Mutants of polygalacturonase and uses thereof

Abstract

The present application belongs to the field of genetic engineering and genetic engineering, and particularly relates to a mutant K106A or Y274A of polygalacturonase and application thereof. The polygalacturonase mutant is obtained by performing K106A or Y274A mutation on a wild-type polygalacturonase with an amino acid sequence as shown in SEQ ID NO: 2. Compared with the wild-type polygalacturonase, the polygalacturonase mutant of the present application significantly enhances the functional adaptability thereof in an acidic environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and genetic engineering, and particularly relates to a mutant of polygalacturonase and application thereof. BACKGROUND

[0002] Pectin is the most common structural polysaccharide in the middle lamella and primary cell wall of plants, and is one of the most complex polysaccharides, mainly composed of homogalacturonan (HG), rhamnogalacturonan-I and rhamnogalacturonan-II. In some structural characterization of pectin, xylogalacturonan and apiose galacturonan were also found. The highest content component and monosaccharide in pectin polysaccharide are HG and galacturonic acid (GalA) respectively. HG is the backbone of pectin, which is also a linear homopolymer of GalA connected by α-1,4-glycosidic bond, accounting for 65% of pectin, and GalA accounts for about 70% of the total content of pectin.

[0003] Pectinase is a general term for a class of enzymes that specifically hydrolyze pectin substrates, and is widely used in food, beverage, feed, papermaking and textile industries. Polygalacturonase (PG) is one of the most widely studied, most clearly characterized and most widely used commercial pectinases, which can cleave the α-1,4-glycosidic bond of pectin backbone (i.e. HG) through hydrolysis.

[0004] In the feed industry, high-temperature pelleting is one of the key processes to improve the quality of feed. This process usually involves high-temperature steam treatment at 70–80℃, aiming to kill pathogenic microorganisms, improve the density and durability of feed pellets, and enhance starch gelatinization to improve digestibility. However, the high-temperature environment significantly damages the activity of functional enzyme preparations (such as pectinase) added in the feed. In addition, the stomach environment of animals (especially monogastric animals) puts strict requirements on the acid resistance adaptability of enzyme preparations. This is because the pH value of animal gastric juice is usually less than 4, and the residence time of feed in this environment is relatively long (about 1–3 hours).

[0005] However, the optimal pH of existing PGs is mostly concentrated in the neutral range (pH 5–7), and the activity is significantly limited in the low pH environment of the stomach, resulting in low decomposition efficiency of pectin in feed, which directly affects the release and absorption of nutrients.

[0006] In summary, for pectinase preparations used in feed, not only proper thermal stability is needed, but also structural stability under strong acidic conditions is needed to efficiently catalyze the substrate.

[0007] Some studies have improved the thermal stability of enzymes through glycosylation or immobilization techniques, but these methods often come at the cost of sacrificing catalytic efficiency or increasing production costs, and have limited effect on improving low pH activity.

[0008] Currently, the development of heat-resistant PG mainly focuses on natural enzymes from thermophilic microorganisms or enhancing the thermal stability through molecular modification. However, the activity of these enzymes in acidic environment is generally insufficient, which cannot meet the dual requirements of high-temperature granulation and gastric digestion.

[0009] Therefore, the industry urgently needs a polygalacturonase that can efficiently catalyze in the strong acidic environment of the animal stomach and has appropriate heat-resistant performance. SUMMARY

[0010] The technical problem to be solved by the present application is how to make the polygalacturonase have strong acid resistance while appropriately considering the heat-resistant performance.

[0011] As described above, although some studies have tried to improve the thermal stability of polygalacturonase, it is difficult to consider the stability in strong acidic environment. In the process of studying the structure and function of the polygalacturonase with the amino acid sequence shown in SEQ ID NO: 2, the inventors rationally designed targeted mutations, obtained multiple mutants through condition exploration and screening, and unexpectedly found that two mutants K106A and Y274A significantly enhanced the catalytic efficiency under acidic conditions, especially at pH 3-4, while moderately reducing the thermal stability of the enzyme, thereby completing the present application.

[0012] Accordingly, the technical solution adopted by the present application to solve its technical problem is as follows.

