 4-(deoxy-beta-D-gluc-4-enuronosyl)-D-galacturonic acid residues (4-Deoxybeta-D-gluc-4-enuronosyl) and pectic acid with reduced molecular weight at its non-reducing end (http: / / www.kegg.jp, Reaction: R08694 and R02361). Pectin lyase breaks the α-1,4 glycosidic bond of pectin polymers through a trans-elimination reaction to form Δ4,5-unsaturated oligogalacturonic acid products. It is the only enzyme among pectinases that can degrade highly esterified pectin without producing methanol (RituSaharan and KantiPrakashSharma, 2019).

[0003] Pectin lyase is a commonly used enzyme catalyst with significant application and economic value in industries such as food, medicine, textiles, agriculture, feed, and waste recycling. Pectin lyase (EC 4.2.2.10), systematically designated (1→4)-6-O-methyl-α-D-galacturonan lyase, removes 6-methyl-Δ-4-5-galacturonan residues from pectin. This enzyme hydrolyzes the α-1-4 glycosidic bonds of highly esterified pectins without the need for pre-action by other enzymes. In juice production, the pulp after pressing is very viscous due to the presence of pectin and cellulose, which reduces juice yield and clarity. Adding pectinase during the production process can reduce pulp viscosity, increase juice yield, improve the sensory experience, and retain more nutrients from the fruit. Pectin lyase derived from Penicillium expansum F16, when added to apple and grape juice, can increase light transmittance and effectively clarify the juice (Cristina F. Mantovani, 2005). In animal feed production, many non-starch polysaccharides (NSPs) are present in animal feed. These NSPs affect the activity of gastrointestinal digestive enzymes, hindering nutrient absorption and utilization. Most animals lack the enzymes required to degrade NSPs, resulting in poor appetite. Adding pectin lyase to feed can improve digestion and absorption (Hoondal G, 2002). In the pulping and papermaking process, the use of pectinase to degrade polygalacturonic acid into galacturonic acid or monomers with a lower degree of polymerization can significantly reduce the cation requirement of white water, from 431 μeq / L to 248 μeq / L (Thornton JW, 1994). In textile production, the traditional treatment method is alkali degumming. This process produces wastewater with high COD (Chemical Oxygen Demand), high BOD (Biochemical Oxygen Demand), high pH, and high salinity, resulting in high energy consumption and severe pollution. Modern processes use alkaline pectinase to degumming cotton and linen textiles. The resulting fibers are highly wettable, clean, have a high crystallinity index, are easily dyed, and have a good feel. This method surpasses alkaline treatment while avoiding the drawbacks of traditional processes (Tzanov T, 2001). Pectin and other biomacromolecules in tobacco leaves produce various harmful components and pungent odors during cigarette smoking. Enzymes such as pectin lyase catalyze the hydrolysis of pectin and other substances to produce flavor compounds or their precursors, altering the chemical composition of the tobacco, enhancing the aroma, reducing odor and irritation, reducing harmful effects, and improving cigarette quality (Zhu Xiaolan, 2015).

[0004] In industrial applications, approximately 50% of pectate lyases are derived from fungi, 35% from bacteria, and the remaining 15% from animals or plants (Demir N et al., 2014). The industrial production of pectate lyases currently relies primarily on microbial fermentation. The microorganisms used primarily include Penicillum, Rhizopus, Aspergillus, Fusarium, Kluyvermyoes, and Geotrichum. Commonly used are Aspergillus fungi, including Aspergillus niger, Aspergillus flavipes, Aspergillus nidμLans, Aspergillus sydowii, and Aspergillus ustus. Pectate lyase-producing bacteria primarily include Xanthomonas, Bacillus, Ervinia, Pseudomaonas, and Clostridium. However, current production strains exhibit low pectate lyase expression levels, resulting in high enzyme production costs. Therefore, research in this field is focused on modifying pectate lyase-producing strains to improve enzyme expression efficiency and promote the development of the enzyme industry.

[0005] Through bioinformatics analysis and experimental verification, we discovered a series of P. expansum effector factors, most of which are hydrolases, including glycosidic bond hydrolases, ester hydrolases, and pectin lyases (Li Guangwei, 2019). Among them, pectin lyase (pel, EC: 4.2.2.10) is a key effector of plant pathogens. During the process of pathogen infection of fruit hosts, it degrades the host cell wall, thereby regulating the physiological processes of host cells to facilitate better infection and growth, or activates the host immune response by promoting cell necrosis, providing nutrition for saprotrophic pathogens. Therefore, pectin lyase plays an important role in the process of pathogen infection of plants (Li, W. et al., 2024).

