Mutant gene of poria cocos endo-chitinase and preparation method and application of recombinant protein of mutant gene

Abstract

The invention provides a recombinant bacterium for highly expressing a tuckahoe chitin endonuclease mutant and a preparation method of a protein of the recombinant bacterium. The method comprises the following steps: integrating an optimally designed tuckahoe chitin endonuclease target gene mutant SEQ ID NO.1 into a pichia pastoris expression vector, and then transforming into a pichia pastoris expression strain. A recombinant bacterium for expressing active poria cocos chitin endonuclease is obtained through high-Zeocin-resistance YPD plate screening, and a high-expression pichia pastoris transformant is further selected from high-Zeocin-resistance transformants. Enlarged culture is carried out under a shake flask condition, the expression quantity of the recombinant poria cocos chitin endonuclease reaches 214 mg / L, and protein electrophoresis shows a single band. Ammonia sulfate fractional precipitation and ion exchange technologies are adopted, and efficient purification of the enzyme is achieved. The recombinase mutant can more efficiently hydrolyze colloid chitin to generate chitin oligosaccharide, and has important potential value in the fields of functional chitosan oligosaccharide preparation, biological energy development, medicine research, feed industry, environmental protection, agricultural application and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically discloses a method for synthesizing and constructing a chitin endonuclease mutant gene based on Poria cocos, the recombinant expression and secretion of the gene in Pichia pastoris, the efficient purification process of the obtained recombinant protein, and a systematic analysis of its enzymatic properties. Background Technology

[0002] Chitin is a linear polymer of N-acetylglucosamine linked by β-1,4 glycosidic bonds. As the second most abundant renewable resource in nature after cellulose, it is widely distributed in the shells of crustaceans, fungal cell walls, and insect exoskeletons. Endochitinases (EC 3.2.1.14) can specifically hydrolyze the β-1,4 glycosidic bonds within chitin molecules, and have important applications in biodegradation, industrial production, and biocontrol. These enzymes are widely found in microorganisms, plants, insects, and arthropods. Current research focuses on using metagenomics to discover novel endochitinase genes resistant to extreme conditions from extreme environments such as the deep sea and polar regions.

[0003] The main applications of chitin endonucleases are as follows: (1) Antifungal agents: By degrading the chitin components in the cell walls of pathogenic fungi (such as gray mold and rice blast fungus), they can effectively inhibit the growth of pathogens. For example, endonucleases produced by Trichoderma harzianum have been used in the development of biopesticides. (2) Promote plant immunity: They can induce systemic acquired resistance in plants and enhance the disease resistance of crops. (3) Chitosan oligosaccharide preparation: Chitosan oligosaccharides are produced by enzymatic degradation of chitin. The latter has a variety of biological activities such as antibacterial, antioxidant and immunomodulatory effects. (4) Wound healing: Chitosan oligosaccharides can promote tissue repair and have application potential in the development of medical dressings. (5) Food processing: They are used for shelling of crustaceans and extraction of flavor substances.

[0004] Despite the significant application potential of chitin endonucleases, their industrialization process still faces many challenges: (1) Low efficiency of expression systems: In prokaryotic expression systems (such as E. coli), inclusion bodies are easily formed, and enzyme activity is significantly lost after refolding; in eukaryotic expression systems (such as yeast and insect cells), differences in protein modification patterns may affect enzyme stability. (2) Difficulty in optimizing fermentation processes: Enzyme-producing strains are easily inhibited by carbon and nitrogen sources, and the control of parameters such as dissolved oxygen and pH during fermentation is complex, resulting in unstable yields after large-scale scaling. (3) High cost of downstream purification: The crude enzyme solution contains a high content of impurity proteins, requiring purification through multiple steps of chromatography (such as ion exchange chromatography and affinity chromatography), with recovery rates often below 50%. In addition, the short shelf life of enzyme preparations and freeze-drying or immobilization technologies further increase production costs.

[0005] Natural chitin endonucleases generally suffer from low yield, difficult extraction, and unstable enzyme activity. Existing recombinant expression systems (such as *E. coli* and *Saccharomyces cerevisiae*) also have drawbacks such as low secretion efficiency, complex products, and high purification costs. Traditional fermentation processes are difficult to achieve high-density cultivation and large-scale production. The Pichia pastoris methanol-induced expression system has significant limitations: its expression strictly relies on toxic methanol as an inducer, which not only increases production safety risks but also limits its application in the pharmaceutical / food fields; a staged cultivation strategy (such as glycerol growth followed by methanol induction) is required, making the operation complex and requiring precise control of dissolved oxygen, pH, and methanol concentration gradients, which is difficult for industrial scale-up; methanol metabolism relies on the peroxisomal pathway, causing cell growth to stagnate during the induction phase, with more energy used for target protein expression rather than biomass accumulation, limiting overall fermentation efficiency.