[0013] The first aspect of the present application provides a mutant of polygalacturonase, wherein the amino acid sequence of the polygalacturonase is shown in SEQ ID NO: 2, and the mutation of the mutant is K106A or Y274A.

[0014] The second aspect of the present application provides an isolated or synthetic nucleic acid molecule encoding the mutant of polygalacturonase of the first aspect of the present application.

[0015] In some embodiments, the nucleotide sequence of the isolated or synthetic nucleic acid molecule is shown in SEQ ID NO: 3.

[0016] In some embodiments, the nucleotide sequence of the isolated or synthetic nucleic acid molecule is shown in SEQ ID NO: 4.

[0017] The third aspect of the present application provides a recombinant vector comprising the isolated or synthetic nucleic acid molecule of the second aspect of the present application.

[0018] The fourth aspect of the present application provides a host cell comprising the isolated or synthetic nucleic acid molecule of the second aspect of the present application or the recombinant vector of the third aspect of the present application.

[0019] A fifth aspect of the present invention provides a feed composition comprising a mutant of the polygalacturonase of the first aspect of the present invention.

[0020] In some implementations, the feed composition is used for monogastric animals.

[0021] The sixth aspect of this invention provides a method for producing polygalacturonase, comprising the following steps:

[0022] Preparation of recombinant vectors comprising the present invention;

[0023] Transform the recombinant vector into host cells;

[0024] Host cells were cultured by fermentation to isolate and purify polygalacturonase.

[0025] Page 7 of this invention provides the application of a mutant of polygalacturonase from the first aspect of this invention in the preparation of feed.

[0026] In some implementations, the feed is that of monogastric animals.

[0027] The beneficial effects of this invention are that, by obtaining a recombinant polygalacturonase mutant through site-directed mutagenesis, the problem of the influence of high-temperature granulation on enzyme activity can be solved, while further ensuring the high efficiency of the enzyme in the highly acidic environment such as the animal stomach, and verifying its stability when used for long-term treatment, providing the feed industry with a novel enzyme preparation solution that combines process adaptability and functionality. Attached Figure Description

[0028] The accompanying drawings, which are provided to further illustrate embodiments of the invention and constitute a part of this invention, are not intended to limit the scope of the invention.

[0029] Figure 1 Electrophoresis diagrams of the construction results of various mutants of polygalacturonase MlPG28A to verify the results.

[0030] Figure 2 The results of verifying the expression of polygalacturonase MlPG28A and its mutant proteins are shown in the figure.

[0031] Figure 3 The curves showing the optimal reaction temperature for polygalacturonase MlPG28A and its mutants are shown.

[0032] Figure 4 The graph shows the stability of polygalacturonase MlPG28A and its mutant.

[0033] Figure 5The optimal reaction pH curves for polygalacturonase MlPG28A and its mutants are shown. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The following definitions are supplementary to those definitions in the art and relate to this application, but are not extrapolated to any relevant or unrelated circumstances, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in the practical testing of this valve, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0036] The terms "comprising," "including," and "having" in this invention are open-ended descriptions, encompassing the specified steps described, as well as other steps that do not materially affect them, and are optional and not excluded. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of said sequence, or may have additional amino acids or nucleotides at one or both ends of said protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0037] The term "about" in this invention is used to indicate the standard deviation allowed by the numerical value and the apparatus or method for determining the numerical value.

[0038] The first aspect of the present invention provides a mutant of polygalacturonase, wherein the mutant is mutated to K106A.

[0039] In an alternative implementation, a mutant of polygalacturonase is provided, the mutant being mutated to Y274A.

[0040] The "polygalacturonase" (EC 3.2.1.15, abbreviated as PG) referred to in this invention is the common name for poly-α-1,4-galacturonide glycanohydrase, which is a type of enzyme that can decompose pectin or pectin acid. It is also known as pectic enzyme or pectin depolymerase and has the activity of hydrolyzing the D-galacturonic acid α-1,4-glycosidic bonds that make up pectin acid.

[0041] The nucleotide sequence of the polygalacturonase of the present invention is shown in SEQ ID NO:1.