[0006] Currently, most pectin lyases are room-temperature enzymes. Improving the thermal stability of pectin lyase in industry can reduce the number of enzyme additions and the amount of enzyme used. By replacing the amino acid sequence of the first 17, first 31, first 58, and 34 to 58 positions of the N-terminus of the pectin lyase from alkalophilic Bacillus, the mutant significantly improved the enzyme activity while maintaining the optimal pH and optimal temperature at 11.0 and 70°C, reaching a specific activity of 160,000 U / mg, a 1.24-fold increase over the original enzyme. Based on the obtained mutant, three groups of mutation sites were selected to introduce disulfide bonds. The mutant had a pH increase of 0.5 compared to the original enzyme, the optimal temperature was reduced to 65°C, and the enzyme activity reached 55,640.2 U / mL. (Ma Jun, 2023) Based on the L253 and G254 sites of the recombinant Bacillus subtilis pectate lyase (BspPel-th), reverse mutations were performed to obtain the single mutants BspPel-th / L253I and BspPel-th / G254V. After incubation at 60°C for 1 hour, the residual enzyme activities were 71% and 66.4%, respectively, which were 54.8% and 50.2% higher than those of BspPel-th, respectively. The thermal stability of both mutants was also improved (Wei Xiaofeng, 2023). Chinese patent document CN108588061A mutated glutamic acid and lysine at positions 184 and 185 of the wild-type pectate lyase to aspartic acid and serine, respectively. The mutants significantly improved the specific enzyme activity and thermal stability of the enzyme. The above sequence optimization was obtained in a random manner, which is relatively random. It is often necessary to screen a large number of mutants to obtain the ideal optimized product. Therefore, it is still necessary to use sequence editing methods to improve the performance of a specific sequence.

[0007] Chroma is an artificial intelligence (AI) model that designs and samples new protein structures and sequences. By adjusting variable parameters, it generates proteins with specific structures and functions. It will be widely used in research fields such as protein drugs, antibodies, vaccines, and novel synthetic biology components (Ingraham JB et al., 2023). The protein space is complex. Using Chroma, protein design problems are represented using composable building blocks, from which a variety of all-atom protein structures can be automatically generated. As a joint model of structure and sequence, Chroma can also be used for common protein modeling tasks, such as generating sequences from a given backbone, packing side chains, and scoring designs. Summary of the Invention

[0008] In order to improve the thermal stability of pectin lyase, the present invention provides the following technical solution:

[0009] The present invention cloned the P. expansum gene PEX2_107230 to obtain a full-length DNA sequence (SEQ ID NO.1). The obtained DNA sequence was predicted to have a complete reading frame, and the expressed protein sequence was shown in SEQ ID NO.2. The protein sequence was predicted to contain a secretory protein signal peptide, and the signal peptide was predicted to be located at the N-terminal 1-22 amino acids.

[0010] To determine the enzyme activity, the first 1-22 amino acids were removed to obtain a protein sequence (SEQ ID NO.3), and the codons were optimized based on this protein sequence to synthesize a nucleic acid sequence (SEQ ID NO.4), which was expressed in Escherichia coli to obtain a recombinant expression strain.

[0011] By using protein computational biology technology, based on SEQ ID NO. 3, optimization and screening were performed to obtain an improved pectin lyase OptPePeL8, whose amino acid sequence is any of the following:

[0012] 1) the sequence shown in SEQ ID NO.5;

[0013] 2) The sequence of SEQ ID NO. 5 is subjected to one or more amino acid substitutions and / or deletions and / or additions, and the amino acid sequence related to pectin cleavage is obtained.

[0014] The nucleotide sequence of the gene encoding the improved pectin lyase OptPePeL8 is any one of the following:

[0015] 1) the sequence shown in SEQ ID NO. 6;

[0016] 2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO. 6 and encodes a nucleotide sequence with the same protein function.

[0017] The present invention also provides a method for obtaining pectin lyase, which uses Alphafold (https: / / alphafold.com / ) and Chroma models to optimize the sequence of pectin lyase, comprising at least the following steps:

[0018] 1) Based on the pectate lyase protein sequence, the protein structure was predicted using Alphafold (https: / / alphafold.com / ) to obtain the PDB file.

[0019] 2) Construct a conditioner file to restrict the amino acid sequence at positions 92-298 to change, but not to change the domain.

[0020] 3) Use the Chroma model for protein sequence optimization, setting the parameters as langevin_factor = 7, inverse_temperature = 6, sde_func = "langevin".

[0021] 4) Sort the output results according to the residue confidence (pLDDT) and the standard error (PAE), and take the top 10 sequences for verification and screening.

[0022] The sequence identity of pectin lyase before and after optimization was 28.5%, and the similarity was 44.7%. In terms of spatial structure, the alpha helix and beta stack were relatively conserved.

[0023] Through the sequence optimization of the present invention, the optimum pH of the improved pectin lyase is increased by 1, and the optimum reaction temperature is increased by 15° C. compared with the wild-type pectin lyase.

[0024] The present invention provides use of pectin lyase OrgPePeL8 in reducing pectin.

[0025] The present invention also provides use of the improved pectin lyase OptPePeL8 in reducing pectin.

[0026] The present invention also provides a method for reducing pectin in tobacco, which comprises using improved pectin lyase OptPePeL8 and / or pectin lyase OrgPePeL8, preferably using OptPePeL8 enzyme solution to degrade pectin at 65° C. and 35% relative humidity.

[0027] The invention improves the thermal stability of pectin lyase, expands the use conditions of pectin lyase, and makes the pectin lyase have wider application prospects in the fields of food, textile, papermaking, tobacco, feed and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Prediction map of the PePeL8 signal peptide excision site.