[0006] In contrast, the Pichia pastoris constitutive expression system demonstrates significant industrial application potential by eliminating methanol dependence, simplifying the process, and improving metabolic compatibility. Utilizing constitutive promoters (such as the GAP promoter), this system can continuously express target genes under conventional carbon source conditions such as glycerol or glucose, without methanol induction, significantly reducing safety risks. The fermentation process can be completed in a single stage (using a consistent carbon source throughout), simplifying operation and shortening the production cycle, making it more suitable for large-scale production. With continuous optimization of promoter engineering, host strain modification, and fermentation strategies, the expression efficiency of this system is expected to rival that of methanol-induced systems, making it an ideal platform for the safe and efficient production of recombinant chitinases and other proteins.

[0007] A chitin endonuclease gene was identified from the *Wolfiporia cocos* strain "Xiangjing 28," and the corresponding protein sequence is shown in SEQ ID NO. 3. The expression product of this enzyme in *Pichia pastoris* can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharides (COS), showing significant application potential in the preparation of functional chitosan oligosaccharides, bioenergy production, pharmaceutical development, feed processing, environmental protection, and agriculture. However, the protein encoded by the nucleotide sequence shown in SEQ ID NO. 3, after expression in *Pichia pastoris*, exhibits a heterogeneous double-band phenomenon in electrophoresis. This invention achieves efficient secretion and expression of a uniformly active recombinant protein of this enzyme in a constitutive expression system. Summary of the Invention

[0008] Therefore, the objective of this invention is to provide a chitin endonuclease mutant gene from Poria cocos that enables high-level protein expression with uniform bands and excellent activity, while also covering its recombinant vector, protein preparation method, and related applications.

[0009] In view of this, one of the objectives of the present invention is to provide a highly expressed chitin endonuclease mutant gene of Poria cocos, the nucleotide sequence of which is shown in SEQ ID NO.1, or the amino acid sequence encoded by which the gene is shown in SEQ ID NO.2.

[0010] One of the objectives of this invention is to provide biological materials containing the genes described above, wherein the biological materials include recombinant vectors, expression cassettes, or recombinant bacteria.

[0011] Furthermore, the recombinant expression vector is composed of an empty vector and the aforementioned target gene inserted into the empty vector to form a chitin endonuclease mutant gene of Poria cocos, wherein the empty vector is a constitutive expression vector.

[0012] Preferably, the constitutive expression vector is pGAPZB.

[0013] The second objective of this invention is to provide a method for preparing recombinant Poria cocos chitin endonuclease, comprising the following steps:

[0014] 1) The genes described above are constructed into a constitutive expression vector to obtain a recombinant expression vector;

[0015] 2) Transform the recombinant expression vector obtained in step 1) into Pichia pastoris host cells to obtain recombinant bacteria;

[0016] 3) Ferment the recombinant bacteria obtained in step 2), and the resulting supernatant contains recombinant Poria cocos chitin endopeptidase.

[0017] Furthermore, the fermentation cycle in step 3) is 4 days;

[0018] Furthermore, in step 3), the fermentation process requires the addition of glycerol and potassium phosphate buffer.

[0019] Preferably, the volume of the added glycerin is 1% of the total volume;

[0020] Preferably, the concentration of the potassium phosphate buffer is 1 mol / L, and the volume of the supplemented potassium phosphate buffer is 10% of the total volume;

[0021] Furthermore, the process includes a protein purification step: precipitating the target protein with 90% saturated ammonium sulfate, followed by dialysis desalting with 10 mM Tris-HCl buffer at pH 8.3; equilibrating the DEAE chromatography column with 10 mM Tris-HCl buffer at pH 8.3, then loading the sample, dialysis, centrifuging, and filtering the filtrate; washing the column with a buffer containing 20 mM sodium dihydrogen phosphate at pH 6.0; and finally eluting the target protein with a buffer containing 200 mM NaCl at pH 6.0 to obtain a high-purity product.

[0022] Furthermore, the constitutive expression vector is pGAPZB, and the Pichia pastoris host strain is strain X33.

[0023] A third objective of this invention is to provide the application of the above-described Poria cocos chitin endonuclease mutant gene in the preparation of recombinant Poria cocos chitin endonuclease.

[0024] The fourth objective of this invention is to provide the recombinant enzyme prepared by the above-described preparation method and its application in the hydrolysis of polysaccharides or in the preparation of functional oligosaccharides, bioenergy, feed, medicine, environmental protection and agriculture.

[0025] Preferably, the polysaccharide is chitin;

[0026] Preferably, the preparation of functional oligochitosan is achieved by hydrolyzing polysaccharides.