[0042] SEQ ID NO.1:

[0043]

[0044] In some embodiments, the nucleotide sequence of polygalacturonase is a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO.1.

[0045] The term "homology" in this invention refers to the ability of those skilled in the art to adjust sequences, whether protein or nucleotide sequences, according to actual work needs, so that, compared to sequences obtained by prior art, it has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, and 39%. 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity.

[0046] The amino acid sequence of the polygalacturonase of the present invention is shown in SEQ ID NO:2.

[0047] SEQ ID NO.2:

[0048] TTCTVAKDASDDATTITAAFNACKNGGTVVFTKGQTYNLKSLVSVSGLKNVNVQFYGTVNLPAYNTKFDGESSYFLIKGDNIHWDGNNVGGFVGGGQDWWNAQDKKAPSVLRITATHSSFINFKISQSPRAHLGVTSSDDVLLQHITLHSVSSNSNLPKNTDALDISNSKNIVVQNS DFTVGDDCLAINGNVSNVTLSDVTCTTNGHGFSVGSLGKGGETDVVKDITVQNSACINCQNGVRIKTWPGGKGSVSNVKFKNVNLPSVENAVLITTHYCDNNQMSYCNGKDDASLTISDVNISGLTGSMSGSNPMVNINCSTNTPCSGFSLSGITISKNSKTKANVCTNLNGASSISYC

[0049] In some embodiments, the amino acid sequence of polygalacturonase is a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO.2.

[0050] The term "mutant" in this invention refers to a polypeptide having polygalacturonase activity and containing alterations (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution refers to replacing an amino acid occupying a position with a different amino acid; deletion refers to removing an amino acid occupying a position; and insertion refers to adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0051] Taking the mutant “K106A” as an example, the mutant “K106A” means that the mutant obtained by replacing the 106th lysine (K) with alanine (A) on the basis of the starting sequence.

[0052] In some implementations, the mutant is mutated to K106A.

[0053] In some implementations, the mutant is mutated to Y274A.

[0054] The present invention also provides an isolated or synthesized nucleic acid molecule that encodes a mutant of the polygalacturonase of the present invention.

[0055] In some implementations, the nucleotide sequence of the isolated or synthesized nucleic acid molecule is shown in SEQ ID NO:3.

[0056] SEQ ID NO:3

[0057] ACTACTTGTACTGTTGCTAAGGATGCTTCTGATGATGCCACTACTATCACTGCTGCTTTTAACGCTTGTAAGAACGGTGGTACTGTTGTTTTTACCAAGGGTCAAACCTACAACTTGAAGTCTTTGGTTTCCGTCTCTGGTCTGAAGAACGTTAACGTTCAATTTTACGGTACTGTTAACTTGCCAGCCTACAACACTAAGTTTGACGGCGAATCTTCCTACTTTTTGA TTAAGGGTGACAACATTCATTGGGATGGTAACAACGTTGGTGGTTTTGTTGGTGGTGGTCAAGATTGGTGGAACGCTCAAGATAAGGCTGCTCCATCTGTTCTGCGTATTACTGCCACCCACTCTTCCTTCATTAACTTTAAGATTTCTCAATCTCCAAGAGCCCATTTGGGTGTTACTTCTTCTGATGATGTTTTGTTGCAACATATCACCTTGCATTCTGTCTCTTC TAACTCTAACTTGCCAAAGAACACTGATGCCTTGGATATTTCTAACTCCAAGAACATTGTCGTCCAAAACTCCGATTTTACTGTCGGTGATGATTGTTTGGCTATCAACGGTAACGTTTCTAACGTTACTTTGTCTGACGTTACTTGTACTAACGGTCATGGTTTTTCTGTTGGTTCTTTGGGTAAGGGTGGTGAAACTGACGTCGTTAAGGATATTACTGTTCAAAACTCCGCTTGCATTAACTGTCAGAACGGTGTTAGAATTAAGACTTGGCCAGGTGGTAAGGGTTCTGTTTCTAACGTTAAGTTTAAGAACGTTAACTTGCCCTCTGTTGAGAACGCTGTTTTGATTACTACTCACTACTGCGATAACAACCAAATGTCCTACTGCAACGGTAAGGATGACGCTTCTTTGACTATTTCTGATGTCAACATTTCTGGCTTGACTGGTTCTATGTCTGGTCTAACCCCATGGTCAACATTAACTGTTCTACTAACACTCCATGTTCTGGTTTTTCCTTGTCTGGTATCACTATTTCCAAGAACTCTAAGACTAAGGCCAACGTCTGCACTAACTTGAACGGTGCTTCTTCTATTTCTTACTGTTGA