[0029] Figure 2 .OrgPePeL8 evolutionary analysis with known pectate lyases, where the numbers are patent or database numbers.

[0030] Figure 3 .Sequence alignment of pectin lyase before and after optimization ( Figure 3 A), the sequence identity before and after optimization was 28.5%, and the similarity was 44.7% ( Figure 3 A), and the spatial structure before and after optimization ( Figure 3 B, Figure 3 C), alpha helix and beta stack are relatively conserved ( Figure 3 D).

[0031] Figure 4 Protein SDS-PAGE electrophoresis. Among them: the first lane is the protein molecular weight marker, lanes 2 and 3 are the proteins before and after optimization, and lane 4 is the empty control ( Figure 4 A). Figure 4 B is a protein Western blotting image, using Anti-His as the primary antibody (Beijing Quanshijin Biotechnology Co., Ltd.) and goat anti-mouse as the secondary antibody (Beijing Quanshijin Biotechnology Co., Ltd.). Lane 1 is a protein molecular weight marker, lane 2 is an empty control, and lanes 3 and 4 are the proteins before and after optimization, respectively.

[0032] Figure 5 A. Thermostability of pectin lyase from Penicillium before and after optimization; Figure 5 B. Effect of pH on pectin lyase activity before and after optimization.

[0033] Figure 6 .OptPePeL8 enzyme degradation of pectin in tobacco ( Figure 6 A) Reduce the pectin methyl ester content ( Figure 6 B) and acetylation ( Figure 6 C) The activity was significantly higher than that of OrgPePeL8 (p<0.05). DETAILED DESCRIPTION

[0034] The present invention utilizes the most common molecular biology techniques, such as gene cloning, vector construction, protein expression, and purification. Reference can be made to any classic molecular biology reference, such as J. Sambrook's Molecular Cloning: A Laboratory Manual (USA). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art may employ other methods, protocols, and reagents in the art based on the technical solutions described herein, without being limited to the specific embodiments of the present invention.

[0035] The present invention is described in detail below with reference to specific embodiments.

[0036] Example 1 Obtaining OrgPePeL8

[0037] According to the sequencing results of the Penicillium expansum genome (Accession: PRJNA255744) in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), P. expansum contains 11 pel genes. The P. expansum gene PEX2_107230 was cloned and the full-length DNA sequence was obtained (SEQ ID NO.1). The obtained DNA sequence was predicted to have a complete reading frame, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. Cloning and sequencing revealed that the protein sequence encoded by this gene contains an extracellular secretion signal peptide located at the N-terminal 1-22 amino acids ( Figure 1 To determine the enzyme activity, the protein encoded by this gene after removing the signal peptide (OrgPePeL8 (SEQ ID NO.3)) was subjected to evolutionary analysis with reported pectin lyases ( Figure 2 The results showed that the protein encoded by this gene has a 67.2% identity and a 79.0% similarity to the pectin lyase (A2QFN7.1|PELB_ASPNC) reported to be the most similar in structure to it in prior art. It also has a 62.9% identity and a 77.5% similarity to the reported patent sequences (CN108102934A and CN108004153A). This high degree of structural similarity and identity further confirms that the protein encoded by this gene, OrgPePeL8, is a novel pectin lyase.

[0038] Example 2 Extraction of Penicillium RNA and cDNA Synthesis

[0039] (1) Infect apple tissue with Penicillium expansum, take 100-200 mg of apple tissue from the rotten border, place it in a grinder, and quickly add liquid nitrogen and 100 mg of quartz sand, and grind it.

[0040] (2) Take the ground tissue and add 1.5 mL of Trizol reagent for every 100 mg of apple tissue. Incubate at 15-30°C for 15 min.

[0041] (3) Centrifuge at 12,000 rpm and 4°C for 10 min, collect the supernatant, mix the supernatant with chloroform at a volume ratio of 4:1, shake vigorously for 15 s, and incubate at room temperature for 2–3 min.

[0042] (4) Centrifuge at 12000 rpm and 4°C for 10 min, and add the aqueous phase to an RNase-free EP tube.

[0043] (5) Mix the above liquid with isopropanol at a volume ratio of 2:1 and incubate at room temperature for 10 minutes.

[0044] (6) Centrifuge at 12000 rpm and 4°C for 10 min and discard the supernatant.

[0045] (7) Add 1 mL of 75% ethanol to wash the precipitate.

[0046] (8) Centrifuge at 12,000 rpm and 4°C for 10 min, discard the supernatant, and air-dry at room temperature for 5–10 min.

[0047] (9) Add 30 μL of RNA-free water to dissolve the RNA and store at -80°C, or proceed with the following reverse transcription procedure.

[0048] (10) RNA was reverse transcribed into cDNA. The following reagents were added to a 20 μL reaction system:

[0049]

[0050] Reverse transcription: incubate at 70°C for 5 min and terminate the reaction at 4°C to obtain P. expansum cDNA.

[0051] All the above reagents and consumables need to be treated with RNase-free.

[0052] Example 3 PePeL8 gene cloning

[0053] The cDNA obtained in Example 2 was amplified by PCR as follows:

[0054] (1) The reaction system (50 μL) contains:

[0055]

[0056] Add purified water to a total volume of 50 μL and mix thoroughly.