[0027] The fifth objective of this invention is to provide a method for preserving the recombinant Poria cocos chitin endonuclease prepared by the above-described method, comprising the steps of freezing the recombinant enzyme at -80°C and then freeze-drying it.

[0028] The technical solution provided by this invention has the following advantages: First, the expression method described in this technical solution can secrete, express, and purify recombinant Poria cocos chitin endonuclease mutant protein with uniform molecular weight and high bioactivity. The mutant exhibits increased specific activity and effectively prevents degradation of the expression product by the host bacteria, reducing the metabolic burden on host cells and the toxicity of the expression product to the host. Second, by utilizing the self-signal peptide on the yeast vector pGAPZB-Poria cocos chitin endonuclease mutant to guide the secretory expression of the target protein gene, a large amount of the target protein is secreted into the culture medium, and it can form accurate... The spatial structure of the protein was optimized to maintain the natural activity of the chitin endonuclease in Poria cocos. Thirdly, yeast transformants capable of stable and high-level secretion expression of the chitin endonuclease mutant were obtained through screening. Fourthly, a method for expressing the chitin endonuclease mutant using the eukaryotic host Pichia pastoris and a rapid and efficient method for purifying the chitin endonuclease mutant were developed, which can reduce costs and achieve mass production. Fifthly, the expressed recombinant protein can be rapidly purified using a two-step method of ammonium sulfate precipitation and DEAE chromatography. The purified protein exhibits strong bioactivity in hydrolyzing colloidal chitin and generating chitin oligosaccharides. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the construction of the expression vector pGAPZB-Poria chitin endonuclease mutant in an embodiment of the present invention;

[0030] Figure 2The results of DNS color development after centrifugation of the supernatant and colloidal chitin in the yeast transformants of 12 strains of Poria cocos with high Zeocin resistance according to this invention were obtained by culturing the supernatant and the supernatant. The darker the color, the stronger the enzyme activity.

[0031] Figure 3 This invention uses SDS-PAGE to analyze the supernatant of the most strongly stained transformants in DNS to verify the expression of the target protein;

[0032] Figure 4 The DNS colorimetric results of the supernatant after centrifugation and reaction of the supernatant with colloidal chitin at different times after shake-flask feeding culture of the transformants with the highest protein expression in this invention;

[0033] Figure 5 In this invention, after fed-batch culture of high-expression transformants in shake-flask culture, the expression of the target protein is analyzed by SDS-PAGE.

[0034] Figure 6 The results of SDS-PAGE identification of the target recombinant protein of this invention after purification, dialysis and concentration.

[0035] Figure 7 The nanoLC-MS / MS spectrum of the purified target recombinant protein is shown below.

[0036] Figure 8 The above are the SDS-PAGE results of the purified product of the comparative sequence of this invention expressed in Pichia pastoris;

[0037] Figure 9 This invention presents HPLC results of the hydrolysis of colloidal chitin by a recombinant Poria cocos chitin endonuclease mutant to generate chitin oligosaccharides. Detailed Implementation

[0038] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available.

[0039] The Pichia pastoris strain and the integrative expression plasmid pGAPZB used in this invention were both purchased from Invritrogen, USA.

[0040] The culture medium formula used is as follows:

[0041] 1) YPD medium

[0042] Completely dissolve 10 g of yeast extract and 20 g of peptone, bring the volume to 900 ml, autoclave at 121 °C for 15-20 min, cool to approximately 70 °C, and then add 100 ml of 20% sterile glucose solution. Adding 1.8% agar to this solution yields YPD solid medium.

[0043] 2) YPG medium

[0044] Completely dissolve 10 g yeast extract, 20 g peptone, and 20 g glycerin, bring the volume to 1000 ml, and autoclave at 121 ℃ for 15-20 min.

[0045] 3) 1 M pH 6.0 potassium phosphate buffer

[0046] Take 132 mL of 1M dipotassium hydrogen phosphate (KH₂PO₄) solution and 868 mL of 1M potassium dihydrogen phosphate (KH₂PO₄) solution, mix them, adjust the pH to 6.0 with KOH, and finally bring the volume to 1000 mL with distilled water. After preparation, autoclave (121°C, 20 min) and store at 4°C for later use.

[0047] Example 1

[0048] This embodiment discloses an optimized, artificially synthesized chitin endonuclease mutant gene from Poria cocos, the nucleotide sequence of which is detailed in SEQ ID No. 1. The recombinant protein sequence expressed by this gene is shown in SEQ ID No. 2. After removing the signal peptide with 25 amino acid residues from the N-terminus, the resulting mature protein contains 504 amino acid residues. The optimized DNA sequence was compared with the natural sequence by NCBI and showed no significant similarity.

