A recombinant Poria cocos chitin endonuclease and its gene, preparation method and application

Abstract

This invention discloses a chitin endonuclease gene, recombinant vector, recombinant bacteria, recombinant enzyme, preparation method, and applications of high expression of Poria cocos chitin endonuclease. The nucleotide sequence of the gene is shown in SEQ ID No. 1. The nucleotide sequence shown in SEQ ID No. 1 can be used to achieve high-level recombinant secretory expression of the target protein in Pichia pastoris host bacteria using a Pichia pastoris inducible expression vector. Under high-density fermentation conditions, the total secretory expression level of the target protein can reach 10.7 g / L. The recombinant enzyme is purified by DEAE ion exchange chromatography, and this recombinant enzyme can efficiently hydrolyze colloidal chitin to produce chitin oligosaccharides. The recombinant Poria cocos chitin endonuclease prepared by this invention has important application value and broad application prospects in the preparation of functional chitosan oligosaccharides, antibacterial, antitumor, environmental protection, and agriculture.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically involving the construction of an artificially synthesized chitin endonuclease gene from Poria cocos, optimization of the Pichia pastoris expression system, high-level secretory expression under high-density fermentation conditions, development of efficient purification methods, and analysis and application of enzymatic characteristics. Background Technology

[0002] This invention belongs to the field of bioengineering technology. Specifically, it relates to an innovative method for the efficient production of a recombinant Poria cocos chitin endonuclease based on a Pichia pastoris methanol-induced expression system, and the application of this method in several important fields, including but not limited to biodegradation, agricultural disease control, and the development of medical biomaterials.

[0003] Chitin is an N-acetylglucosamine polymer linked by β-1,4 glycosidic bonds. As the second most abundant renewable resource in nature after cellulose, it is widely distributed, primarily found in the exoskeletons of crustaceans, the cell walls of fungi, and the exoskeletons of insects. The biodegradation of chitin relies on a specific hydrolytic enzyme—endochitinase (EC 3.2.1.14)—which specifically cleaves the β-1,4 glycosidic bonds within the chitin molecular chain, thus degrading high-molecular-weight chitin into low-polymerization oligosaccharides. Numerous studies have shown that these oligosaccharides possess various biological activities, including significant antibacterial activity, excellent antioxidant properties, and unique immunomodulatory functions, thus demonstrating significant application prospects in multiple fields such as the development of agricultural antifungal agents, the preparation of medical wound healing materials, and the processing of functional foods. However, the current industrial production of endonucleases mainly relies on natural enzyme-producing strains such as Trichoderma spp., which faces many technical bottlenecks, such as generally low enzyme yield (usually below 1 g / L), complex and cumbersome extraction and purification processes, and poor enzyme activity stability.

[0004] Existing recombinant expression technologies face numerous challenges in the large-scale production of chitin endonucleases. First, in prokaryotic expression systems (such as *E. coli*), exogenous proteins readily form insoluble inclusion bodies, resulting in a 60%-80% loss of enzyme activity even after complex refolding processes. Furthermore, due to the lack of post-translational modification capabilities unique to eukaryotes, the final product activity is often significantly lower than that of the natural enzyme. Second, while the *Pichia pastoris* expression system possesses advantages over eukaryotic expression, traditional constitutive expression systems suffer from insufficient expression levels, failing to meet industrial-scale demands. Simultaneously, in conventional fermentation processes, enzyme-producing strains are susceptible to inhibition by carbon and nitrogen source metabolism, and precise control of dissolved oxygen levels and pH is highly complex, leading to enzyme yield fluctuations exceeding 30% during large-scale production. Finally, downstream purification processes are costly, with crude enzyme solutions typically containing over 50% contaminating proteins, requiring multiple complex purification steps, resulting in a final yield of less than 40%, and the resulting enzyme preparations generally exhibiting short half-lives.

[0005] To address the aforementioned issues, there is an urgent need in this field to develop a highly efficient recombinant expression system suitable for industrial-scale production. The Pichia pastoris methanol-induced expression system, as a highly efficient platform for exogenous protein production, offers several significant advantages. Based on the methanol-induced alcohol oxidase (AOX1) promoter, this system is strongly activated in the presence of methanol, driving high-level expression of the target protein, making it ideal for large-scale industrial production. Its regulatory mechanism is extremely stringent; under non-inducible conditions (such as using glycerol or glucose as a carbon source), the promoter is almost inactive, effectively avoiding the metabolic burden or toxic effects on host cells caused by premature exogenous protein synthesis, ensuring the stability of high-density culture. Pichia pastoris can grow to extremely high cell densities in low-cost inorganic salt basal media, and methanol induction significantly increases yield per unit volume, substantially reducing overall production costs. As a eukaryotic expression system, Pichia pastoris possesses complete post-translational modification capabilities, including protein glycosylation and disulfide bond formation, enabling the correct folding of complex eukaryotic proteins, making it particularly suitable for the production of pharmaceutical proteins or enzyme preparations requiring specific modifications. Furthermore, this system supports secretory expression strategies, guiding the target protein to be secreted into the extracellular culture medium via a signal peptide, greatly simplifying the subsequent purification process and significantly reducing host protein contamination. Methanol, as an inducer, is not only inexpensive but also easy to control precisely. Combined with the system's adaptability to high-density fermentation, it offers both economic efficiency and scalability in industrial applications. Compared to mammalian cell culture, Pichia pastoris is simpler to operate and has a shorter culture cycle; compared to prokaryotic expression systems such as Escherichia coli, it expresses eukaryotic proteins with higher activity and a wider range of applications. Therefore, this system is of great significance for achieving efficient and low-cost industrial production of chitin endonuclease. This invention will provide a method for efficiently producing recombinant Poria cocos chitin endonuclease and its multi-field applications.

