Recombinant poria cocos chitin endonuclease as well as gene, preparation method and application thereof

Abstract

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the construction of an artificially synthesized chitin endonuclease gene from Poria cocos, optimization of the Pichia pastoris expression system, high-level secretory expression under shake-flask culture conditions, efficient purification process, and analysis and application of its enzymatic characteristics. Background Technology

[0002] Chitin, a substance of vital importance in nature, is an N-acetylglucosamine polymer formed by β-1,4 glycosidic bonds. It is worth emphasizing that chitin is the second most abundant renewable resource in nature, widely found in various biological structures, such as crustacean exoskeletons, fungal cell walls, and insect exoskeletons. The biodegradation of chitin relies on a specific hydrolase—endochitinase (EC 3.2.1.14). This enzyme breaks the β-1,4 glycosidic bonds within the chitin chain, generating low-polymerization-degree chitosan oligosaccharides. Numerous studies have shown that chitosan oligosaccharides possess multiple important functions, such as antibacterial properties that inhibit the growth of harmful bacteria; antioxidant properties that help resist oxidative stress; and immunomodulatory effects that effectively regulate the body's immune system. Given these functions of chitosan oligosaccharides, they have significant application value in multiple fields. In agricultural antifungal preparations, they can help crops resist fungal attacks; in medical healing materials, they can promote wound healing; and they can also play a role in food processing. Currently, industrial production mostly uses natural strains such as Trichoderma to produce chitinase. However, this production method has many problems, such as low enzyme yield; complex extraction processes that require a lot of time and effort; and poor enzyme activity stability, with enzyme activity easily affected by different environmental conditions.

[0003] Currently, in the field of chitin-related research, most chitinases obtained through recombinant expression technology exhibit a relatively typical characteristic. Specifically, these chitinases typically only possess a certain degree of hydrolytic activity towards colloidal chitin, promoting a certain degree of decomposition during interaction with colloidal chitin. However, when faced with chitin or other types of polysaccharides, these chitinases show almost no degradation ability, failing to substantially alter the chemical structure of chitin and other polysaccharides. Given the broad application prospects of chitin in numerous fields, such as agriculture, medicine, and food, there is an urgent need for the effective utilization of chitin. Therefore, actively exploring and developing a novel high-activity endochondrinase gene and its recombinant protein preparation method is particularly important. This novel high-activity endochondrinase needs to possess high activity, capable of efficiently catalyzing the hydrolysis of chitin under relatively mild conditions. Simultaneously, it should possess the advantage of broad selectivity for polysaccharide substrates, exhibiting not only good hydrolysis of colloidal chitin but also degradation of chitin and other polysaccharides. This would significantly improve chitin utilization, providing stronger technical support for chitin applications in various fields and promoting the further development of related industries.

[0004] In the large-scale production of chitin endonucleases, existing recombinant expression technologies face a series of complex technical challenges, specifically in the following key aspects. First, prokaryotic expression systems, typically based on *E. coli*, readily form inclusion bodies during expression. After inclusion body formation and refolding, enzyme activity loss can reach 60%–80%. Furthermore, prokaryotic expression systems lack eukaryotic post-translational modification capabilities, resulting in significantly lower activity of their expressed products compared to natural enzymes. Third, downstream purification costs remain high, with crude enzyme solutions containing over 80% contaminating proteins, necessitating multi-step purification processes to meet certain purity standards. Even then, the yield remains below 40%, and the enzyme preparation has a short active half-life, negatively impacting practical application and storage.

[0005] To overcome the aforementioned technical challenges, there is an urgent need to develop a highly efficient recombinant expression system suitable for industrial production. The Pichia pastoris methanol-induced expression system, as a highly efficient platform for the production of exogenous proteins, possesses numerous significant advantages. Firstly, this system relies on a methanol-induced alcohol oxidase (AOX1) promoter, which can be strongly activated in the presence of methanol, promoting high expression levels of the target protein, particularly meeting the needs of large-scale industrial applications. The system's regulation is extremely strict; under non-inducible conditions (such as using glycerol or glucose as carbon sources), there is almost no leakage expression of the promoter, avoiding premature synthesis of exogenous proteins that could burden host cell metabolism or cause toxicity, thus ensuring the stability of high-density culture. Pichia pastoris can grow to extremely high cell densities in low-cost media, and after methanol induction, the yield per unit volume is significantly increased, greatly reducing production costs. As a eukaryotic expression system, Pichia pastoris possesses post-translational modification capabilities (such as 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, guiding the target protein to be secreted extracellularly via a signal peptide, simplifying subsequent purification steps and reducing host protein contamination. Methanol, as an inducer, is inexpensive and easy to control, and its adaptability to high-density fermentation makes this system both economical and scalable for industrial applications. Compared to mammalian cell culture, Pichia pastoris is simpler to operate and has a shorter cycle; compared to prokaryotic systems such as Escherichia coli, it expresses eukaryotic proteins with higher activity and has a wider range of applications. Therefore, this system has significant value in the efficient and low-cost industrial production of chitin endonucleases.