[0058] In some embodiments, the nucleotide sequence of the isolated or synthesized nucleic acid molecule is a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO.3.

[0059] In some implementations, the nucleotide sequence of the isolated or synthesized nucleic acid molecule is shown in SEQ ID NO:4.

[0060] SEQ ID NO:4

[0061]

[0062] In some embodiments, the nucleotide sequence of the isolated or synthesized nucleic acid molecule is a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO.4.

[0063] The present invention also provides a recombinant vector comprising the isolated or synthesized nucleic acid molecules of the present invention.

[0064] In some embodiments, the recombinant vector is a nucleic acid molecule isolated or synthesized according to the present invention or a recombinant plasmid expressing a mutant of the present invention.

[0065] The term "expression" in this invention includes any step involving the production of polygalacturonase or mutants thereof, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0066] In some implementations, the recombinant plasmids include the pPICZ series plasmids.

[0067] The present invention also provides a host cell containing the isolated or synthesized nucleic acid molecule of the present invention or the recombinant vector of the present invention.

[0068] In this invention, the term "host cell" refers to any cell type that is readily transformable, transfected, transduced, etc., using a nucleic acid construct or recombinant vector containing the nucleic acid molecules of this invention. The term "host cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations occurring during replication.

[0069] The host cell can be any cell useful in the recombinant production of polygalacturonase mutants, such as prokaryotic or eukaryotic cells.

[0070] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Bacillus macrocephala, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0071] The host cell can also be a eukaryotic cell, such as an insect, plant, or fungal cell.

[0072] In some implementations, the host cell is a Pichia pastoris cell.

[0073] The present invention also provides a feed composition comprising a mutant of polygalacturonase of the first aspect of the present invention.

[0074] In some implementations, the feed composition is used for monogastric animals.

[0075] Monogastric animals have a highly acidic stomach throughout, while the foregut (rumen / reticulum) of ruminants is nearly neutral, with only the abomasum being highly acidic. Therefore, the feed composition of the present invention is particularly suitable for monogastric animals.

[0076] In some implementations, monogastric animals include pigs, horses, poultry (e.g., chickens, ducks, geese), and rabbits.

[0077] In some implementations, monogastric animals do not include marine animals.

[0078] In some implementations, the stomach of a monogastric animal is an acidic environment; for example, the pH value of the stomach of a monogastric animal is less than or equal to 4, less than or equal to 3.9, less than or equal to 3.8, less than or equal to 3.7, less than or equal to 3.6, less than 3.5, less than or equal to 3.4, less than or equal to 3.3, less than or equal to 3.2, less than or equal to 3.1, or less than 3; or the pH value of the stomach of a monogastric animal is greater than or equal to 2, greater than or equal to 2.1, greater than or equal to 2.2, greater than or equal to 2.3, greater than or equal to 2.4, greater than 2.5, greater than or equal to 2.6, greater than or equal to 2.7, greater than or equal to 2.8, greater than or equal to 2.9, or greater than 3.

[0079] The present invention also provides a method for producing polygalacturonase, comprising the following steps:

[0080] Prepare a recombinant vector comprising the isolated or synthesized nucleic acid molecule of the present invention; transform the recombinant vector into a host cell; ferment the host cell to isolate and purify polygalacturonase.

[0081] In some implementations, methods for converting recombinant vectors into host cells are well known in the art, such as electroporation and CaCl2 conversion.

[0082] In some implementations, culturing host cells involves culturing the host cells to the logarithmic growth phase and inducing expression with a suitable inducer.

[0083] In some implementations, the culture medium includes BMGY medium or BMMY medium.