[0006] Poria cocos is a brown-rot fungus. Through differential expression transcriptomics analysis, the inventors discovered a novel chitin endonuclease expressed at high abundance in Poria cocos under culture conditions using colloidal chitin as the sole carbon source. Sequence alignment analysis revealed that no known sequence with more than 70% homology to this enzyme was found in the Uniprot database, demonstrating significant novelty. Currently, no mature technical method has been reported for achieving efficient secretory expression of this chitin endonuclease using a Pichia pastoris expression system. Summary of the Invention

[0007] Based on this, one objective of the present invention is to provide a chitin endonuclease gene from Poria cocos, the nucleotide sequence of which is shown in SEQ ID No. 1, and the amino acid sequence encoded by which the gene is shown in SEQ ID No. 2. The open reading frame of the sequence SEQ ID No. 1 contains 1260 nucleotides, including a stop codon TAA that does not encode any amino acid. The sequence SEQ ID No. 2 is a sequence consisting of 419 amino acid residues.

[0008] A second objective of this invention is to provide a biological material containing the above-mentioned Poria cocos chitin endonuclease gene, wherein the biological material includes a recombinant expression vector, an expression cassette, or a recombinant bacterium.

[0009] Furthermore, the recombinant expression vector consists of an empty vector and the above-mentioned Poria cocos chitin endonuclease gene inserted into the empty vector, wherein the empty vector is a secretory expression vector.

[0010] Furthermore, the secretory expression vector is pPICZαA.

[0011] Specifically, the recombinant vector is constructed by inserting the target gene sequence as shown in SEQ ID No. 1 into the pPICZαA secretory expression vector.

[0012] The third objective of this invention is to provide a method for preparing recombinant Poria cocos chitin endonuclease, comprising the following steps:

[0013] 1) The above-mentioned chitin endonuclease gene of Poria cocos was constructed into the above-mentioned secretory expression vector to obtain the recombinant expression vector;

[0014] 2) The recombinant expression vector obtained in step 1) was transformed into Pichia pastoris host cells to obtain recombinant bacteria;

[0015] 3) Screen the recombinant bacteria obtained in step 2) to obtain transformants with high expression levels;

[0016] 4) The transformants obtained in step 3) are fermented and cultured, and the supernatant is collected, which contains recombinant Poria cocos chitin endonuclease.

[0017] Furthermore, in step 4), the fermentation conditions are as follows: continuous cultivation at 28 °C for 96-120 hours, with intermittent addition of methanol as a carbon source. The methanol addition rate is controlled in conjunction with dissolved oxygen, with dissolved oxygen set at 30%. The pH is adjusted using concentrated ammonia; the pH is set to 6.0 during the glycerol addition stage and 5.0 during the methanol induction stage.

[0018] Furthermore, the procedure also includes the purification of the supernatant obtained in step 4) using a nickel affinity chromatography column: First, the target protein is precipitated using PEG8000 with a relative saturation of 12%, followed by dialysis with 10 mM Tris-HCl at pH 8.5 to remove salt ions. The DEAE chromatography column is first equilibrated with 10 mM Tris-HCl buffer at pH 8.5, and then the dialysate supernatant after centrifugation and filtration is passed through the column. The column is first washed and equilibrated with 100 ml of ultrapure water, then rinsed with 100 ml of 10 mM Tris-HCl buffer containing 10 mM NaCl at pH 6.8, and finally eluted with 50 ml of 20 mM PBS containing 200 mM NaCl at pH 6 to obtain purified recombinant Poria cocos chitin endonuclease.

[0019] Furthermore, the secretory expression vector includes pPICZαA; the Pichia pastoris host strain includes any one of GS115, X33, or SMD1168.

[0020] Preferably, the secretory expression vector is pPICZαA, the Pichia pastoris host strain is GS115, and high-density fermentation is carried out using a fermenter to obtain a large amount of recombinant Poria cocos chitin endonuclease.

[0021] The fourth objective of this invention is to provide a recombinant enzyme obtained by the above-described method for preparing recombinant Poria cocos chitin endonuclease, and its applications in the preparation of functional chitosan oligosaccharides, antibacterial, antitumor, environmental protection, or agricultural fields. This recombinant enzyme exhibits high activity and can efficiently hydrolyze colloidal chitin to generate chitin oligosaccharides, possessing significant application value in environmental protection and agriculture. For better results, it is recommended to apply this recombinant enzyme at a pH of 4-5 and a temperature of 40-50°C.

[0022] Preferably, the recombinase has an optimal pH of 5 and an optimal temperature of 50 °C.