[0049] The DNA sequence, optimized based on the chitin endonuclease gene characteristics of Poria cocos and yeast codon bias, was cloned into the Pichia pastoris expression vector pGAPZB to construct a recombinant vector. Subsequently, following the Invitrogen manual, the recombinant vector was introduced into the Pichia pastoris host strain X-33 using the lithium chloride transformation method. After transformation, the vector was plated on YPD plates containing 100 µg / mL Zeocin antibiotic for initial screening.

[0050] Transformants grown on 100 µg / mL Zeocin plates were picked, resuspended in sterile water, and then sequentially plated onto YPD plates containing 250 µg / mL and 500 µg / mL Zeocin for gradient screening. By gradually doubling the Zeocin concentration, highly resistant Pichia pastoris transformants that could stably grow on 2 mg / mL Zeocin YPD plates were finally obtained.

[0051] The selected highly resistant transformants were inoculated into 100 mL Erlenmeyer flasks containing 5 mL of YPG medium and cultured at 28 °C and 250 rpm until OD500. 600 ≈10. Centrifuge and collect the supernatant. Take 500 µL of the supernatant and mix it with 400 µL of colloidal chitin (5 mg / mL) and 100 µL of 50 mM pH 5.0 citrate-phosphate buffer. Incubate at 50℃ for 60 minutes, then cool and centrifuge (12000 g, 5 minutes) to remove unhydrolyzed particles. Add 1 mL of DNS reagent to terminate the reaction. After color development in a boiling water bath, measure the absorbance at 540 nm. The transformant with the highest absorbance value is the strain with the optimal chitin endonuclease expression level in Poria cocos.

[0052] Example 2

[0053] This embodiment provides a method for preparing Poria cocos chitin endonuclease protein, which specifically includes the following steps:

[0054] S1: Construction of expression vector and transformation: The DNA sequence (SEQ ID No. 1) optimized based on the gene sequence characteristics and yeast codon preference described in Example 1 was cloned into the Pichia pastoris constitutive expression vector pGAPZB to construct the recombinant plasmid pGAPZB-Poria cocos chitin endonuclease mutant. Figure 1 A schematic diagram illustrating the construction process of the recombinant plasmid pGAPZB-Poria cocos chitin endonuclease mutant is shown. The main steps of vector construction are as follows:

[0055] (1) Using Xho and Xba The plasmid containing the synthetic chitin endonuclease mutant gene of Poria cocos was digested with two enzymes to obtain the target fragment. The reaction system was as follows (all endonucleases and buffers were purchased from Beyotime Biotechnology): 15 μL plasmid containing the synthetic chitin endonuclease mutant gene of Poria cocos, 5 μL 10×M buffer, Xho 5 U, Xba Add 5 U of sterile water to a final volume of 50 μL.

[0056] (2) Using Xho and Xba pGAPZB was double-digested to obtain the vector fragment. The reaction system was as follows (all restriction enzymes and buffers were purchased from Beyotime Biotechnology): 15 μL of plasmid pGAPZB, 5 μL of 10×M buffer, Xho 5 U, Xba Add 5U of sterile water to a final volume of 50μL.

[0057] (3) The target fragment and vector fragment obtained in steps (1) and (2) are recovered using a DNA gel recovery kit, which was purchased from Beyotime Biotechnology. The specific operation is performed according to the kit instructions.

[0058] (4) The target fragment and vector recovered in step (3) were ligated using T4 DNA ligase (purchased from Beyotime Biotechnology). The target gene was accurately inserted into the reading frame of the constitutive vector. The reaction system was as follows: 1 μL of vector pGAPZB fragment, 3 μL of target fragment, 1 μL of 10× buffer, 0.5 μL of T4 ligase, and sterile water to 10 μL.

[0059] S2: Transformation of the recombinant plasmid: The recombinant vector pGAPZB-Poria cocos chitin endonuclease mutant was transformed using Avr... Linearization was achieved through single enzyme digestion. Following the lithium chloride transformation method provided in the Invitrogen manual, the recombinant vector was transformed into Pichia pastoris host cells; in this example, X-33 was used. After transformation, selection was performed using YPD plates containing 100 µg / mL Zeocin antibiotic.

[0060] S3: Screening and protein expression of high-level Pichia pastoris transformants: After transformation, the transformants were initially screened on YPD plates containing 100 µg / mL Zeocin. The grown transformants were collected, washed with sterile water, and then plated onto YPD plates containing 250 µg / mL Zeocin. Subsequently, the surviving transformants were sequentially transferred to YPD plates containing 500 µg / mL, 1000 µg / mL, and 2000 µg / mL Zeocin. By gradually increasing the antibiotic concentration, transformants that could stably grow on 2 mg / mL Zeocin YPD plates were finally selected.