[0006] This invention provides a recombinant chitinase from *Wolfiporia cocos*, its gene, preparation method, and applications. Transcriptome sequencing analysis of the hyphae of strain "Xiangjing 28" from *Wolfiporia cocos*, a brown-rot fungus, revealed a highly abundant chitinase gene. The natural cDNA sequence of this enzyme (as shown in SEQ ID NO:3) was aligned to NCBI nucleotide sequence homology using BLASTN, and no known full-length homologous sequences were found. Therefore, the chitinase from *Wolfiporia cocos* identified in this invention is a novel enzyme that has not been previously reported. Currently, a mature technical solution for achieving efficient secretory expression of this enzyme based on the *Pichia pastoris* expression system has not yet been established. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a chitin endonuclease gene of 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.

[0008] A second objective of this invention is to provide a biological material containing the genes described above, 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 aforementioned gene *Poria cocos* chitin endonuclease gene inserted into the empty vector, wherein the empty vector is a secretory expression vector.

[0010] Preferably, the secretory expression vector is any one of pPICZαA, pPIC9K, and pHIL-S1.

[0011] The third objective of this invention is to provide a method for preparing recombinant Poria cocos chitin endonuclease, comprising the following steps:

[0012] 1) The genes described above are constructed into a secretory expression vector to obtain a recombinant expression vector;

[0013] 2) Transform the recombinant expression vector obtained in step 1) into Pichia pastoris host cells to obtain recombinant bacteria;

[0014] 3) Ferment the recombinant bacteria obtained in step 2), and the resulting supernatant contains recombinant Poria cocos chitin endopeptidase.

[0015] Furthermore, after obtaining the recombinant bacteria in step 2), the process also includes screening the recombinant bacteria to obtain transformants with high-level expression, and then fermenting the obtained transformants.

[0016] Furthermore, the fermentation process described above requires the addition of methanol.

[0017] Preferably, the amount of methanol is 1.5% of the total volume of the culture system, and the culture system is a liquid culture medium.

[0018] Furthermore, the fermentation conditions were maintained at 28°C for 4-5 days.

[0019] Furthermore, the process includes a protein purification step: the supernatant obtained in step 3) is purified using a nickel affinity chromatography column, the column is equilibrated with equilibration buffer, the sample is loaded, and the column is washed with a pH 8.0 buffer containing 10 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole. Finally, the target protein is eluted with a pH 6.0 buffer containing 20 mM sodium dihydrogen phosphate, 100 mM NaCl and 150 mM imidazole.

[0020] Preferably, the secretory expression vector is any one of pPICZαA, pPIC9K, and pHIL-S1; and the Pichia pastoris host strain is any one of strains X33, GS115, SMD1168, and KM71.

[0021] Preferably, the secretory expression vector is pPICZαA; and the Pichia pastoris host strain is strain X33.

[0022] 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.

[0023] Preferably, the recombinase is used in a pH range of 4-6;

[0024] Preferably, the recombinase is used at a pH of 5;

[0025] Preferably, the recombinase is used at a temperature below 50°C;

[0026] Preferably, the recombinase is used at a temperature of 50°C.

[0027] Preferably, the polysaccharide is any one or more of chitin, shrimp shell, crab shell, chitin, agar, agarose and pectin.

[0028] Furthermore, the preparation of functional oligochitosan oligosaccharides is achieved by the recombinase breaking down chitin and / or polysaccharides.