[0023] In summary, the high-expression Poria cocos chitin endonuclease gene provided by this invention, with its nucleotide sequence shown in SEQ ID No. 1, can achieve high-level recombinant secretory expression of the target protein in Pichia pastoris host bacteria through a Pichia pastoris inducible expression vector. Under high-density fermentation conditions, the total secretory expression level of recombinant Poria cocos chitin endonuclease can reach 10.7 g / L; the recombinant enzyme purified by DEAE ion exchange chromatography can efficiently hydrolyze colloidal chitin to generate chitin oligosaccharides. The recombinant Poria cocos chitin endonuclease prepared by this invention has important application value and broad application prospects in the fields of functional oligosaccharide preparation, antibacterial, antitumor, environmental protection, and agriculture. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the construction of the expression vector pPICZαA-Poria chitin endonuclease in an embodiment of the present invention;

[0025] Figure 2 This is the SDS-PAGE detection result of the yeast transformant culture supernatant of the high Zeocin-resistant Poria cocos chitin endonuclease gene in this embodiment of the invention;

[0026] Figure 3 This is the SDS-PAGE detection result of the target protein expression at different time points under shake flask culture conditions in this embodiment of the invention;

[0027] Figure 4 This is the SDS-PAGE detection result of the target protein expression under high-density fermentation culture conditions in this embodiment of the invention;

[0028] Figure 5 This is the SDS-PAGE detection result of the target protein after DEAE ion exchange in an embodiment of the present invention;

[0029] Figure 6 This is a chromatogram showing the LC-MS / MS identification results of the purified protein of this invention;

[0030] Figure 7 The above are the SDS-PAGE results of proteins in the methanol-induced supernatant of Pichia pastoris transformants in the comparative example of this invention.

[0031] Figure 8 The above are the SDS-PAGE results of the supernatant protein from the methanol-induced Pichia pastoris transformant in Example 3 of this invention.

[0032] Figure 9 This is the DNS colorimetric result of the hydrolysis of colloidal chitin by recombinant Poria cocos chitin endonuclease to generate reducing sugars according to the present invention;

[0033] Figure 10The results of the colorimetric experiment on the generation of chitin oligosaccharides from colloidal chitin hydrolyzed by recombinant Poria cocos chitin endonuclease according to the present invention are shown. Detailed Implementation

[0034] The present invention will be illustrated in detail below through examples. These examples are for illustrative purposes only and do not limit the scope of application of the present invention. The present invention is not limited to the following implementation methods or examples. Any improvements and modifications that do not violate the core ideas of the present invention should be included within the protection scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following examples are all purchased commercially.

[0035] The Pichia pastoris strain and secretory expression plasmid used in this invention were purchased from Invitrogen, USA.

[0036] The culture medium formula used is as follows:

[0037] 1) YPD medium: Dissolve 10 g yeast extract and 20 g peptone, bring the volume to 900 mL, autoclave at 121 °C for 15 min, and add 100 mL of 20% sterile glucose solution when cooled to about 70 °C. If preparing YPD solid medium, add 1.8% agar.

[0038] 2) Yeast growth medium (BMGY): Dissolve 10 g yeast extract and 20 g peptone, bring the volume to 800 mL, autoclave at 121 °C for 15 min, cool to room temperature, and add 100 mL 1 M potassium phosphate solution, 100 mL YNB, 2 mL 500× biotin and 20 mL 50% sterile glycerol.

[0039] 3) Yeast induction medium (BMMY): Dissolve 10 g yeast extract and 20 g peptone, bring the volume to 800 mL, autoclave at 121 °C for 15 min, cool to room temperature, and add 100 mL 1 M potassium phosphate solution, 100 mL YNB, 2 mL 500× biotin and 10 mL methanol.

[0040] 4) YPG medium: Dissolve 10 g yeast extract and 20 g peptone, bring the volume to 900 mL, autoclave at 121 °C for 15 min, cool to about 70 °C and add 100 mL of 20% sterile glycerol solution.

[0041] 5) Yeast inorganic salt culture medium: Each liter of culture medium contains 6 g potassium sulfate, 5 g magnesium sulfate, 1 g potassium hydroxide, 9 mL concentrated phosphoric acid, 30 g glycerol, appropriate amount of defoamer and 0.3 g calcium sulfate. Adjust the pH to 5.5 with ammonia water, sterilize by moist heat and cool to room temperature. Add 5 mL trace element solution per liter at the time of inoculation.

[0042] 6) Trace element solution: Each liter contains 65 g FeSO4·7H2O, 24 g MoNa2O4·2H2O, 20 g ZnCl2, 6 g CuSO4·5H2O, 3 g MnSO4·H2O, 0.5 g CoCl2, 0.2 g biotin, 0.1 g KI, 0.05 g H3BO3 and 5.0 mL concentrated H2SO4. After filtration through a 0.22 µm bacterial filter, it is stored at 4 ℃ for later use.

[0043] Example 1

[0044] This embodiment presents an optimized, artificially synthesized chitin endonuclease gene from Poria cocos, the nucleotide sequence of which is shown in SEQ ID No. 1 and consists of 1260 deoxynucleotides. This sequence covers the full-length reading frame of the stable and mature protein of Poria cocos chitin endonuclease and the stop codon TAA, and the encoded protein has the amino acid residue sequence shown in SEQ ID No. 2.