[0061] Transformants grown on 2 mg / mL Zeocin-resistant YPD plates were inoculated into 100 mL Erlenmeyer flasks containing 5 mL YPG medium and cultured with shaking at 28°C and 250 rpm until OD (October Expiratory Scale) was reached. 600 The absorbance reached 10. Centrifuge at 12,000 g for 10 minutes and collect the supernatant. Take 500 µL of the supernatant and mix it with 400 µL of colloidal chitin (5 mg / mL) and 100 µL of 50 mM citrate-phosphate buffer (pH 5.0). Incubate at 50°C for 60 minutes, then immediately stop the reaction by placing it on ice. Centrifuge at 12,000 g for 5 minutes to remove unhydrolyzed substrate. Add 1 mL of DNS reagent to the supernatant, incubate in a boiling water bath for 10 minutes, and measure the absorbance at 540 nm. Figure 2The degree of color development was positively correlated with chitinase activity: YPG cultures of 6 empty vector transformants and 6 single colonies of X33 originating bacteria showed no color development, while 12 highly resistant transformants all showed significant color development, confirming the expression of an active chitinase mutant protein. The strain with the highest absorbance was preliminarily identified as the engineered strain with the optimal level of expression of the Poria cocos chitinase mutant. Two 20 μL samples of the culture supernatant from the three transformants with the highest absorbance were selected for SDS-PAGE analysis. Figure 3 The results showed that a single band specific to the target protein was detected in the supernatant of all three high-expression transformants, while no such band was observed in the empty vector control. This demonstrates the successful screening of multiple Pichia pastoris transformants that efficiently secrete and express the recombinant Poria cocos chitin endonuclease mutant. Note: Fermentation parameters can be optimized and adjusted according to experimental requirements.

[0062] The transformant with the highest absorbance was selected as a strain highly expressing the chitin endonuclease mutant from Poria cocos. This strain was cultured in YPG form until OD... 600 When the pH reaches 10-15, it is used as the seed culture medium, and then transferred to fresh YPG medium at an inoculation ratio of 10% (v / v). During the culture period, glycerol is added to the culture system every 24 hours, with the volume of glycerol added being 1% of the total volume, and 10% of the total volume of 1 mol / L potassium phosphate buffer (pH 6.0) is added at the same time. This feeding operation is performed three times (i.e., four days of culture).

[0063] It should also be noted that the DNS colorimetric method was used to analyze the expression of target proteins at different time points under fed culture conditions, and the colorimetric results are as follows: Figure 4 As shown in the figure, the three repeated experiments all showed that after 24 hours of culture, the activity of chitin-in-the-core enzymes was significantly increased. With the extension of fermentation time, the expression level of the target enzyme was significantly increased. However, after 3 days of culture, the expression of the target recombinant enzyme was close to the highest level on the fourth day. This indicates that under fed-batch conditions, the optimal expression can be obtained when the fermentation cycle is about 4 days. The specific results of the total protein concentration of each supernatant are shown in Table 1 below.

[0064] Table 1 Total Protein Content

[0065]

[0066] SDS-PAGE analysis was performed on the supernatant of the fermentation broth after 1-4 days of culture. The results are as follows: Figure 5As shown, a significant single band of the target protein was present at approximately 66 kDa. With continuous glycerol supplementation, the total yield of the target recombinase mutant protein was significantly increased. SDS-PAGE showed that the proportion of the target protein in the total protein increased with increasing culture time. By day 3, the expression level of the target recombinase protein was close to the peak level reached on day 4. It was finally determined that under fed-batch culture conditions, a fermentation cycle of approximately 4 days yielded optimal protein expression. Colloidal grayscale scanning results confirmed the proportion of the target protein in the total protein of the supernatant; detailed data are shown in Table 2.

[0067] Table 2. Percentage of target protein in supernatant obtained at different induction times.

[0068]

[0069] The calculated content of the target protein is shown in Table 3 below.

[0070] Table 3 Total Target Protein

[0071]

[0072] Preferably, after step S3, the following step of purifying the protein is further included:

[0073] S4: Centrifuge the supernatant of the fermentation broth obtained in S3, stir in an ice-water bath and slowly add ammonium sulfate powder to achieve a solution concentration of 40% saturation. Incubate overnight at 4°C, then centrifuge (≥12000 g, 20 min). Collect the supernatant, stir again in an ice-water bath and slowly add ammonium sulfate powder to achieve 90% saturation. Incubate overnight at 4°C, then centrifuge (≥12000 g, 20 min). Dissolve the precipitate in 10 mM pH 8.3 Tris-HCl buffer, dialyze using a 10 kDa dialysis bag in the same buffer, centrifuge again (≥12000 g, 20 min), and collect the supernatant. Equilibrate the DEAE chromatography column with 2–4 column volumes of pH 8.3 Tris-HCl buffer (10 mM), then load the sample.