[0057] Forward primer: 5'-atgaagatcacagccatccttg-3'

[0058] Reverse primer: 5'-tcaaagcttaccctggccagcatt-3'

[0059] (2) PCR reaction conditions

[0060] Denaturation at 95°C for 3 min, denaturation at 95°C for 20 s, annealing at 59°C for 20 min, and extension at 72°C for 40 s were performed for 30 cycles.

[0061] (3) Electrophoresis identification of PCR results

[0062] The PCR results were subjected to agarose gel electrophoresis, and the target band size was approximately 1134 bp, which was consistent with the predicted molecular size.

[0063] (4) Cloning and sequencing of target genes

[0064] The target band was cloned into the pUC19 vector (GenScript Biotech Co., Ltd.) and transformed into DH5alpha Escherichia coli (Beijing Quanshijin Biotechnology Co., Ltd.). Clones were selected and identified by enzyme digestion. Clones with the correct band size were sequenced. The ligation, enzyme digestion, and sequencing methods used were based on the "Molecular Cloning Manual" (J. Sambrook), which are standard procedures in molecular biology. Sequencing results were compared with the NCBI database, and the cloned gene sequence was consistent with the NCBI database, indicating that the PEX2_107230 gene sequence was successfully obtained.

[0065] Example 4OrgPePeL8 gene expression

[0066] (1) Based on the PEX2_107230 protein sequence (SEQ ID NO. 2), the N-terminal 1-22 amino acids were removed, Met was added at the first position, and the codons were optimized to artificially synthesize the OrgPePeL8 gene sequence (SEQ ID NO. 4). This was then inserted into the expression vector pET22b(+) (GenScript Biotech Co., Ltd.) between NdeI and XhoI, and fused to the vector C-terminal His tag to obtain the expression vector. The expression vector was transformed into Escherichia coli BL21(DE3) (Beijing Quanshijin Biotechnology Co., Ltd.) and plated on an LB plate containing ampicillin resistance (10 g / L tryptone, 5 g / L yeast extract, 10 g NaCl, 15 g / L agar powder, sterilized at 121°C for 20 min).

[0067] (2) Pick 5-10 monoclonal bacteria, add 2 mL of ampicillin-resistant LB medium (10 g / L tryptone, 5 g / L Lyeast extract, 5 g NaCl, sterilized at 121°C for 20 min), and culture at 37°C with a shaker at 200 rpm until the OD600 absorbance is about 1.0.

[0068] (3) Take 0.5 mL of bacterial solution and add sterile glycerol to a final glycerol concentration of 25%, and store the strain at -80°C.

[0069] (4) The remaining bacterial solution was centrifuged at 4000 rpm for 10 min at room temperature, the supernatant was discarded, and fresh LB medium containing 0.8 mM IPTG was added. The culture was induced on a shaker at 200 rpm and 18°C for 16 h.

[0070] (5) Take 40 μL of induced bacterial solution and add 10 μL of protein 5× loading buffer (1 M pH 6.8 Tris-HCl 25%, sodium dodecyl sulfate 10%, bromophenol blue 0.5%, glycerol 42%, 5 M DTT 10%) for PAGE-SDS electrophoresis and Western blotting for protein identification.

[0071] (6) The clones with higher protein expression were selected for expansion culture. The culture medium used was the autoinduction medium AIM (Tryptone 16.0 g / L, Yeast Extract 10.0 g / L, MgSO4 0.15 g / L, (NH4)2SO4 3.30 g / L, KH2PO4 6.80 g / L, Na2HPO4 7.10 g / L, Glucose 0.50 g / L, Alpha Lactose 2.00 g / L, pH 7.0).

[0072] (7) The bacterial suspension was inoculated into AIM medium and cultured overnight on a shaker at 200 rpm and 37°C.

[0073] The results of PAGE-SDS electrophoresis and Western-blotting showed that ( Figure 4 A, 4B), the molecular weight of the protein was about 42 KDa, which was consistent with the expected molecular weight, indicating that a high-efficiency expression strain had been obtained, and the strain could efficiently produce OrgPePeL8 protein.

[0074] Example 5OrgPePeL8 protein purification

[0075] (1) Ni-NTA columns (GenScript Biotech Co., Ltd.) were used for gravity purification of expressed proteins. 3 g of bacterial cells were added to 30 mL of buffer (50 mM PBS, pH 7.4, 0.5 M NaCl) and ultrasonicated for cell wall disruption. The disruption conditions were: 300 W, 10 s on, 10 s off, and 20 min of operation.

[0076] (2) The cell wall lysate was centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0077] (3) Equilibrate 5 column volumes with equilibration buffer (PBS buffer containing 0.5 M NaCl, 50 mM pH 7.4) at a controlled flow rate of 2 mL / min;

[0078] (4) The treated cell lysate was loaded at a flow rate of 1 mL / min;

[0079] (5) The impurities were eluted in stages using a buffer solution (50 mM PBS buffer, pH 7.4, containing 0.5 M NaCl and 20 mM imidazole) with an elution volume of 5 column volumes and a flow rate of 1 mL / min.