[0045] The DNA sequence of SEQ ID No. 1 was directly ligated into the Pichia pastoris secretory expression vector pPICZαA to obtain the recombinant vector. Subsequently, the recombinant vector was transformed into the Pichia pastoris host strain GS115 using the lithium chloride transformation method. After transformation, selection was performed using YPD plates containing 100 µg / mL Zeocin antibiotic. Next, the Pichia pastoris transformants grown on the plate were washed off with sterile water and spread onto YPD plates containing 500 µg / mL Zeocin antibiotic. Then, the transformants grown on the plates containing 500 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 1000 µg / mL Zeocin antibiotic. After that, the transformants grown on the plates containing 1000 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 1500 µg / mL Zeocin antibiotic. Finally, the transformants grown on the plates containing 1500 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 2000 µg / mL Zeocin antibiotic, thus obtaining highly resistant Pichia pastoris transformants that could grow normally on these plates.

[0046] The selected high-Zeocin-resistant transformants were placed in 50 mL centrifuge tubes containing 6 mL of BMGY culture medium and incubated at 28 ℃ and 250 rpm until OD was reached. 600The bacterial cell count reached 10-15. Cells were collected by centrifugation, and then 1.5 mL of BMMY medium was added. Induction culture was performed at 28 ℃ and 250 rpm for 3 days. From the time of BMMY medium addition, 20 µL of methanol was added to the centrifuge tube approximately every 24 hours, for a total of two additions. After induction culture, the supernatant was obtained by centrifugation, and 20 µL of the supernatant was analyzed by SDS-PAGE to determine the expression status of the target protein. The results showed that the target protein was expressed.

[0047] Example 2

[0048] This embodiment proposes a method for preparing chitin endonuclease protein, the specific steps of which are as follows:

[0049] S1: Construction of recombinant expression vector: The DNA shown in SEQ ID No.1 in Example 1 was ligated to the Pichia pastoris inducible secretory expression vector pPICZαA to obtain the recombinant vector pPICZαA-Poria cocos chitin endonuclease. Figure 1 This diagram illustrates the construction of the pPICZαA-Poria cocos chitin endonuclease recombinant expression vector. The main vector construction steps are as follows:

[0050] (1) Using Xho and Xba The artificially synthesized plasmid containing the synthetic chitin endonuclease gene of Poria cocos was digested with two enzymes (this plasmid contains the chitin endonuclease gene of Poria cocos, and the gene is divided into Xho at both ends). and Xba The target fragment was obtained by detecting the restriction enzyme sites. The reaction system was as follows (all restriction enzymes and buffers were purchased from Takara Bio): 12 µL of plasmid containing the synthesized Poria cocos chitin endonuclease gene, 4 µL of 10×M buffer, Xho 4 U, Xba Add 4 U of sterile water to a final volume of 40 µL;

[0051] (2) Using Xho and Xba Double digestion of pPICZαA yielded the vector fragment. The reaction mixture consisted of: 12 µL of plasmid pPICZαA, 4 µL of 10×M buffer, and Xho. 4 U, Xba Add 4 U of sterile water to a final volume of 40 µL;

[0052] (3) Use the DNA gel recovery kit (purchased from Takara Bio) to recover the target fragment and vector fragment obtained in steps (1) and (2). The specific operation is carried out according to the kit instructions.

[0053] (4) Use T4 DNA ligase (purchased from Takara Bio) to perform a ligation reaction on the target fragment and vector fragment recovered in step (3) so that the target gene is accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor. The reaction system is as follows: 2 µL of vector pPICZαA fragment, 6 µL of target fragment, 2 µL of 10× buffer, 1 µL of T4 ligase, and sterile water to 20 µL.

[0054] S2: Transformation of recombinant plasmid: Transform the recombinant vector pPICZαA-Poria cocos chitin endonuclease using Pme Linearization was achieved through single enzyme digestion. The recombinant vector was then transformed into Pichia pastoris host cells using the lithium chloride conversion method; strain GS115 was selected in this example. After transformation, selection was performed using YPD plates containing 100 µg / mL Zeocin antibiotic. Subsequently, the Pichia pastoris transformants grown on the plate were washed off with sterile water and spread onto YPD plates containing 500 µg / mL Zeocin antibiotic; then, the transformants grown on the plate containing 500 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 1000 µg / mL Zeocin antibiotic; next, the transformants grown on the plate containing 1000 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 1500 µg / mL Zeocin antibiotic; finally, the transformants grown on the plate containing 1500 µg / mL Zeocin antibiotic were washed off with sterile water and spread onto YPD plates containing 2000 µg / mL Zeocin antibiotic, thus obtaining highly resistant Pichia pastoris transformants that could grow normally on the plate.

[0055] S3: Screening and low-level expression of yeast transformants with high-level secretory expression: Transformants showing high Zeocin resistance as verified by PCR were screened and expressed. 6 mL of BMGY culture medium was added to 50 mL centrifuge tubes and cultured at 28 ℃ and 250 rpm until OD... 600 = 15, collect bacterial cells by centrifugation at 2000 g for 10 min, then add 1.5 mL of BMMY medium and induce culture at 28 ℃ and 250 rpm for 4 days, adding 20 µL of methanol to the centrifuge tube every 24 hours (for a total of 3 times). After induction, centrifuge and take 20 µL of supernatant for SDS-PAGE analysis to screen out several transformants expressing recombinant Poria cocos chitin endonuclease at high secretory levels.