[0074] S5: After washing the DEAE column with pH 6.0 buffer containing 20 mM sodium dihydrogen phosphate for 50 column volumes, elution was performed using pH 6.0 buffer containing 20 mM sodium dihydrogen phosphate and 200 mM NaCl. The eluent was collected, concentrated in a 10 kDa ultrafiltration centrifuge tube, and then analyzed by SDS-PAGE. The results are as follows. Figure 6 As shown, a single target protein band is visible at approximately 66 kDa.

[0075] The SEQ ID No. 1 sequence provided by this invention, using pGAPZB as the expression vector and X33 as the expression strain, can purify approximately 15 mg of the target protein per 100 mL of fermentation broth. The final recovery rate of the target protein can reach nearly 69%, and the purity is above 95%. It can be seen that the Pichia pastoris expression system using the sequence SEQ ID No. 1 provided by this invention, with pGAPZB as the expression vector and X33 as the expression strain, expresses a target protein with high expression levels, few contaminating proteins, and is easy to purify. The relevant purification results are shown in Table 4.

[0076] Table 4. Results of purification of fermentation supernatant protein

[0077]

[0078] The LC-MS / MS identification and analysis steps for the purified recombinant Poria cocos chitin endonuclease mutant protein are as follows: Protein samples eluted with 150 mM imidazole buffer were separated by SDS-PAGE, and the target band at approximately 66 kDa was excised. This gel band was then sequentially reduced with 5 mmol / L dithiothreitol for 40 min at room temperature, followed by alkylation with 15 mmol / L iodoacetamide in the dark for 40 min. Subsequently, the alkylated protein was digested overnight at 37°C with chymotrypsin (Promega) at a 1:50 enzyme-substrate ratio. The digested products were acidified with 1% trifluoroacetic acid, desalted using a self-made C18 desalting column, and the peptides were concentrated and dried under vacuum to prepare nanoLC-MS / MS analysis samples.

[0079] The analytical system parameters were configured as follows: an Easy-nLC 1000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) paired with a self-made nanocolumn (100 μm × 10 cm, filled with 3 μm, 120 Å ReproSil-Pur C18-AQ reversed-phase resin, Dr. Maisch GmbH, Germany), and a Q Exactive mass spectrometer (Thermo Fisher Scientific). Using Proteome Discoverer software to analyze the raw data and perform database searches, 79 peptide fragments of this recombinant protein were successfully identified. Figure 7 The secondary mass spectrum of the representative peptide SLTAAVSAGSANY (corresponding to positions 205-217 of the protein) is shown, confirming that the purified protein is the target recombinant Poria cocos chitin endonuclease mutant protein.

[0080] Preferably, after step S5, the following step of preserving the protein is further included:

[0081] S6: The ultrafiltration concentrate obtained after concentration in a 10 kDa ultrafiltration centrifuge tube was rapidly frozen at -80°C and then freeze-dried to prepare lyophilized protein powder. Approximately 1 mg of the lyophilized powder was dissolved in 500 µL of 50 mM citrate-phosphate buffer (pH 5.0) and centrifuged at 12,000 g for 20 minutes at 4°C. 100 µL of the supernatant was mixed with 400 µL of colloidal chitin suspension (5 mg / mL) and 500 µL of 50 mM citrate-phosphate buffer (pH 5.0) and reacted at 50°C for 60 minutes. After centrifugation at 12,000 g for 5 minutes to remove unhydrolyzed substrate, 1 mL of DNS reagent was added to the supernatant for color development in a boiling water bath for 10 minutes, ultimately yielding a dark brown product. This phenomenon confirms that the treatment process of this recombinant mutant enzyme did not lead to protein denaturation or degradation.

[0082] Since constitutive transformants do not require the use of flammable and harmful substances such as methanol during cell growth, the key is to achieve constitutive secretory expression of this novel Poria cocos chitin endonuclease gene in Pichia pastoris and establish a high-density fermentation culture system.

[0083] Comparative Example

[0084] 1) In this invention, mutations were first eliminated at the K30R, K145R, V262S, and L331T sites in the chitin endonuclease gene of *Poria cocos* (SEQ ID NO.4). The gene was then transformed into *Pichia pastoris* X33 using the secretory expression plasmid pPICZαA, and positive transformants were obtained through screening. After methanol-induced fermentation, the culture supernatant was collected for enzyme activity assay, and the results showed that the activity of the target enzyme was almost undetectable.