[0080] (6) The OrgPePeL8 protein was eluted in stages using a buffer solution (50 mM PBS buffer, pH 7.4, containing 0.5 M NaCl and 300 mM imidazole).

[0081] (7) The BCA (bicinchonininc acid) method was used to determine the protein content of purified OrgPePeL8.

[0082] (8) Wash 5 column volumes with pure water and then 3 column volumes with 20% ethanol at a flow rate of 1 mL / min. Store the column in a low-temperature environment.

[0083] Example 6 Improved Pectin Lyase OptPePeL8 Protein Sequence Optimization and Expression 6.1 Model Construction

[0084] (1) Based on the OrgPePeL8 protein sequence (SEQ ID NO. 3), the PEX2_107230 protein structure was predicted using the Alphafold database (https: / / alphafold.com / ) and the PDB file OrgPexPel8_1.pdb was downloaded;

[0085] (2) Construct a conditioner file. The conditioner file is a Pytorch.nn module known in the prior art. It accepts the system state (i.e., structure, energy, and diffusion time) and outputs a possibly updated structure and energy. The conditioner file mainly restricts the pectin lyase conserved domain (positions 92-298) from changing, but its sequence can change.

[0086] The main parameters of Conditioner are as follows:

[0087]

[0088]

[0089] Obtain the Conditioner restriction file through parameter optimization.

[0090] 6.2 Protein Sequence Design

[0091] (1) The PDB file was used as the input optimization target and the conditioner file as the optimization condition. Chroma parameters were optimized with the following parameters: langevin_factor = 7, inverse_temperature = 6, and sde_func = "langevin". Protein sequences were simulated using Alphafold (https: / / github.com / google-deepmind / alphafold) and ranked by per-residue confidence (pLDDT) and per-residue error (PAE).

[0092] 6.3 Functional verification of proteins

[0093] (1) Sort the results predicted in 6.2 and select the top 10 sequences for verification. Based on their protein sequences, optimize and artificially synthesize the nucleic acid sequences into the pET22b(+) vector, and use Escherichia coli BL21(DE3) as the expression host.

[0094] (2) Screening clones and performing expression identification. Cloning, screening, and enzyme activity identification were performed in the same manner as in Example 4. Improved proteins were screened based on high enzyme activity, high optimum temperature, and high optimum pH to obtain an improved pectin lyase OptPePeL8. The amino acid sequence of the improved pectin lyase is shown in SEQ ID NO. 5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 6.

[0095] Example 7 Determination of the optimum temperature of pectin lyase

[0096] The optimal temperature of pectin lyases OrgPePeL8 and OptPePeL8 was detected by the following method:

[0097] To 900 μL of enzyme reaction solution (0.2% polygalacturonic acid, 50 mM phosphate buffer, pH 7.5, 1 mM CaCl₂), add 100 μL of purified pectin lyase and incubate at 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, and 85°C for 10 min. The reaction was terminated by adding 250 μL of 50 mM HCl, and the absorbance was measured at 232 nm. Purified pectin lyase OrgPePeL8, inactivated at 100°C for 10 min, was added to the 900 μL enzyme reaction solution as a control. Enzyme activity is defined as the amount of enzyme required to achieve a one-unit change in absorbance at 232 nm per minute (Li Guangwei, 2019).

[0098] The specific activities of wild-type pectin lyase (OrgPePeL8) and improved pectin lyase (OptPePeL8) under different temperature conditions were compared, and the relative enzyme activity was calculated with the maximum enzyme specific activity as 100%. Figure 5 As shown in A, the optimum temperature of OptPePeL8 is 65°C, which is 15°C higher than that of the wild-type pectate lyase OrgPePeL8.

[0099] Example 8 Determination of the optimal pH of pectin lyase

[0100] Polygalacturonic acid was added to 50mM acetic acid / sodium acetate buffer (pH 4.0, pH 4.5, pH 5.0, pH 5.5 and pH 6.0), 50mM phosphate buffer (pH 6.5, pH 7.0 and pH 7.5), 50mM Tris-HCl buffer (pH 8.0, pH 8.5 and pH 9.0) and other buffers, with the final concentration of polygalacturonic acid being 0.2%, and then OrgPePeL8 and OptPePeL8 were added to purify the protein respectively.

[0101] The optimal pH of wild-type pectin lyase and improved pectin lyase was determined at their respective optimal temperatures, and the relative enzyme activity was calculated with the maximum enzyme specific activity as 100%. Figure 5 As shown in B, the optimum pH of OrgPePeL8 is around pH 7; the optimum pH of OptPePeL8 is around pH 8, which is 1.0 unit higher than that of the wild-type pectate lyase OrgPePeL8.