[0029] The technical solution provided by this invention has the following advantages: By continuously optimizing the natural sequence SEQ ID NO.3 of the Poria cocos chitin endonuclease, the nucleotide sequence SEQ ID NO.1 was obtained. A recombinant plasmid and recombinant vector were constructed using the gene shown in SEQ ID NO.1, which can be transformed into Pichia pastoris to obtain recombinant bacteria. Fermentation with the recombinant bacteria can achieve high-level recombinant secretory expression of the target protein. This lays a solid foundation for the application of recombinant Poria cocos chitin endonuclease. Attached Figure Description

[0030] 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;

[0031] Figure 2 The results of this invention are as follows: 10 yeast transformants of Poria cocos with high Zeocin resistance to chitin endonuclease gene were induced with methanol, the supernatant of the culture medium was reacted with colloidal chitin, the supernatant was collected by centrifugation, and then the supernatant was developed by DNS colorimetry; the darker the DNS color, the stronger the chitin endonuclease activity.

[0032] Figure 3 For the present invention Figure 2 SDS-PAGE results of total protein in the supernatant of the three most darkly stained transformants after methanol induction;

[0033] Figure 4The results of the reaction of culture supernatant with colloidal chitin and DNS colorimetric analysis after different methanol induction times in shake flask culture of the highest protein expression transformant of the present invention were used to determine the optimal induction time.

[0034] Figure 5 The image shows the SDS-PAGE results of the target protein expression in the supernatant of the culture medium after different methanol induction times in shake-flask culture of the highest protein expression transformant of the present invention.

[0035] Figure 6 This is an SDS-PAGE image of the target protein after nickel affinity purification and ultrafiltration concentration in an embodiment of the present invention.

[0036] Figure 7 The identification results of the target protein of this invention;

[0037] Figure 8 To illustrate this invention, the DNS colorimetric results of the supernatant of Pichia pastoris expression transformants with three other nucleotide sequences and SEQ ID No. 1 transformant on the hydrolytic activity of colloidal chitin were compared.

[0038] Figure 9 To demonstrate the results of supernatant hydrolysis of colloidal chitin and DNS colorimetric analysis of transformants selected from Pichia pastoris by various expression vectors containing the gene of SEQ ID No. 1 after methanol induction culture, the present invention presents the results of supernatant hydrolysis of colloidal chitin and DNS colorimetric analysis of transformants.

[0039] Figure 10 This is a TLC result of the recombinant Poria cocos chitin endonuclease hydrolyzing colloidal chitin to generate chitin oligosaccharides according to the present invention.

[0040] Figure 11 The results of the colorimetric assay of the products of recombinant Poria cocos chitin endonuclease hydrolyzing shrimp and crab shells, colloidal chitin, chitin and other macromolecular polysaccharides with DNS are shown. Detailed Implementation

[0041] 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 all conventional commercially available products.

[0042] The Pichia pastoris strain and secretory expression plasmid used in this invention were both purchased from Invritrogen, USA.

[0043] The culture medium formula used is as follows:

[0044] 1) Yeast growth medium (BMGY)

[0045] Completely dissolve 10 g of yeast extract and 20 g of peptone, and bring the volume to 800 mL. Autoclave at 121 °C for 15-20 min, cool to room temperature, and add 100 mL of 1 M potassium phosphate solution, 100 mL of YNB, 2 mL of 500× biotin, and 20 mL of 50% sterile glycerol.

[0046] 2) Yeast induction medium (BMMY)

[0047] Completely dissolve 10 g of yeast extract and 20 g of peptone, and bring the volume to 800 mL. Autoclave at 121 °C for 15-20 min, cool to room temperature, and add 100 mL of 1 M potassium phosphate solution, 100 mL of YNB, 2 mL of 500× biotin, and 10 mL of methanol.

[0048] 3) YPD medium

[0049] 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 1.6-1.8% agar will yield YPD solid medium.

[0050] 4) YPG medium

[0051] Dissolve 10 g of yeast extract and 20 g of peptone, bring the volume to 900 mL, autoclave at 121 °C for 15 min, cool to about 70 °C, and then add 100 mL of 20% sterile glycerol solution.

[0052] Example 1

[0053] This invention discloses an optimized, artificially synthesized chitin endonuclease gene from *Poria cocos* and its expression vector, and successfully screened high-level secretory transformants from *Pichia pastoris*. The gene sequence, as shown in SEQ ID No. 1, is a full-length 1533 deoxynucleotides, synthesized based on the sequence characteristics of the *Poria cocos* chitin endonuclease gene itself and the codon bias of *Pichia pastoris*. This sequence contains the complete reading frame, His tag sequence, and stop codon of the mature *Poria cocos* chitin endonuclease protein, and does not contain restriction enzyme sites used for molecular cloning. Its encoded product is a protein with the amino acid sequence shown in SEQ ID No. 2.