[0084] In some implementations, the inducing agent includes methanol.

[0085] In some implementations, purification includes collecting the induction supernatant, purifying the supernatant, and collecting the polygalacturonase protein.

[0086] In some implementations, purification includes dialysis and / or ion exchange chromatography.

[0087] The present invention also provides the application of the mutant of the polygalacturonase of the present invention in the preparation of feed.

[0088] In some implementations, the feed is that of monogastric animals.

[0089] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0090] Experimental materials:

[0091] Plasmids and strains: The full-length PG gene MlPG28A (corresponding protein KAF1803089.1) was obtained from the genome of *Mucor lusitanicus* (formerly known as *Mucor circinelloides*) screened from a marine environment. The gene encoding MlPG28A was synthesized by Azenta Life Sciences (Suzhou, China); the host strain *Pichia pastoris* X33 and the vector pPICZαA were obtained commercially.

[0092] Experimental instruments: centrifuge (Eppendorf); electroporator (BIO-RAD); protein electrophoresis apparatus (BIO-RAD); PCR instrument (BIO-RAD); microplate reader (BIO-RAD); protein purification instrument (GE Healthcare).

[0093] Enzyme activity assay method: Add 20 μL of appropriately diluted enzyme solution to 200 μL of 0.05% polygalacturonic acid solution (pH 5.0). React at 30℃ for 20 min. Then, take 128 μL of the above solution and add 96 μL of DNS to terminate the reaction. The control group was treated with 20 μL of deionized water instead of the enzyme solution. The reaction system was treated in a boiling water bath for 10 min, immediately cooled in an ice-water bath, centrifuged for 5 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance at 540 nm was measured, and the enzyme activity was calculated as follows: A standard curve was plotted by measuring the absorbance values ​​of DNS corresponding to the standard concentration of GalA. The reducing sugar content was calculated from the standard curve, thus determining the enzyme activity. Definition of enzyme activity (U): The amount of enzyme required to release 1 μmol of galacturonic acid per minute at 30℃ is defined as one enzyme activity unit. Three replicates were used for each group, and the average value was taken as the final result.

[0094] Example 1: Construction of recombinant vector

[0095] The strain *Mucor lusitanicus* was inoculated onto a PDA plate and cultured until spores were produced. Spores were scraped off and inoculated onto YPD medium and cultured for 48 hours. Mycelium was collected, placed in a mortar, and flash-frozen in liquid nitrogen. RNA was extracted and reverse transcribed to obtain cDNA.

[0096] Primers MlPG28A-F1 / R1 (sequences shown in SEQ ID NO.5 and SEQ ID NO.6, respectively) were designed based on the endoglucuronidase gene published in the NCBI database.

[0097] SEQ ID NO.5:

[0098] GAAGAAGGGGTATCTCTCGAGAAAAGATCCAAGACTTGCACAGCTA AGTCCGGTAC

[0099] SEQ ID NO.6:

[0100] CAAACTCAATGATGATGATGATGATGCTGCTTGCAATAGGAGATCTTG TCGGAT

[0101] The full-length gene containing the signal peptide was amplified, named MlPG28A, and sent for sequencing. Based on the sequencing results, the signal peptide was predicted using the website SignalP 5.0 (http: / / www.cbs.dtu.dk / services / SignalP). Primers MlPG28A-F2 / R2 (sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively) were designed.

[0102] SEQ ID NO.7:

[0103] GAAGAAGGGGTATCTCTCGAGAAAAGAACTACTTGTACTGTTGC

[0104] SEQ ID NO.8:

[0105] CTAAGGCTACAAACTCAATGATGATGATGATGATGAC

[0106] The gene MlPG28A (nucleotide sequence shown in SEQ ID NO.1) was amplified and constructed between the EcoRI and SalI restriction sites of the pPICZαA vector. The successfully constructed plasmid was named pPICZαA-MlPG28A.