[0085] 2) The cDNA sequence corresponding to the mature protein (SEQ ID NO.4, including a tag consisting of 6 histidine residues and a stop codon TAA that does not encode any amino acids, with the N-terminal 25 amino acid signal peptide removed) was inserted into the multiple cloning site of the Pichia pastoris secretory expression vector pPICZαA. The recombinant Poria cocos chitinase gene fragment and the pPICZαA vector were double-digested with Xho I and Xba I, respectively, and the recombinant expression vector was constructed through ligation. The resulting recombinant plasmid pPICZαA-Poria cocos chitinase was subjected to Sac... After linearization by single enzyme digestion, the enzyme was introduced into Pichia pastoris host cells using lithium chloride transformation, and positive transformants were obtained through Zeocin resistance selection. Highly resistant transformants, verified by PCR, were streaked onto YPD plates containing 2000 μg / mL Zeocin. Well-grown transformants were selected and cultured in 50 mL YPG medium at 28°C and 250 rpm for 18 hours to prepare a seed culture. Subsequently, the seed culture was transferred to 500 mL BMGY medium at a 1:10 (v / v) inoculation rate and cultured under the same conditions (28°C, 250 rpm) until OD. 600 The value reached approximately 15. Bacterial cells were collected by centrifugation at 1500 g at room temperature and resuspended in 100 mL of BMMY induction medium containing 1% (v / v) methanol. The resuspended medium was inducing expression at 28℃ and 250 rpm, with 1.5 mL of methanol added every 24 hours for a total of 4 days. SDS-PAGE analysis was used to analyze the expression of the target protein at different induction time points. Figure 8 The results showed that the protein expressed by SEQ ID NO.4, which did not eliminate mutations at sites such as K55R, K170R, V287S, and L356T, exhibited two bands with heterogeneous molecular weights, one of which was significantly larger than 66 kDa. This comparison indicates that the SEQ ID NO.1 sequence can express a mutant recombinase with a single band on SDS-PAGE.

[0086] Example 3

[0087] The specific activity of the recombinant Poria cocos chitin endonuclease mutant was determined using the DNS method. The specific steps and results are as follows:

[0088] 1) The optimal pH for recombinant Poria cocos chitin endopeptidase was determined using the DNS method.

[0089] First, colloidal chitin was prepared: chitin was dissolved in concentrated hydrochloric acid, stirred under ice bath conditions, then centrifuged and washed until neutral, and finally dispersed by sonication in 0.1 M, pH 3-8 citrate-phosphate buffer. Simultaneously, DNS reagent was prepared, containing 3,5-dinitrosalicylic acid, sodium hydroxide, and potassium sodium tartrate.

[0090] In establishing the standard curve, glucose was used as the reducing sugar standard, and a concentration gradient series from 0 to 1.0 mg / mL was prepared. 1 mL of each sample was mixed with an equal volume of DNS reagent, heated in a boiling water bath for 5 minutes, and the absorbance at 540 nm was measured. The standard curve was then plotted based on this measurement.

[0091] The total volume of the reaction system was 1 mL, containing 500 μL of colloidal chitin substrate (5 mg / mL), 400 μL of phosphate buffer (50 mM, pH 7.0), and 100 μL of enzyme solution (1 μg / μL). The mixture was incubated in a 50°C water bath for precisely 30 minutes, as this temperature and pH are the optimal reaction conditions for this endonuclease. Immediately after the reaction was complete, 1 mL of DNS reagent was added to terminate the enzymatic reaction, and the mixture was heated in a boiling water bath for 10 minutes to ensure complete color development.

[0092] After the samples cooled to room temperature, they were centrifuged at 12000 g for 5 minutes using a high-speed centrifuge to remove unhydrolyzed chitin particles and any impurities. The absorbance of the clear supernatant was measured at 540 nm. Three replicates were performed for each sample, and the average value was used to improve data reliability. The measured absorbance values ​​were converted to reducing sugar concentrations (in glucose equivalents) using a pre-plotted glucose standard curve (concentration range 0.1–2.0 mg / mL). Enzyme activity units (U) are defined as the amount of enzyme required to catalyze the formation of 1 μmol of reducing sugar per minute from a substrate at 50°C and pH 7.0. Specific activity is expressed as the enzyme activity per unit mass of enzyme protein (U / mg).

[0093] The measured data showed that the specific activity of the modified recombinant Poria cocos chitin endonuclease mutant reached approximately 9.2 U / mg. Under identical experimental conditions, parallel measurements were performed on the recombinant enzyme of SEQ ID NO:4 sequence without the elimination of mutations at K55R, K170R, V287S, and L356T sites, and its specific activity was approximately 6.8 U / mg. These results confirm that rationally designed mutations at K55R, K170R, V287S, and L356T sites significantly enhance the catalytic efficiency of the enzyme protein. The specific activity of the mutant was approximately 35% higher than that of the original enzyme, fully validating the effectiveness of the mutation strategy in enhancing the function of Poria cocos chitin endonuclease.