[0056] The Pichia pastoris transformants that achieved the highest expression level were subjected to low-level expression in shake flasks: 200 mL of BMGY culture medium was added to a 1 L Erlenmeyer flask and cultured at 28 ℃ and 250 rpm until OD was reached. 600Approximately 15 cells were collected by centrifugation at 2000 g for 10 min, resuspended in 40 mL of BMMY medium, and then transferred to a 250 mL Erlenmeyer flask. The flask was incubated at 28 ℃ and 250 rpm for 4 days. Approximately 500 µL of methanol was added to the flask every 24 hours (a total of 3 additions). A 500 µL sample was taken every 24 hours, centrifuged, and the supernatant was collected. After induction, 20 µL of the supernatant from each sample was used for SDS-PAGE analysis to determine the relationship between the target protein secretion level and methanol induction time under shake-flask conditions.

[0057] S4: High-density fermentation expression of recombinant enzymes: The transformants with the highest expression of chitin endonuclease from Poria cocos selected in S3 were cultured in YPG medium until OD500. 600 12 were used as seed culture. The seed culture was inoculated into a bioreactor containing inorganic salt medium at a volume ratio of 1:5-10 for fermentation. During fermentation, the culture was continued at 28℃, with 50% glycerol added as a carbon source. The glycerol replenishment rate was linked to dissolved oxygen, maintaining dissolved oxygen at 30%. The pH was adjusted to 6.0 using concentrated ammonia until the cell wet weight reached above 260 g / L. Subsequently, methanol was added to induce expression. The methanol replenishment rate was set to be linked to dissolved oxygen; when dissolved oxygen exceeded 30%, methanol was automatically added to maintain dissolved oxygen around 30%. The pH was adjusted to 5.0 using concentrated ammonia. After 5 days of induction culture, a large amount of recombinant Poria cocos chitin endonuclease was obtained from the fermentation supernatant.

[0058] It should be noted that transformants with high Zeocin resistance were screened for expression using BMMY medium. SDS-PAGE analysis revealed several stable yeast transformants with high levels of secretion of Poria cocos chitin endonuclease from high-resistant transformants with resistance levels not lower than 2000 µg / mL Zeocin. Transformants on Zeocin YPD plates with resistance levels below 200 µg / mL showed significantly weaker target protein secretion ability than high-resistant transformants, and the enzyme activity assay results were consistent. SDS-PAGE results of target protein secretion from some high-Zeocin-resistant transformants are shown below. Figure 2 As shown.

[0059] It is particularly important to emphasize that the induction effects of different culture times were compared and analyzed when conducting small-scale protein expression experiments using shake flasks. The study showed that after 1-4 days of methanol-induced culture, timely addition of methanol to maintain the induction conditions significantly increased the total protein expression. The effectiveness of this optimized culture protocol was reflected in a significant increase in the content of soluble protein in the supernatant. Detailed quantitative data have been systematically compiled and are presented in Table 1, which clearly shows the specific effects of different culture times and methanol addition strategies on the total protein content in the supernatant. The experimental results show that this staged methanol addition induction culture method can more efficiently promote the expression and accumulation of the target protein.

[0060] Table 1 Total protein content in small-scale protein expression experiments

[0061]

[0062] SDS-PAGE was used to systematically analyze the expression levels of the target protein at different induction time points. Figure 3 This paper presents SDS-PAGE electrophoresis patterns of the supernatant protein from the fermentation broth after 1 to 4 days of methanol induction under small-scale shake-flask culture conditions. The images clearly show distinct protein bands at the expected molecular weight positions, indicating successful expression of the target protein. Quantitative analysis of the electrophoresis results revealed a gradual increase in the expression level of the target protein with increasing methanol induction time (from 1 to 4 days), demonstrating that continuous methanol supplementation effectively promotes the accumulation of the target protein. Notably, the expression level of the target protein reached an extremely high level on day 4 of induction. To further evaluate the expression efficiency of the target protein, we also determined the proportion of the target protein in the total protein of the supernatant; detailed data are shown in Table 2. These results fully demonstrate the effectiveness of the methanol-induced expression system, providing important evidence for the large-scale production of the target protein.

[0063] Table 2. Percentage of target protein in supernatant obtained at different induction times.

[0064]

[0065] Calculations show that the target protein can reach a concentration of approximately 128 mg / L after 4 days of induction.

[0066] Furthermore, it is particularly important to emphasize that in the large-scale expression preparation of recombinant Poria cocos chitin endonuclease using a high-density fermentation process, a methanol induction culture phase lasting 1 to 5 days, with methanol added in a gradient manner as an inducer, significantly improved the expression level of the target protein. Specifically, compared with shake flask culture conditions, the optimized high-density fermentation system not only promoted the accumulation of cell biomass but, more importantly, greatly enhanced the expression level of the recombinant protein. SDS-PAGE electrophoresis and Bradford protein quantification revealed that the total amount of soluble protein in the supernatant reached its peak level on day 5 of induction culture. Detailed protein expression data and their trends over time are shown in Table 3.

[0067] Table 3 Total protein content under high-density fermentation conditions

[0068]

[0069] The supernatant of the fermentation broth from 0-5 days of induction culture was analyzed by SDS-PAGE, and the results are as follows: Figure 4 As shown, significant target protein expression was observed in the 66-95 kDa range. Supplementing with methanol resulted in a more substantial total amount of target protein obtained during culture. Gray-scale scanning of the SDS-PAGE gel revealed a positive correlation between the target protein's proportion of the total supernatant protein and culture time. Furthermore, the detailed results for the target protein's proportion of the total supernatant protein are shown in Table 4.

[0070] Table 4. Percentage content of target protein in supernatant under high-density fermentation conditions.