[0054] This embodiment describes a chitin endonuclease expression vector for Poria cocos and its recombinant expression transformant method in Pichia pastoris. The detailed steps are as follows:

[0055] S1: Construction and transformation of expression vector: The DNA shown in SEQ ID No. 1 was ligated into the Pichia pastoris inducible secretory expression vector pPICZαA to obtain the recombinant vector pPICZαA-Poria cocos chitin endonuclease. Figure 1 This is a schematic diagram illustrating the construction of the eukaryotic expression vector pPICZαA-Poria cocos chitin endonuclease in an embodiment of the present invention. The main vector construction steps are preferably as follows:

[0056] (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 restriction enzyme sites were identified to obtain the target fragment. The reaction system was as follows (all restriction enzymes and buffers were purchased from Beyotime Biotechnology): 12 μL plasmid containing the synthesized Poria cocos chitin endonuclease gene, 4 μL 10×M buffer, Xho 4 U, Xba Add 4 U of sterile water to a final volume of 40 μL;

[0057] (2) Using Xho and Xba Double digestion of pPICZαA yielded the vector fragment. The reaction mixture consisted of: 12 μL pPICZαA plasmid, 4 μL 10×M buffer, and Xho... 4 U, Xba Add 4 U of sterile water to a final volume of 40 μL;

[0058] (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.

[0059] (4) The target fragment and vector recovered in step (3) are ligated using T4 DNA ligase (purchased from Beyotime Biotechnology). The target gene is accurately inserted into the reading frame of the secretory vector containing the secretory 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.

[0060] S2: Transformation of the recombinant plasmid: The recombinant vector pPICZαA-Poria cocos chitin endonuclease protein was transformed using Sac... Linearization was achieved through single-enzyme digestion. The recombinant vector was then transformed into *Pichia pastoris* host cells using the lithium chloride conversion method; in this example, X33 was selected. After transformation, selection was performed using YPD plates containing 100 µg / mL Zeocin antibiotic. *Pichia pastoris* transformants were progressively inoculated from 100 µg / mL Zeocin YPD plates to higher concentrations: first to 500 µg / mL, then to 1000 µg / mL, 1500 µg / mL, and finally to 2000 µg / mL Zeocin YPD plates. After sterile washing and plating, highly resistant transformants that grew normally at 2000 µg / mL Zeocin were obtained.

[0061] S3: Screening of yeast transformants with high levels of secretory expression: PCR-verified high Zeocin resistance transformants were inoculated into 50 mL centrifuge tubes (containing 6 mL of BMGY medium) and cultured at 28°C and 250 rpm until OD500. 600 15. Collect bacterial cells by centrifugation, resuspend in 1.5 mL BMMY medium, and induce at 28°C and 250 rpm for 72 hours (adding 25 μL of methanol every 24 hours, for a total of two additions). After induction, centrifuge at 12,000×g for 10 minutes to collect the supernatant. Mix 500 μL of supernatant with 500 μL of colloidal chitin solution (5 mg / mL), react at 50°C for 30 minutes, and then centrifuge. Take 0.5 mL of the reaction solution, add an equal volume of DNS reagent to terminate the reaction, and develop the color in a boiling water bath for 10 minutes.

[0062] Figure 2 The staining depth was positively correlated with chitin endonuclease activity: three empty vector transformants showed no staining, while ten highly resistant transformants all showed significant staining, confirming the expression of highly active enzymes. The three transformants with the deepest staining were selected, and 20 μL of the induction supernatant was used for SDS-PAGE. Figure 3 The results showed that a specific band of the target protein was detected in the supernatant of the three high-expression transformants, while no such band was found in the empty vector control, proving that multiple yeast transformants that efficiently secrete and express recombinant Poria cocos chitin endonuclease were successfully screened. Note: Fermentation parameters can be optimized and adjusted according to experimental needs.