[0102] Example 9: Degradation of pectin in tobacco by improved pectin lyase OptPePeL8

[0103] OrgPePeL8 and OptPePeL8 enzyme solutions were prepared in Tris-HCl buffer (containing 1 mM CaCl2, 20 mM Tris-HCl buffer, pH 7.5) with an enzyme activity of 1000 U / mL. These solutions were then dispensed into a small sprayer and used immediately. Yunnan flue-cured tobacco was shredded after conditioning, and each 40 g portion was evenly sprayed with 2 mL of the enzyme solution. The tobacco was then sealed in a sealed bag and placed in a constant temperature chamber at 65°C, maintaining a relative humidity of approximately 35%, and subjected to enzymatic hydrolysis for 3 hours. Following enzymatic hydrolysis, the tobacco was heated to 85°C for 30 minutes to inactivate the enzyme. Tris-HCl buffer without enzyme was used as a control. The experiment was repeated three times.

[0104] The tobacco cuts after enzymatic hydrolysis were dried at 35℃ and ground into powder for determination of pectin content. The determination method of pectin was carried out according to the method in the literature (Xiaolan Zhu, 2014). Figure 6 As shown, the results showed that the improved pectin lyase OptPePeL8 enzyme was effective in degrading pectin ( Figure 6 A) Reduce the pectin methyl ester content ( Figure 6 B) and acetylation ( Figure 6 C), the activity was significantly higher than that of the wild-type pectin lyase OrgPePeL8 (p<0.05), and it could effectively degrade tobacco pectin.

[0105] References

[0106] 1.Kanchan Yadav,Shruti Dwivedi,Supriya Gupta,Aiman Tanveer,SangeetaYadav,Pramod K.Yadav,Gautam Anand,Dinesh Yadav.Recent insights into microbialpectin lyases:Areview.Process Biochemistry,2023,134(1),199-217.

[0107] 2. Ritu Saharan, Kanti Prakash Sharma. Production, purification and characterization of pectin lyase from Bacillus subtilis isolated from moongbeans leaves (Vigna radiata). Biocatalysis and AgricμLtural Biotechnology, 2019, 21, 101306.

[0108] 3. Cristina F. Mantovani, Mercedes P. Geimba & Adriano Brandelli. Enzymatic Clarification of Fruit Juices by Fungal Pectin Lyase, Food Biotechnology, 2005, 19(3): 173-181, DOI: 10.1080 / 08905430500316284.

[0109] 4. Demir N, Nadaroglu H, Demir Y, et al. Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillusborstelensis(P35) and its applications in fruit juice and oil extraction. European Food Research and Technology, 2014, 239(1):127-135.

[0110] 5. Demir N, Nadaroglu H, Demir Y, et al. Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillusborstelensis(P35) and its applications in fruit juice and oil extraction. European Food Research and Technology, 2014, 239(1):127-135.

[0111] 6. Li Guangwei. Study on AchGLK gene improving tomato fruit quality and LysM family genes of Penicillium[D]. Hefei University of Technology, 2019.

[0112] 7.Li, W., Li, P., Deng, Y., Situ, J., He, Z., Zhou, W., Li, M., Xi, P., Liang, X., Kong, G., & Jiang, Z. Aplant cell death-inducing protein from litchiinteracts withPeronophythora litchii pectate lyase and enhances plant resistance.Naturecommunications,2024,15(1),22.https: / / doi.org / 10.1038 / s41467.

[0113] 8. Ma Jun. Molecular modification of pectin lyase and directed evolution of its expression host. Qilu University of Technology: Master's thesis, 2023.

[0114] 9. Wei Xiaofeng. Study on the Alkali Resistance Mechanism of Pectin Lyase and Its High-Efficiency Expression System. Qilu University of Technology: Master's Thesis. 2023.

[0115] 10.Hoondal G, Tiwari R, Tewari R, et al. Microbial alkaline pectinases and their industrial applications: a review. Applied Microbiology and Biotechnology 2002, 59 (4-5): 409-418

[0116] 11. Ingraham JB, Baranov M., Costello Z. et al. Illuminating proteinspace with a programmable generative model. Nature, 2023, 623, 1070–1078. https: / / doi.org / 10.1038 / s41586-023-06728-8.

[0117] 12. Xiaolan Zhu, Baizhan Liu, Saijing Zheng and Yun Gao. Quantitative and structural analysis of pectin in tobacco by 13C CP / MAS NMRspectroscopy. Anal. Methods, 2014, 6, 6407-6413. https: / / doi.org / 10.1039 / C4AY01156B.

[0118] 13. Thornton J W. Enzymatic degradation of polygalac-turonic acids released from mechanical pulp during peroxide bleaching. Tappi Journal, 1994, 77(3): 161-167.

[0119] 14.Tzanov T,Calafell M,Guebitz GM,et al.Bio-preparation of cottonfabrics.Enzyme and Microbial Technology2001,29(6):357-362.

[0120] 15. Zhu Xiaolan, Liu Baizhan, Wang Weimiao, Gao Yun. Study on the degradation of tobacco pectin using bioenzymes. Journal of Hunan Agricultural University (Natural Science Edition), 2015, 41(3): 257–262.