[0071]

[0072] The calculated content of the target protein is detailed in Table 5 below. After 5 days (120 h) of methanol-induced expression, the total amount of the target protein reached approximately 10.7 g / L.

[0073] Table 5 Total amount of target protein

[0074]

[0075] Preferably, after step S4, the following step of purifying the protein is further included:

[0076] S5: The culture medium after S4 fermentation was centrifuged, and the target protein was precipitated using PEG8000 with a relative saturation of 12%. Subsequently, dialysis was performed using 10 mM Tris-HCl buffer at pH 8.5 to remove salt ions. First, the DEAE chromatography column was equilibrated with 10 mM Tris-HCl buffer at pH 8.5, and then the dialysis supernatant after centrifugation and filtration was passed through the column. The column was first washed with 100 ml of ultrapure water, then rinsed with 100 ml of 10 mM Tris-HCl buffer containing 10 mM NaCl at pH 6.8. Finally, the target protein was eluted with 20 mM PBS buffer containing 200 mM NaCl at pH 6, thus obtaining high-purity target protein. The SDS-PAGE analysis results of the eluted protein are shown below. Figure 5 As shown in Table 6, after scale-up culture in a fermenter, 175 mg of the target protein could be purified from every 20 mL of fermentation broth. The final recovery rate of the target protein reached 81%, with a purity higher than 97%, indicating that this system has significant advantages in both efficient expression and simple purification.

[0077] Table 6. Results of protein purification from fermentation broth supernatant

[0078]

[0079] S6: The purified recombinant Poria cocos chitin endonuclease protein was identified by nanoLC-MS / MS. The analytical procedure was as follows: The purified protein sample was separated by SDS-PAGE, and the target protein band in the range of 66-95 kDa was excised. The gel band was first reduced with 5 mmol / L dithiothreitol for 40 minutes at room temperature, and then alkylated with 15 mmol / L iodoacetamide in the dark for 40 minutes. Next, the alkylated protein was digested with chymotrypsin at a 1:50 enzyme-substrate ratio overnight at 37°C. The digestion product was acidified with 1% trifluoroacetic acid, desalted using a self-made C18 desalting column, and finally the peptides were concentrated and dried under vacuum to prepare the nanoLC-MS / MS analysis sample.

[0080] The system parameter configuration and analysis are as follows: An Easy-nLC 1000 ultra-high performance liquid chromatography system (ThermoFisher Scientific) was used, paired with a 100 µm × 10 cm nanocolumn filled with 3 µm, 120 Å ReproSil-Pur C18-AQ reversed-phase resin, and a Q Exactive (ThermoFisher Scientific) mass spectrometer was selected. After analyzing the raw data and completing the database search using Proteome Discoverer software, 45 polypeptide fragments of this recombinant protein were successfully identified (see Table 7 for details), and the coverage of the recombinant Poria cocos chitin endonuclease sequence reached 60%. Figure 6 The secondary mass spectrum of the representative peptide LGPDGDPVTDVSGF (corresponding to positions 179-192 of the protein) is displayed to verify that the purified product is the target recombinant Poria chitin endonuclease protein.

[0081] Table 7. Peptides identified by secondary mass spectrometry

[0082]

[0083] The present invention provides SEQ ID No. 1, which uses pPICZαA as the expression vector and GS115 as the expression strain. Through high-density fed-batch fermentation in a fermenter, 175 mg of the target protein can be purified from every 20 mL of fermentation broth, with a final target protein recovery rate exceeding 81% and a purity higher than 97%. This demonstrates that using the Pichia pastoris expression system with pPICZαA as the vector and GS115 as the strain results in high target protein expression levels and ease of purification.

[0084] Comparative example:

[0085] After obtaining data from the NCBI database, primer design was carried out. The target gene was amplified using RT-PCR technology and ligated into a cloning vector. The natural sequence of the target gene is shown in SEQ ID No. 3. In this study, several optimized artificial DNA sequences were also synthesized, including three sequences: SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6.

[0086] Following the methods described in Example 2 regarding vector construction, transformation, screening, and induction, the recombinant Poria cocos chitin endonuclease gene sequence was double-digested with XhoI and XbaI, and then ligated into the pPICZαA expression vector, which had also undergone double digestion with XhoI and XbaI. Pme was used... Single enzyme digestion linearized the recombinant vector pPICZαA-recombinant Poria cocos chitin endonuclease, and then introduced it into Pichia pastoris host cells via lithium chloride conversion. Positive clones were obtained by Zeocin selection.

[0087] PCR-verified high-resistance Pichia pastoris transformants were streaked onto YPD plates containing 2000 µg / mL Zeocin. Transformants with normal growth were selected and placed in 50 mL centrifuge tubes containing 6 mL of BMGY medium. The tubes were incubated at 28 ℃ and 250 rpm until OD (Occurrence Limit) was reached. 600 =15. After collecting the bacterial cells, centrifuge them, add 1.5 mL of BMMY, and continue culturing at 28℃ and 250 rpm for 4 days, adding 20 µL of methanol every 24 hours. After induction, centrifuge and take 20 µL of the supernatant for SDS-PAGE detection. The SDS-PAGE results of the target protein (e.g., ...) Figure 7 The results indicate that only a weak band of the target protein appeared at the target location. This suggests that only by transforming the DNA sequence shown in SEQ ID No. 1 of the sequence listing of this invention into Pichia pastoris can high-level secretory expression of the target protein be achieved.