[0063] Example 2

[0064] This invention provides a method for the expression and purification of recombinant protein from Poria cocos chitin endonuclease, the detailed steps of which are as follows:

[0065] S4: One transformant with the highest chitin endonuclease activity from the S3 stage was cultured in 50 mL YPG medium at 28℃ and 250 rpm for 18 hours to prepare seed culture. Subsequently, the seed culture was transferred to 500 mL BMGY medium at an inoculum ratio of 1:10 (v / v) and cultured under the same conditions (28℃, 250 rpm) until the culture effluent OD... 600 The value reached approximately 15. The bacterial cells were collected by centrifugation at 1500 g at room temperature. The bacterial cells were resuspended in 100 mL of BMGY induction medium containing 1% (v / v) methanol. The resuspended culture was incubated at 28°C and 250 rpm for induction expression. Subsequently, 1.5 mL of methanol was added to the culture system every 24 hours. After approximately 5 days of induction culture, the fermentation supernatant contained a large amount of recombinant Poria cocos chitin endonuclease.

[0066] It should be noted that this study used the DNS method to detect the activity of chitin endonuclease in the supernatant of the culture medium after methanol induction for different times. Specifically, 500 μL of supernatant was added to a system containing 500 μL of colloidal chitin (5 mg / mL), and incubated at 50℃ for 30 minutes. After centrifugation, 0.5 mL of the supernatant was collected, and 0.5 mL of DNS reagent was immediately added to terminate the reaction, followed by color development in a boiling water bath. The results are shown below. Figure 4 As shown, the color depth is positively correlated with chitin endonuclease activity, meaning that the deeper the color, the stronger the enzyme activity. Experiments showed that DNS color development reached its peak on day 4 of methanol-induced expression, indicating peak enzyme activity; further extension of the induction time did not result in a significant increase in enzyme activity.

[0067] In addition, the expression of the target protein at different induction time points was analyzed using SDS-PAGE. The electrophoresis results are shown in [Figure number missing]. Figure 5 This figure shows the SDS-PAGE results of target protein expression at different time points after methanol induction. The total protein content after methanol induction for 1 to 5 days is detailed in Table 1.

[0068] Table 1 Total protein content at different induction times

[0069]

[0070] SDS-PAGE grayscale analysis showed that, under shake-flask culture conditions, a clear target protein expression band could be detected after one day of methanol induction. With prolonged induction time, the target protein expression level increased accordingly, but it reached its peak on day 4, and further extension of the induction time did not show a significant increase. Furthermore, the proportion of the target protein to the total protein in the supernatant is detailed in Table 2.

[0071] Table 2. Percentage of target protein in supernatant obtained at different induction times.

[0072]

[0073] Calculations show that after 4 days of induction, the total amount of target protein can reach approximately 300.3 mg / L (total protein concentration on day 4 × percentage of target protein on day 4).

[0074] Preferably, after step S4, the following step of purifying the protein is also included:

[0075] S5: Centrifuge the culture medium after fermentation in S4, take the supernatant and adjust the pH to 8.0 using Tris base, centrifuge at a speed of 12000 g or higher for 10 minutes, add the obtained supernatant to a nickel affinity chromatography column equilibrated with pH 8.0 Tris-HCl buffer, and wash the nickel affinity chromatography column with 10-15 column volumes of pH 8.0 buffer containing 10 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole;

[0076] S6: Elute the nickel affinity chromatography column using a pH 6.0 buffer containing 20 mM disodium hydrogen phosphate, 100 mM NaCl, and 150 mM imidazole. The SDS-PAGE results of the eluted proteins are shown below. Figure 6 As shown in the figure. The results indicated that when eluted with buffer containing 150 mM imidazole, the protein exhibited two bands due to differences in the degree of glycosylation modification, both of which were specifically eluted with high purity. The eluent containing the target protein was collected and dialyzed in 10 mM pH 6.0 sodium dihydrogen phosphate buffer using a dialysis bag with a molecular weight cutoff of 10 kDa. Subsequently, the recombinant protein was concentrated by ultrafiltration. The protein purity and recovery results for each purification step are shown in Table 3.

[0077] Table 3. Results of protein purification from fermentation broth supernatant

[0078]

[0079] The SEQ ID No. 1 provided by this invention uses pPICZαA as the expression vector and X33 as the expression strain. After shake-flask amplification and induction culture, 21.8 mg of target protein can be purified per 100 mL of fermentation broth. The final recovery rate of the target protein is greater than 70% and the purity is above 95%. It can be seen that using Pichia pastoris as the expression system, pPICZαA as the expression vector, and X33 as the expression strain, the expression level of the target protein is high and it is easy to purify.