[0121] Sequence Listing

[0122] SEQ ID NO. 1PEX2_107230

[0123] atgaagatcacagccatccttgcagcactcgcagtgcgagctgccgccgtcggtgtctcc

[0124] ggcgccgcagagggtttcgccaagggcgtcactggcggtggctctgccacccccgtctac

[0125] cccagcaccacagccgagctggtctcgtacctgggtgactcttccccccgcgtgatcatc

[0126] ttgaccaagaccttcgatttcaccggctctgagggaaccaccaccagcaccggctgcgcc

[0127] ccttggggtactgccgctgcttgccaggttgcaatcaaccaggacaactggtgcaagaac

[0128] tacgagcccaacgctccttccacccctgtcaagtacgacaacgccggtaccctcggcata

[0129] atcgtccagtccaacaagtccatcgttggctccggcagcgccggtatcatcaagggcaag

[0130] ggtctccgtattgtcagcggcgcaaacaacgttatcatccagaacgttgccatcaccgac

[0131] atcaacgccaagtacgtctggggcggtgatgccatcaccatcaacaacgccgacatggtc

[0132] tggattgaccacgtcaccactgcccgcatctctcgccagcacattgttctcggcacccag

[0133] gcctccaagcgtgtcactatttccaacagcttcatcaacggtgccagcgactactccgct

[0134] acctgcaacggataccactactggggtatctaccttgatggatccagcgatctcgttacc

[0135] ctcaagggcaactacatctaccacaccagtggacgtgcccccaaggtccagggcaacact

[0136] cttctgcacgccgtcaacaactactggtacgagaactccggccacgccttcgagatcggt

[0137] gccggcggttacgtcctcgctgagggcaatgtgttccagaacattgttgcttccgtcgag

[0138] acccccattgccggacagctgttctcctcgcccgataccaccaccaatgctgtctgcagc

[0139] tctaaccttggtcgtgcctgccaggtcaacggattcgggtcttccggaaacctcggtggc

[0140] cgcgctgatactggattcttggccaactttgctggaaagaacattgcctctgctgctgct

[0141] tacaccactgttgtttccagcgtcactgccaatgctggccagggtaagctttga

[0142] SEQ ID NO.2PePeL8

[0143] MKITAILAALAVRAAAVGVSGAAEGFAKGVTGGGSATPVYPSTTAELVSYLGDSSPRVII

[0144] LTKTFDFTGSEGTTTSTGCAPWGTAAACQVAINQDNWCKNYEPNAPSTPVKYDNAGTLGI

[0145] IVQSNKSIVGSGSAGIIKGKGLRIVSGANNVIIQNVAITDINAKYVWGGDAITINNADMV

[0146] WIDHVTTARISRQHIVLGTQASKRVTISNSFINGASDYSATCNGYHYWGIYLDGSSDLVT

[0147] LKGNYIYHTSGRAPKVQGNTLLHAVNNYWYENSGHAFEIGAGGYVLAEGNVFQNIVASVE

[0148] TPIAGQLFSSPDTTTNAVCSSNLGRACQVNGFGSSGNLGGRADTGFLANFAGKNIASAAA

[0149] YTTVVSSVTANAGQGKLSEQ ID NO.3 signal peptide removed PePeL8 (OrgPePeL8)

[0150] MAEGFAKGVTGGGSATPVYPSTTAELVSYLGDSSPRVIILTKTFDFTGSEGTTTSTGCAP

[0151] WGTAAACQVAINQDNWCKNYEPNAPSTPVKYDNAGTLGIIVQSNKSIVGSGSAGIIKGKG

[0152] LRIVSGANNVIIQNVAITDINAKYVWGGDAITINNADMVWIDHVTTARISRQHIVLGTQA

[0153] SKRVTISNSFINGASDYSATCNGYHYWGIYLDGSSDLVTLKGNYIYHTSGRAPKVQGNTL

[0154] LHAVNNYWYENSGHAFEIGAGGYVLAEGNVFQNIVASVETPIAGQLFSSPDTTTNAVCSS

[0155] NLGRACQVNGFGSSGNLGGRADTGFLANFAGKNIASAAAYTTVVSSVTANAGQGKLSEQ ID NO.4 Codon-optimized OrgPePeL8