[0088] Example 3

[0089] This example illustrates the expression of the SEQ ID No. 1 sequence in other Pichia pastoris secretory expression vectors and strains.

[0090] Using pPICZαA as the expression vector and GS115 as the expression strain, efficient expression of the SEQ ID No. 1 sequence in Pichia pastoris can be achieved. Furthermore, the sequence of SEQ ID No. 1 can also achieve high-level induced secretory expression in other expression strains such as X33 and SMD1168, and is not limited to GS115. Due to the numerous combinations, they cannot all be shown in the examples. Only the X33 and SMD1168 expression strains are used as examples for illustration. The simplified steps are as follows: The sequence of SEQ ID No. 1 is cloned into the induced secretory expression vector pPICZαA. Following the Invitrogen expression instructions, the recombinant vector pPICZαA-Poria cocos chitin endonuclease gene is transformed into Pichia pastoris competent cells X33 and SMD1168 by electroporation. Transformants with a resistance level of 2 mg / mL are selected using Zeocin. After PCR verification, the transformants are inoculated into 50 mL centrifuge tubes containing 6 mL of BMGY medium and cultured at 28 ℃ and 250 rpm until OD. 600 =10~15, centrifuge to collect bacterial cells and add 1.5 ml of BMMY medium, continue culturing at 28 ℃ and 250 rpm for 3 days, adding 20 µL of methanol to the centrifuge tube every 24 hours for induction. After induction, centrifuge and collect 20 µL of supernatant, add to sample loading buffer, and the SDS-PAGE detection results of the target protein are as follows. Figure 8As shown in the figure. The results indicate that the sequence of SEQ ID No. 1 was not only efficiently expressed in GS115, but also achieved high-level secretory expression in X33 and SMD1168. Therefore, the sequence of SEQ ID No. 1 can also achieve high-level secretory expression in other secretory expression vectors and other Pichia pastoris host strains.

[0091] Example 4

[0092] 1) The purified Poria cocos chitin endonuclease was found to possess the bioactivity of hydrolyzing colloidal chitin. The specific operation and results are as follows: The prepared colloidal chitin was ultrasonically dispersed in 0.1 M pH 4 citrate / phosphate buffer. Using colloidal chitin at a final concentration of 3 mg / mL as the substrate, recombinant Poria cocos chitin endonuclease was added at final concentrations of 0, 50, 100, and 200 µg / mL. After reacting at 45 °C for 1 hour, the reaction was immediately terminated by heating in a boiling water bath for 5 minutes. Subsequently, samples from each time point were added to an equal volume of DNS reagent (containing 3,5-dinitrosalicylic acid, NaOH, and potassium sodium tartrate) for color development in a boiling water bath. The results are as follows: Figure 9 As shown, the color intensity is positively correlated with the amount of enzyme added, which clearly indicates that the recombinant Poria cocos chitin endonuclease has the activity of hydrolyzing colloidal chitin.

[0093] 2) The optimal pH for recombinant Poria cocos chitin endonuclease was determined using the DNS method. Colloidal chitin was ultrasonically dispersed in 0.1 M citrate / phosphate buffer (pH 3-8), and DNS reagent was prepared simultaneously. For constructing the standard curve, glucose was used as the reducing sugar standard, prepared in a concentration gradient of 0-1.0 mg / mL. 1 mL of each gradient was mixed with the same volume of DNS reagent, incubated in a boiling water bath for 5 minutes, and the absorbance at 540 nm was measured to plot the curve. The enzyme reaction system contained 500 µL colloidal chitin (5 mg / mL), 400 µL buffer, and 100 µL enzyme solution (the blank group used inactivated enzyme solution). The mixture was incubated at 50 °C for 30 minutes, 1 mL of DNS reagent was added to stop the reaction, and the mixture was then subjected to boiling water incubation for color development. After cooling, the mixture was centrifuged at 12000 rpm for 5 minutes to remove unhydrolyzed particles. The supernatant was measured at 540 nm, and the absorbance was converted to glucose concentration using the standard curve. The relative activities under different pH conditions were calculated, and the results are shown in Table 8.

[0094] Table 8 Determination of Optimal pH

[0095]

[0096] The data in Table 8 show that the optimal pH for this recombinant enzyme is approximately 5, and it maintains high enzyme activity within the pH range of 4-6.

[0097] 3) The optimal temperature for recombinant Poria cocos chitin endonuclease was determined using the DNS method. Following the experimental procedure and steps in 2), colloidal chitin was ultrasonically dispersed in 0.1 M pH 5 citrate / phosphate buffer. The enzyme reaction system contained 500 µL of colloidal chitin (5 mg / mL), 400 µL of buffer, and 100 µL of enzyme solution (the blank group used inactivated enzyme solution). After incubation at 35-60 °C for 30 minutes, 1 mL of DNS reagent was added to terminate the reaction, followed by color development in a boiling water bath. After cooling, the mixture was centrifuged at 12000 rpm for 5 minutes to remove unhydrolyzed particles, and the absorbance at 540 nm was measured using the supernatant. The absorbance was converted to glucose concentration using a standard curve, and the relative activities under different temperature conditions were calculated. The results are shown in Table 9.