[0080] The LC-MS / MS identification and analysis procedure for recombinant Poria cocos chitin endonuclease protein is as follows: Protein samples eluted with 150 mM imidazole buffer were separated by SDS-PAGE, and two target bands at the 66 kDa position were excised. These gel bands were then sequentially reduced with 5 mmol / L dithiothreitol for 40 min at room temperature and alkylated 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.

[0081] 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 DSSKKELVSYDTPHIASL (corresponding to positions 339-353 of the protein) is shown, confirming that the purified product is the target recombinant Poria cocos chitin endonuclease protein.

[0082] Comparative Example

[0083] Specific primers were designed based on the sequence of SEQ ID NO.3, and the target gene fragment was amplified by RT-PCR and cloned into a vector. The natural nucleotide sequence of the target gene, after amplification, is provided in SEQ ID NO.3, which has had the signal peptide with the N-terminal 25 amino acid residues removed. Several codon-optimized artificial DNA sequences were also synthesized in this study. Two representative sequences (SEQ ID NO.4 and SEQ ID NO.5) were ligated after enzyme digestion and inserted into the multiple cloning site of the Pichia pastoris secretory expression vector pPICZαA. Following the vector construction, transformation, screening, and induction methods described in Example 1, the above-mentioned recombinant Poria cocos chitin endonuclease gene fragment and pPICZαA vector were double-digested with Xho I and Xba I, respectively, and ligated into pPICZαA, which was also double-digested with Xho I and Xba I, to construct the recombinant expression vector. The recombinant plasmid pPICZαA-Poria cocos chitin endonuclease was linearized by Sac I single-enzyme digestion and then introduced into Pichia pastoris host cells using the lithium chloride conversion method. Positive transformants were obtained through Zeocin resistance selection. Highly resistant transformants, verified by PCR, were streaked onto YPD plates containing 2000 μg / mL Zeocin. Transformants in good growth condition were selected and inoculated into 50 mL centrifuge tubes containing 6 mL BMGY medium, and cultured at 28℃ and 250 rpm until OD600. 600 The value reached 10-15. After centrifugation to collect the bacterial cells, they were resuspended in 1.5 mL of BMMY medium and induced to express the enzyme under the same culture conditions, with 20 μL of methanol added every 24 hours. After 72 hours of induction culture, 500 μL of the supernatant was mixed with an equal volume of colloidal chitin solution (5 mg / mL) and incubated at 50°C for 30 minutes to perform the enzyme reaction. After the reaction was terminated, the mixture was centrifuged, and 0.5 mL of the supernatant was added to 0.5 mL of DNS reagent. The amount of reducing sugar produced was detected by colorimetric assay in a boiling water bath. The enzyme activity detection results are as follows: Figure 8 As shown, only transformants carrying the DNA sequence shown in SEQ ID NO.1 achieved efficient secretory expression of the target protein, exhibiting a dark brown to blackish-brown color reaction in the DNS colorimetric reaction; while transformants with other sequences showed only weak color development. This result indicates that the Pichia pastoris engineered strain containing only the SEQ ID NO.1 sequence possesses significant enzymatic activity for hydrolyzing colloidal chitin and producing detectable reducing sugars.

[0084] Example 3

[0085] Besides using the pPICZαA expression vector and the X33 expression strain to achieve high-efficiency expression of the SEQ ID NO.1 sequence in Pichia pastoris, this sequence is also applicable to other Pichia pastoris secretory expression vectors (such as pPIC9K, pHIL-S1, etc.) and expression strains (such as GS115, KM71, SMD1168, etc.), all of which can achieve high-level induced secretory expression. Given the large number of combinations, not all can be shown in the examples. Therefore, the inducible secretory expression vector pPIC9K and the GS115 and SMD1168 strains are selected as additional examples. The main steps are as follows:

[0086] The sequence SEQ ID NO.1 was cloned into the pPIC9K vector. Following the Invitrogen manual, the recombinant vector pPIC9K-Poria cocos chitin endonuclease gene was introduced into GS115 and SMD1168 Pichia pastoris competent cells via the LiCl method. Transformants with a resistance level of 2.0 mg / ml were selected using G418. After PCR verification, the cells were cultured in 50 mL centrifuge tubes containing 6 ml of BMGY medium at 28°C and 250 rpm until OD500. 600 =10~15. Collect bacterial cells by centrifugation, add 1.5 ml of BMMY medium, and incubate at 28℃ and 250 rpm for 3 days, adding 20 μl of methanol every 24 hours during induction. After induction, centrifuge, take 500 μL of supernatant and add it to a system containing 500 μL of colloidal chitin (5 mg / mL), and incubate at 50℃ for 30 minutes. After centrifugation, take 0.5 mL of supernatant, immediately add 0.5 mL of DNS reagent to terminate the reaction and develop color in a boiling water bath.