[0107] Example 2: Construction of recombinant expression strain

[0108] The plasmid pPICZαA-MlPG28A constructed in Example 1 was linearized using the restriction endonuclease SacI. The linearized plasmid pPICZαA-MlPG28A was then electroporated into Pichia pastoris X-33 competent cells. The specific procedures are as follows:

[0109] Add 1200 μL of anhydrous ethanol to the linearized product, mix well, and incubate at -20°C for at least 10 min. Centrifuge at 12000 rpm for 5 min, discard the supernatant, then add 400 μL of pre-cooled 70% ethanol, centrifuge at 12000 rpm for 5 min, air dry, and then add 10 μL of sterile ddH2O to dissolve the nucleic acid precipitate. [The remaining liquid is then added to 10 μL of the solution.] The nucleic acid precipitate from ddH2O was added to a pre-chilled 2mm electroporation cuvette containing 80μL of Pichia pastoris X-33 competent cells. The mixture was gently tapped and rotated to mix, then quickly transferred to ice and allowed to stand for 5 minutes before electroporation. Immediately after electroporation, 400μL of pre-chilled 1M sorbitol was added to the electroporation cuvette. The liquid in the electroporation cuvette was then thoroughly mixed and transferred to a 1.5mL sterile centrifuge tube. After incubation at 28°C for 1 hour, the tube was centrifuged at 1500rpm for 1 minute. 300μL of supernatant was removed, and the remaining bacterial culture was mixed thoroughly and spread onto YPD solid medium. The culture was then incubated at 28°C for 3 to 5 days until single colonies appeared.

[0110] After culture, single clones were picked, lysed, and genomic DNA was collected. Positive clones were verified by colony PCR using primers 5'-AOX and 3'-AOX (sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively). After colony PCR, the PCR products were analyzed by agarose gel electrophoresis. Clones showing a single bright band were identified as positive clones and named pPICZαA-MlPG28A.

[0111] SEQ ID NO.9:

[0112] GACTGGTTCCAATTGACAAGC

[0113] SEQ ID NO.10:

[0114] GCAAATGGCATTCTGACATCC

[0115] Example 3: Prediction of functional mutation sites in polygalacturonase

[0116] Polygalacturonase MlPG28A was modeled, and computer-aided protein design was performed based on the modeling results. Through sequence analysis, phylogenetic tree construction, substrate specificity, molecular docking, molecular dynamics, and AlphaFold3 structure prediction, potential functional mutation sites were identified at positions 106 (lysine), 160 (aspartic acid), 274 (tyrosine), 275 (cysteine), and 283 (cysteine). These key amino acids were replaced with alanine (Ala) using an alanine scanning method.

[0117] Example 4: Construction of a recombinant vector for a polygalacturonase mutant

[0118] Based on the results of Example 3, a recombinant vector for the above mutant was further constructed. The specific construction process is as follows:

[0119] Using recombinant plasmid pPICZαA-MlPG28A as a template, the mutant sequences were constructed using overlapping PCR with primer sequences shown in Table 1. The obtained PCR products were digested with restriction endonucleases BamHI and XhoI at 37°C for 35 min. Subsequently, gel electrophoresis was performed to separate and recover the correctly sized bands. The digested fragments were ligated into plasmid pPICZαA using the same method as in Example 1, yielding recombinant plasmids for each mutant. The recombinant plasmids were transformed into Trans1-T1 phage-resistant chemoenergetic cells. The transformed plasmids were further validated by PCR, and the validation electrophoresis results are shown below. Figure 1 As shown, each mutant yielded a band of the correct size, indicating that a positive clone mutant was successfully constructed.

[0120] Table 1

[0121]

[0122] Example 5: Induction of expression by recombinant strains

[0123] Transformants of the positive clone mutants obtained in Example 4 were inoculated into 50 mL of BMGY liquid medium (500 mL Erlenmeyer flask) and cultured at 28°C and 200 rpm for 16–18 h until the OD600 reached 2–6. The bacterial cells were centrifuged at 1500 rpm for 5 min at room temperature, the supernatant was removed, and the cells were resuspended in BMGY liquid medium and transferred to 100 mL of the medium (OD600 approximately 1.0). Methanol was added for induction of expression. Negative control strains X-33-pPICZαA (empty plasmid) and X-33-pPICZαA-MlPG28B (wild type) were used for expression simultaneously according to the above two steps. Every 24 h, 1 mL of methanol was added to 100 mL of medium for further induction. After 96 h of induction culture, the supernatant was collected after centrifugation at 12000 rpm for 30 min, and the recombinant protein expression was detected by SDS-PAGE.