[0094] Example 4

[0095] This invention employs high-performance liquid chromatography (HPLC) to determine the activity of a recombinant Poria cocos chitin endonuclease mutant in hydrolyzing colloidal chitin to generate chitin oligosaccharides. This method features high resolution, high sensitivity, and good reproducibility, accurately reflecting the enzyme's hydrolysis kinetics. The specific steps are as follows: 500 μg of purified recombinant enzyme is added to 5 mL of colloidal chitin solution (pH 5.0, concentration 5 mg / mL). The reaction is carried out at 50℃ with shaking at 180 rpm to ensure a homogeneous reaction system and sufficient contact between the enzyme and substrate. At 0, 0.25, 1, 2, and 4 hours of reaction, 1 mL of sample is taken and immediately placed in a 100℃ water bath for 10 minutes to inactivate the enzyme and terminate the reaction. After cooling, the sample is centrifuged at 12000 g for 5 minutes at 4℃ to thoroughly remove unhydrolyzed particulate substrate and precipitate. The supernatant is collected and filtered through a 0.22 μm microporous membrane to remove any remaining small particles, ensuring sample cleanliness and avoiding column clogging or false peaks.

[0096] The chromatographic conditions used for HPLC analysis were as follows: an SRT-C SEC-100 column (7.8 × 300 mm, 5 μm packing material) was used, with 0.1 M ammonium acetate as the mobile phase, a flow rate of 0.6 mL / min, an injection volume of 10 μL, and a column temperature of 30℃. An evaporative light scattering detector (ELSD) was used, which has high sensitivity for non-optical carbohydrates. Chitosan disaccharides to heptaoses were used as standards, and analysis was performed using a Waters 2695 HPLC system. Each sample was injected three times, and the average value was taken to ensure data reliability.

[0097] The results are as follows Figure 9 As shown, no oligosaccharide chromatographic peaks were detected in the unreacted colloidal chitin solution. After 15 minutes of reaction, disaccharide, trisaccharide, and tetrasaccharide peaks were clearly detected in the chromatogram, with the tetrasaccharide showing the highest peak, indicating it was the initial major product. After 1 hour of reaction, the intensity of each oligosaccharide peak significantly increased, indicating rapid hydrolysis. After 2 hours of reaction, the production of disaccharides, trisaccharides, and tetrasaccharides continued to increase, revealing the enzyme's good sustained catalytic ability. By 4 hours of reaction, the increase in oligosaccharide production slowed significantly, suggesting that the substrate was gradually depleted or the reaction was approaching equilibrium. These results indicate that this recombinant endonuclease mutant can efficiently and continuously hydrolyze colloidal chitin, mainly producing chitin oligosaccharides dominated by disaccharides to tetrasaccharides, demonstrating good application potential.

[0098] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A highly expressed chitin endonuclease mutant gene from Poria cocos, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.1, or the amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. A biological material comprising the gene of claim 1, characterized in that, The biological materials mentioned include recombinant vectors, expression cassettes, or recombinant bacteria.

3. The biomaterial as described in claim 2, characterized in that, The recombinant expression vector is composed of an empty vector and the target genome of claim 1 inserted into the empty vector, wherein the empty vector is a constitutive expression vector.

4. The biomaterial as described in claim 3, characterized in that, The constitutive expression vector is pGAPZB.

5. A method for preparing recombinant Poria cocos chitin endonuclease, characterized in that, Includes the following steps: 1) The gene described in claim 1 is constructed into a constitutive expression vector to obtain a recombinant expression vector; 2) Transform the recombinant expression vector obtained in step 1) into Pichia pastoris host cells to obtain recombinant bacteria; 3) Ferment the recombinant bacteria obtained in step 2), and the resulting supernatant contains recombinant Poria cocos chitin endopeptidase.

6. The preparation method according to claim 5, characterized in that, In step 3), fermentation also requires the addition of glycerol and potassium phosphate buffer.

7. The preparation method according to claim 5, characterized in that, The process also includes protein purification steps: the target protein is precipitated with 90% saturated ammonium sulfate, and then desalted by dialyzing with 10 mM Tris-HCl buffer at pH 8.3; the DEAE chromatography column is equilibrated with 10 mM Tris-HCl buffer at pH 8.3, and then the sample is loaded, dialyzed, and centrifuged to obtain the filtrate; the column is washed with buffer containing 20 mM sodium dihydrogen phosphate at pH 6.0; finally, the target protein is eluted with 20 mM sodium dihydrogen phosphate buffer at pH 6.0 containing 200 mM NaCl to obtain a high-purity product.

8. The preparation method according to any one of claims 5-7, characterized in that, The constitutive expression vector is pGAPZB, and the Pichia pastoris host strain is strain X33.

9. The application of the chitin endonuclease mutant gene of Poria cocos according to claim 1 in the preparation of recombinant chitin endonuclease of Poria cocos.

10. The recombinant enzyme prepared by the method of claim 5 is used in the hydrolysis of polysaccharides or in the preparation of functional oligosaccharides, bioenergy, feed, medicine, environmental protection and agriculture.

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