[0087] according to Figure 9 The results showed that the SEQ ID NO.1 sequence carried by the pPIC9K expression vector achieved high-level secretory expression in both Pichia pastoris GS115 and SMD1168 strains, with a clear colorimetric reaction observed by DNS assay, while the control group of empty vector transformants showed almost no color change. In conclusion, this sequence can achieve efficient secretory expression in various secretory expression vectors and Pichia pastoris host strains.

[0088] Example 4

[0089] This embodiment describes the detection of the chitin endonuclease activity of purified Poria cocos. The specific steps and results are as follows:

[0090] 1) The optimal pH for recombinant Poria cocos chitin endopeptidase was determined using the DNS method.

[0091] 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.

[0092] 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.

[0093] The enzyme reaction system consisted of 500 μL colloidal chitin (5 mg / mL), 400 μL buffer, and 100 μL enzyme solution (the blank group was replaced with inactivated enzyme solution). After incubation at 50°C for 30 minutes, 1 mL of DNS reagent was immediately added to terminate the reaction, and the mixture was then placed in a boiling water bath for color development.

[0094] After cooling, the sample was centrifuged at 12,000 rpm for 5 minutes to remove unhydrolyzed particles. The absorbance of the supernatant was measured at 540 nm. The absorbance values ​​were converted to glucose concentration using a standard curve, and the relative enzyme activities under different pH conditions were calculated. The results are summarized in Table 4.

[0095] Table 4. Determination of Optimal pH

[0096]

[0097] Based on the results in Table 4, the optimal pH for this recombinase is approximately 5.

[0098] 2) Simultaneously, the optimal temperature for recombinant Poria cocos chitin endonuclease was determined using the DNS method. Following method 1), chitin was dissolved in concentrated hydrochloric acid, stirred in an ice bath, and then centrifuged and washed until neutral. The product was ultrasonically dispersed in 0.1 M pH 5 citrate-phosphate buffer to form colloidal chitin. The enzyme reaction system contained: 500 μL colloidal chitin (5 mg / mL), 400 μL buffer, and 100 μL enzyme solution (the blank control group used inactivated enzyme solution). After incubation at 35-60℃ for 30 minutes, 1 mL of DNS reagent was immediately added to terminate the reaction, followed by color development in a boiling water bath. After cooling, centrifugation (12,000 rpm, 5 minutes) was performed to remove unhydrolyzed particles, and the absorbance at 540 nm was measured using the supernatant. The absorbance values ​​were converted to glucose concentration according to the glucose standard curve, and the relative enzyme activity at each temperature point relative to the optimal temperature was calculated. The results are shown in Table 5.

[0099] Table 5, Determination of Optimal Temperature

[0100]

[0101] Table 5 shows that the optimal temperature for this recombinase is approximately 50℃. It maintains high enzyme activity at temperatures below 50℃, but its activity is rapidly lost when the temperature exceeds 55℃.

[0102] Specific activity determination of recombinant Poria cocos chitin endonuclease (DNS method). Following the previously described method, chitin was dissolved in concentrated hydrochloric acid, stirred in an ice bath, centrifuged and washed until neutral, and then ultrasonically dispersed in 0.1 M pH 5.0 citrate-phosphate buffer. The enzyme reaction system contained: 500 μL colloidal chitin (5 mg / mL), 400 μL buffer, and 100 μL enzyme solution (1 μg / μL). The reaction was incubated at 50℃ for 30 minutes, and immediately 1 mL of DNS reagent was added to terminate the reaction. Color development was performed in a boiling water bath. After cooling, centrifugation (12,000 rpm, 5 minutes) was performed to remove unhydrolyzed particles, and the supernatant was measured at 540 nm. The absorbance was converted to reducing sugar (glucose equivalent) concentration according to the glucose standard curve. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute. Based on this, the specific activity of the recombinant enzyme was calculated to be approximately 6.8 U / mg.