[0124] Experimental results are as follows Figure 2 As shown, the mutants corresponding to different channels are 1: wild type, 2: K106A, 3: N160A, 4: Y274A, 5: C275A, and 6: C283A. It can be seen that both the wild type and each mutant express the correct size of the protein.

[0125] Example 6: Purification of polygalacturonase MlPG28A and its mutant

[0126] Following the method in Example 5, recombinant bacteria expressing wild-type and various mutants were induced and cultured for 120 h, then centrifuged at 12000 rpm for 30 min at 4 °C to collect the fermentation supernatant. The protein was purified using a Ni-NTA affinity chromatography column, the specific process of which is as follows: The Ni-NTA chromatography column was vertically fixed. The plastic stopper at the bottom of the Ni-NTA chromatography column was removed to allow the 20% ethanol solution preserving the column material to drain completely; ethanol was removed from the chromatography column using 5V ddH2O. The column was equilibrated with a 5V equilibration buffer; the supernatant crude enzyme solution collected by centrifugation was passed through the column, repeated 3 times to ensure sufficient binding of the recombinant protein to the column material; the elution buffer for contaminating proteins was passed through the column at 3V to thoroughly wash away contaminating proteins; the elution buffer for the target protein was passed through the column at 5V, and the column pass solution was collected. The protein wash buffer was passed through the column at 3V, and the column pass solution was collected. Take 40 μL of each column pass solution collected in the preceding steps, add 10 μL of 5× protein electrophoresis loading buffer, boil in water for 10 min, centrifuge (13000 rpm, 1 min), collect the supernatant, and obtain the purified proteins of wild type and each mutant. Perform SDS-PAGE electrophoresis detection, and determine the optimal elution conditions for the obtained target protein based on the SDS-PAGE electrophoresis results.

[0127] Example 7: Determination of the optimal reaction temperature for polygalacturonase MlPG28A and its mutant

[0128] The protein concentrations of MlPG28A and its mutants K106A, N160A, Y274A, C275A, and C283A were diluted to 0.1 mg / mL, and the optimal reaction temperature was determined as follows: Using the enzyme activity assay method described above, the enzyme activity of the purified recombinant enzyme in a substrate at pH 5 and at different temperatures was measured to characterize its optimal temperature, with the highest measured enzyme activity defined as 100%. The purified recombinant enzyme was reacted at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 30 min, respectively. 128 μL of the reaction solution was taken, 96 μL of DNS was added, and the mixture was boiled for 10 minutes. Then, 1376 μL of purified water was added, and the mixture was then subjected to OD... 540 Measure its absorbance value.

[0129] Experimental results are as follows Figure 3 As shown, the wild-type (WT) enzyme still exhibits high relative activity at high temperatures, such as 70-80°C, consistent with its thermostable polygalacturonase properties, indicating that its structure maintains catalytic efficiency under high-temperature conditions. In contrast, although the relative activity of each mutant at high temperatures decreased compared to the wild-type, the wild-type enzyme itself is a thermostable polygalacturonase, capable of efficiently degrading polygalacturonic acid even after a 15-minute boiling water bath (Chinese Patent Application 202110195466.1). Furthermore, although the mutants have lower activity than the wild-type enzyme, they still retain 30% to 60% of their maximum activity, indicating that these mutants still possess appropriate activity at 70-80°C, thus functioning effectively. In other words, compared to conventional pectinases that are inactivated by high-temperature treatment, these mutants still exhibit appropriate thermostable properties.

[0130] Example 8: Stability determination of polygalacturonase MlPG28A and its mutant

[0131] The protein concentrations of MlPG28A and its mutants K106A, N160A, Y274A, C275A, and C283A were diluted to 0.1 mg / mL, and their stability was determined as follows: The enzyme activity of the purified recombinant enzyme in a substrate at pH 5 and at different treatment times was measured to characterize its stability, with the highest measured enzyme activity defined as 100%. The purified recombinant enzyme was incubated at 90℃ for 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, and 210 min, respectively, and its stability was characterized using the enzyme activity assay method described above.