[0098] Table 9 Determination of Optimal Temperature

[0099]

[0100] Table 9 shows that the optimal temperature for this recombinase is approximately 50°C, and it can maintain high activity within the range of 35-60°C.

[0101] 4) The specific activity of recombinant Poria cocos chitin endonuclease was determined using the DNS method. Following the procedure and steps in 2), colloidal chitin was dispersed by sonication in 0.1 M pH 5 citrate / phosphate buffer. The enzyme reaction system consisted of 500 µL colloidal chitin (5 mg / mL), 400 µL buffer, and 100 µL enzyme solution (1 µg / µL). The mixture was incubated at 50 °C for 30 minutes, followed by the addition of 1 mL of DNS reagent to terminate the reaction, and then color development was performed in a boiling water bath. After cooling, the mixture was centrifuged at 12000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm. The absorbance was converted to glucose concentration using a standard curve. One enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 µg of reducing sugar per hour, and the specific activity of the enzyme was calculated accordingly. The results showed that the specific activity of this recombinant enzyme was approximately 11.8 U / mg.

[0102] Example 5

[0103] This embodiment utilizes thin-layer chromatography to detect the activity of purified recombinant Poria cocos chitin endonuclease in hydrolyzing colloidal chitin to generate chitin oligosaccharides. The specific operation procedure and results are presented below:

[0104] The enzyme reaction system consisted of 500 µL of colloidal chitin (5 mg / mL), 400 µL of pH 5.0 buffer, and 100 µL of enzyme solution. This system was incubated at 50 °C for 0, 1, 2, and 4 hours, followed by heating at 95 °C for 10 minutes to inactivate the enzyme. After cooling, the sample was centrifuged at 12000 rpm for 5 minutes to remove unhydrolyzed particles. The silica gel G plate was activated in a 100 °C oven. After cooling to room temperature, 1 µL of sample was applied using a capillary tube. The sample was applied 1.5 cm from the bottom of the silica gel plate and 2-3 cm from the sides, with a spacing of 1-1.5 cm between each sample spot. The developing solvent (isopropanol:pure water:25% ammonia = 280:119:5.3) was added to the chromatography tank, and after equilibration for several minutes, the silica gel plate was placed inside. When the developing agent rises to a distance of 2-3 cm from the top of the silicone plate, remove the silicone plate and blow it dry. Next, evenly spray the color developer (25% sulfuric acid), and develop the color at 200 ℃ for 10-15 minutes, then remove it from the oven.

[0105] Figure 10 The results of the colorimetric experiment are presented. No colored spots were observed in the colloidal chitin sample without the enzyme; after one hour of reaction with the endonuclease, colored spots appeared, and these spots matched the positions of monosaccharides, chitobiose, chitotriose, and chitotetraose in the chitosan oligosaccharide mixed standard. The spots in the chitobiose region showed the most significant color deepening with increasing reaction time. This indicates that the endonuclease promotes the hydrolysis of colloidal chitin, generating a large amount of chitin oligosaccharides such as chitobiose, chitotriose, and chitotetraose. Therefore, it is demonstrated that the recombinant Poria cocos chitin endonuclease prepared in this invention can efficiently hydrolyze colloidal chitin and produce chitin oligosaccharides.

[0106] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art and will not be elaborated further. The above embodiments and / or experimental examples have provided a detailed explanation of the preferred embodiments of the present invention, but 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 chitin endonuclease gene from Poria cocos, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No. 1, and 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 biomaterials include recombinant expression 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 genome of claim 1 inserted into the empty vector, wherein the empty vector is a secretory expression vector.

4. The biomaterial as described in claim 3, characterized in that, The secretory expression vector includes pPICZαA.

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 the secretory expression vector described in claim 3 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) Screen the recombinant bacteria from step 2) to obtain transformants with high-level expression; 4) Ferment the transformants obtained in step 3), and the resulting supernatant contains recombinant Poria cocos chitin endonuclease.

6. The preparation method according to claim 5, characterized in that, In step 4), fermentation is carried out at 28 °C for 96-120 hours, with methanol added as a carbon source as needed. The methanol addition rate is linked to dissolved oxygen, and the fermentation dissolved oxygen is set at 30%. The pH is adjusted using concentrated ammonia, and the fermentation pH is set at 5.

0.

7. The preparation method according to claim 5 or 6, characterized in that, The process also includes a protein purification step: the target protein is precipitated using PEG8000 with a relative saturation of 12%, and then dialyzed with 10 mM Tris-HCl at pH 8.5 to remove salt ions. The DEAE chromatography column is first equilibrated with 10 mM Tris-HCl buffer at pH 8.5, and then the dialysate supernatant after centrifugation and filtration is passed through the column. The column is washed with 10 mM Tris-HCl buffer at pH 6.8 containing 10 mM NaCl, and then the target protein is eluted with 20 mM PBS at pH 6.0 containing 200 mM NaCl to obtain purified recombinant Poria cocos chitin endonuclease.

8. The preparation method according to any one of claims 5-7, characterized in that, The secretory expression vector includes pPICZαA; the Pichia pastoris host strain includes any one of strains GS115, X33, and SMD1168.

9. The preparation method according to claim 8, characterized in that, The secretory expression vector is pPICZαA; the Pichia pastoris host strain is strain GS115.

10. The use of the recombinase obtained by any of the preparation methods according to claims 5-9 in the preparation of chitin oligosaccharides using colloidal chitin as a substrate.

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