[0103] Example 5

[0104] This experiment used thin-layer chromatography to detect the activity of purified recombinant Poria cocos chitin endonuclease in hydrolyzing colloidal chitin to generate chitin oligosaccharides. The specific procedures and results are as follows:

[0105] The enzyme reaction system consisted of 500 μL colloidal chitin (5 mg / mL), 400 μL pH 5.0 buffer, and 100 μL enzyme solution. The system was incubated at 50°C for 0.5, 1, 2, 3, and 4 hours, respectively, followed by heat inactivation at 90°C for 10 minutes. After cooling, the enzyme was centrifuged (12000 rpm, 5 minutes) to remove unhydrolyzed particles, and the supernatant was collected.

[0106] After activation in a 100°C oven, the silica gel G plate was cooled to room temperature. Using a capillary tube, 1 μL of sample was spotted at a distance of 1.5 cm from the bottom edge of the silica gel plate and 2-3 cm from each side edge, with a spot spacing of 1-1.5 cm. A developing solvent (ethyl acetate:acetic acid:water = 2:1:1) was added to the chromatography tank for pre-equilibration, followed by the spotted silica gel plate. When the developing solvent front moved to 2-3 cm from the top of the plate, the silica gel plate was removed and dried. A 25% sulfuric acid colorimetric reagent was evenly sprayed, and the plate was placed in a 120°C oven for color development for 10-15 minutes before removal.

[0107] Results of colorimetric experiment ( Figure 10The results showed that no staining spots appeared on the untreated colloidal chitin sample. Colored spots were observed after 0.5 hours of reaction with the endonuclease. Compared with the chitin-chitosan mixed standard, the reaction product clearly contained monosaccharides, chitobiose, chitotriose, and chitotetraose. With prolonged reaction time, the monosaccharide and chitobiose spots deepened in color, while the content of chitotriose and chitotetraose gradually decreased. This result demonstrates that the endonuclease can effectively degrade colloidal chitin, generating various chitin oligosaccharides with different molecular weights. This confirms that the recombinant Poria cocos chitin endonuclease prepared in this invention can efficiently hydrolyze colloidal chitin and generate the desired target oligosaccharide product.

[0108] Example 6

[0109] This embodiment tested the activity of purified recombinant Poria cocos chitin endonuclease in hydrolyzing shrimp shells, crab shells, chitin, agar, agarose, pectin, etc. The specific steps and results are as follows:

[0110] The enzyme reaction system contained 400 μL of 0.5% (w / v) shrimp shell, crab shell, chitin, agar, agarose, or pectin substrate, as well as pH 5.0 buffer and 100 μL (1 mg / mL) recombinant chitin endonuclease. The reaction system was incubated at 50°C for 1 hour, followed by heat inactivation at 90°C for 10 minutes. After cooling, the mixture was centrifuged (12,000 rpm, 5 minutes), and 0.5 mL of the supernatant was added to 0.5 mL of DNS reagent. The mixture was then heated in a boiling water bath for 10 minutes for color development, and the reducing sugar content was measured. The colorimetric results of the enzyme activity assay are shown below. Figure 11 As shown, this recombinant Poria cocos chitinase can not only hydrolyze colloidal chitin to generate reducing sugars, but also effectively hydrolyze various macromolecular polysaccharide substrates such as shrimp shells, crab shells, chitin, agar, agarose, and pectin, indicating its broad substrate adaptability. In the fields of functional oligosaccharide preparation, bioenergy production, pharmaceutical development, feed, environmental protection, and agricultural applications, this enzyme shows greater application potential than traditional chitinases.

[0111] 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 chitin endonuclease 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 biomaterial containing 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 is any one of pPICZαA, pPIC9K, and pHIL-S1.

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 secretory 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), methanol needs to be added to the fermentation process.

7. The preparation method according to claim 5, characterized in that, The process also includes a protein purification step: the supernatant obtained in step 4) is purified using a nickel affinity chromatography column, the column is equilibrated with equilibration buffer, the sample is loaded, and the sample is washed with a pH 8.0 buffer containing 10 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole. Finally, the target protein is eluted with a pH 6.0 buffer containing 20 mM sodium dihydrogen phosphate, 100 mM NaCl and 150 mM imidazole.

8. The preparation method according to any one of claims 5-7, characterized in that, The secretory expression vector is any one of pPICZαA, pPIC9K, and pHIL-S1; the Pichia pastoris host strain is any one of strains X33, GS115, SMD1168, and KM71.

9. The preparation method according to claim 8, characterized in that, The secretory expression vector is pPICZαA; the Pichia pastoris host strain is strain X33.

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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