[0132] Experimental results are as follows Figure 4As shown, the wild-type (WT) and various mutants exhibit strong stability during high-temperature incubation. In the initial stage of incubation (e.g., the first 30 minutes), the activity of each group remained close to 100%. Even with extended treatment time (e.g., 150 minutes), the activity of each group still retained a high proportion (approximately 60%). This demonstrates that the wild-type and various mutants showed similar trends during high-temperature incubation and maintained good stability even after prolonged high-temperature treatment.

[0133] Example 9: Determination of the optimal reaction pH for polygalacturonase MlPG28A and its mutant

[0134] The protein concentrations of MlPG28A and its mutants K106A, N160A, Y274A, C275A, and C283A were diluted to 0.1 mg / mL. The optimal reaction temperature was determined as follows: The enzyme activity of the purified recombinant enzyme was measured at different pH substrates to characterize its optimal pH, with the highest measured enzyme activity defined as 100%. The purified recombinant enzyme was reacted in buffers of different pH values ​​at 30°C for 30 min. The enzyme activity was then tested using the above-described enzyme activity assay method.

[0135] Experimental results are as follows Figure 5 As shown, the wild-type (WT), as a thermostable polygalacturonase, exhibits the highest activity (nearly 100%) in neutral to slightly acidic environments (pH 5-7). This characteristic is closely related to the stability of its native conformation under neutral conditions. Within this pH range, the binding efficiency of the enzyme's active site to the substrate polygalacturonic acid is highest, allowing the catalytic reaction to proceed efficiently. However, when the ambient pH deviates from neutral, the activity of WT decreases significantly: under extremely acidic conditions (pH < 3), its activity is almost completely lost.

[0136] Surprisingly, among the mutants, the K106A and Y274A mutants obtained through alanine scanning reverse design showed significantly enhanced functional adaptability in low pH environments. Figure 3 It is observed that the activity peaks of the K106A and Y274A mutants shift towards the acidic region, while other mutants retain the same trend as the wild type, showing no shift towards the acidic region. The optimal pH for the K106A mutant is 4. Its activity reaches approximately 70% at pH 3 (compared to only 10-20% for the wild type). The optimal pH for the Y274A mutant is 4, and its activity is comprehensively superior to that of the wild type and all other mutants except K106A within the pH range of 2-4. Therefore, the enhanced activity of the K106A and Y274A mutants at low pH levels makes them suitable enzymes for animal feed processing or the acidic environment of the stomach, and their functional optimization is highly compatible with industrial needs.

[0137] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely illustrative of the method and its central idea, and are not intended to limit the process. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. A mutant of polygalacturonase, characterized in that, Mutants of polygalacturonase were obtained by mutating the wild-type polygalacturonase, whose amino acid sequence is shown in SEQ ID NO:2, into K106A or Y274A.

2. A nucleic acid molecule that is isolated or synthesized, characterized in that, The isolated or synthesized nucleic acid molecule encodes a mutant of the polygalacturonase of claim 1.

3. The isolated or synthesized nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequences of the isolated or synthesized nucleic acid molecules are shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. A recombinant vector, characterized in that, The recombinant vector comprises the isolated or synthesized nucleic acid molecule as described in claim 2 or 3.

5. A host cell, characterized in that, The host cell contains the isolated or synthesized nucleic acid molecule as described in claim 2 or 3, or the recombinant vector as described in claim 4.

6. A feed composition, characterized in that, The feed composition includes a mutant of the polygalacturonase as described in claim 1.

7. The feed composition according to claim 6, characterized in that, The feed composition is for monogastric animals.

8. A method for producing a mutant of polygalacturonase, characterized in that, Includes the following steps: Prepare the recombinant vector according to claim 4; The recombinant vector was transformed into host cells; The host cells were fermented to isolate and purify the mutant of polygalacturonase.

9. The use of the mutant of polygalacturonase according to claim 1 in the preparation of feed.

10. The application according to claim 9, characterized in that, The feed is for monogastric animals.

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