[0156] atggctgaaggctttgcgaaaggtgtgactggtggtggttctgcaactcctgtctacccg

[0157] tccaccactgcggaactggtctcctacctgggtgactcctccccgcgtgttatcatcctg

[0158] actaagaccttcgactttactggctccgaaggcactactacgagcactggttgtgctccg

[0159] tggggtactgctgctgcatgccaggtagcaatcaaccaggacaactggtgtaagaactac

[0160] gagccgaacgctccgagcaccccagtgaaatatgataacgccggtaccctgggcatcatc

[0161] gtccagtctaacaaatccatcgttggcagcggttccgcgggtatcattaagggcaagggc

[0162] ctgcgtatcgtatctggcgctaacaacgttatcatccagaacgtcgctatcaccgacatc

[0163] aacgccaaatacgtctggggcggcgacgcgatcactattaataacgcggacatggtctgg

[0164] attgatcacgtaactactgctcgtatcagccgtcagcatatcgttctgggtactcaagca

[0165] agcaaacgtgtaacgatctctaacagcttcatcaacggcgcttctgattactccgctacc

[0166] tgcaacggctatcactactggggtatctacctggatggtagctctgatctggttaccctg

[0167] aagggcaactacatttatcacacctccggtcgcgctccgaaagtacagggtaacactctg

[0168] ctgcacgcggtaaataactattggtatgagaacagcggccacgcgttcgaaattggtgca

[0169] ggtggttacgttctggcagaaggtaacgtatttcagaacatcgtggcaagcgtggaaacc

[0170] ccaattgccggtcagctgttttccagcccggatactaccaccaatgcggtatgttcctct

[0171] aacctgggccgtgcgtgccaagttaatggcttcggctctagcggcaatctgggtggccgt

[0172] gcagacaccggttttctggcgaacttcgcgggcaaaaacatcgcgagcgctgcggcgtac

[0173] accaccgttgtgtcctctgttaccgccaacgctggtcaaggcaagctg

[0174] SEQ ID NO.5 Improved Pectate Lyase OptPePeL8

[0175] MLGGFAKSYTGAGDEKPVYATTPEQFRELLTSDKRANIIITKDLDFGNALGTIEATGVGP

[0176] AGKDKSSLVYIDKNGFVSKTAPTTSPQKVKISTEAAVPLSVNSDTTVIGSGTQAKISGVG

[0177] LSFSNGTSNVIVEECTIEGVAPDVFNTGSPITINDADNVYLKKVTFKNINGSAVKVDPES

[0178] AKDVVIENCYIDLRHPKDLNSAGTNNAQVLIASKDAYTTIKNCLFTSANDANPKVTANNL

[0179] VVINDCYFTDISSNFLEVGAGTVVIVDNSLFVSIKEAVKKPIEFKLFDASNEEAAKKAKE

[0180] YLGKPLKPNVFIGSKDLGGIFDEDFLEKIKGKNIPEIKDPKEKLAELKGTVGPNNESEQ ID NO.6 improved pectin lyase encoding gene OptPePeL8

[0181] atgctgggcggtttcgcaaaaagctataccggtgctggtgacgagaaaccggtgtacgcg

[0182] actaccccggaacagttccgtgaactgctgaccagcgacaaacgtgcgaacatcatcatc

[0183] actaaagatctggacttcggcaacgcgctgggtactattgaagcaaccggtgttggtccg

[0184] gctggtaaagataaatctagcctggtgtacatcgataaaaacggtttcgtttccaagacc

[0185] gctccgactaccagcccgcaaaaagtaaaaatttctactgaagctgccgtaccactgtct

[0186] gtgaactccgatactacggtgattggtagcggtacccaggcgaaaatttctggtgtaggc

[0187] ctgagcttctctaatggcaccagcaacgtaatcgtcgaagaatgcacgattgagggtgtc

[0188] gctccggatgtgttcaacaccggtagccctatcactatcaacgatgccgataatgtttac

[0189] ctgaaaaaggtaaccttcaaaaacatcaacggtagcgcagttaaagtggacccggaatct

[0190] gcgaaagacgtggtaatcgaaaactgttatatcgatctgcgtcacccgaaagatctgaac

[0191] tctgctggtaccaataacgcgcaggtgctgatcgcgtctaaagatgcctataccaccatc

[0192] aaaaactgtctgtttactagcgccaacgacgcgaacccgaaagttaccgctaataacctg

[0193] gtagtgatcaacgactgctactttactgacatctccagcaactttctggaagtaggtgca

[0194] ggtactgtggtgatcgtcgataatagcctgttcgtttccatcaaagaggcggttaaaaaa

[0195] ccgatcgagttcaaactgttcgacgcatctaacgaagaagcggcgaagaaagctaaagag

[0196] tacctgggcaaaccgctgaaacctaacgttttcatcggttctaaagacctgggcggtatc

[0197] ttcgacgaggatttcctggaaaaaattaaaggcaaaaacatcccggaaatcaaagatcct

[0198] aaagaaaaactggcggaactgaaaggtacggtgggtccgaacaatgaa

Claims

1. Improved pectin lyase OptPePeL8, the amino acid sequence of which is shown in SEQ ID NO.

5.

2. A gene OptPePeL8 encoding the improved pectin lyase OptPePeL8 according to claim 1.

3. The gene OptPePeL8 according to claim 2, whose nucleotide sequence is shown in SEQ ID NO.

6.

4. An expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising the gene OptPePeL8 according to claim 3.

5. A method for obtaining pectin lyase, comprising the following steps: 1) Based on the pectate lyase protein sequence shown in SEQ ID NO. 3, the protein structure was predicted using Alphafold (https: / / alphafold.com / ) to obtain the PDB file; 2) Construct a conditioner file to restrict the amino acid sequence at positions 92-298 to change, but not to change the domain; The main parameters of conditioner are as follows: selection_string = "(resid 50) around 20" residues_to_design = plane_split_protein(X, C, protein, 0.6).nonzero()[:,1].tolist(); 3) Use the Chroma model for protein sequence optimization, setting the parameters as langevin_factor = 7, inverse_temperature = 6, sde_func = "langevin"; 4) Sort the output results according to residue confidence (pLDDT) and standard error (PAE), and select the top 10 sequences for verification and screening.

6. A method for reducing pectin in a sample, comprising adding the improved pectin lyase OptPePeL8 according to claim 1 to the sample.

7. The method according to claim 6, wherein the sample is tobacco, fruit, feed, paper products, or textiles.

Images