Chitin endonuclease based on artificially optimized cDNA (complementary deoxyribonucleic acid) as well as gene, preparation method and application of chitin endonuclease

Abstract

The invention discloses an artificially optimized cDNA (complementary deoxyribonucleic acid)-based high-expression trametes versicolor chitin endonuclease gene, a recombinant vector, recombinant bacteria, recombinant enzyme and a preparation method thereof. The optimized trametes versicolor chitin endonuclease gene, such as a sequence as shown in SEQ ID NO.1, is constructed into a pichia pastoris expression vector and then converted into a pichia pastoris expression strain; according to the method, recombinant bacteria expressing active trametes versicolor chitin endonuclease are obtained through high-Zeocin-resistance YPD plate screening, high-expression pichia pastoris transformants are further screened from high-Zeocin-resistance transformants, finally fermentation is carried out in a bioreactor, obtained supernate contains a large amount of recombinant active trametes versicolor chitin endonuclease, and the recombinant active trametes versicolor chitin endonuclease can be obtained under the high-density fermentation condition. The expression level of the recombinant trametes versicolor chitin endonuclease reaches 3.1 g / L, and the trametes versicolor chitin endonuclease is efficiently purified through ammonia sulfate fractional precipitation and ion exchange. The recombinase can hydrolyze colloidal chitin to generate chitin oligosaccharide, and has important application value and wide application prospect in the aspects of preparation of functional chitosan oligosaccharide, antibiosis and anti-tumor, agricultural and biological control, environmental protection, industrial catalysis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a chitinase based on artificially optimized cDNA, a gene thereof, a preparation method and an application thereof. Background Art

[0002] Chitin is a polymer of N-acetylglucosamine linked by β-1,4 glycosidic bonds. It is the second largest renewable resource in nature after cellulose and is widely distributed in the shells of crustaceans, fungal cell walls, and insect exoskeletons. Chitinase (EC 3.2.1.14), an enzyme that specifically hydrolyzes β-1,4 glycosidic bonds within chitin, has shown important value in biodegradation, industrial production, and biocontrol. This type of enzyme is widely present in nature, primarily from microorganisms, plants, insects, and arthropods. In recent years, the use of metagenomic technology to discover new endonuclease genes that are resistant to extreme conditions from extreme environments (such as the deep sea and the polar regions) has become a research hotspot.

[0003] The application value of chitinase is mainly reflected in the following aspects: (1) Antifungal agent: By degrading chitin in the cell wall of pathogenic fungi (such as gray mold and rice blast fungus), it effectively inhibits their growth. For example, the endonuclease produced by Trichoderma harzianum has been used in the development of biopesticides; (2) Promoting plant immunity: It can induce plant systemic resistance and enhance crop disease resistance;

[0004] (3) Preparation of chitosan oligosaccharides: Chitosan oligosaccharides are produced by enzymatic degradation of chitin. Chitosan oligosaccharides have multiple biological activities such as antibacterial, antioxidant and immunomodulatory activities. (4) Wound healing: Chitosan oligosaccharides can promote tissue repair and therefore have potential applications in the development of medical dressings. (5) Food processing: They are used for shelling crustaceans and extracting flavor substances.

[0005] Although chitinase has great application potential, its industrial production still faces many challenges:

[0006] (1) Inefficient expression system: Prokaryotic expression systems (such as Escherichia coli) are prone to forming inclusion bodies, resulting in significant loss of enzyme activity after renaturation; while eukaryotic systems (such as yeast and insect cells) have differences in glycosylation modifications, which may affect enzyme stability; (2) Difficulty in optimizing the fermentation process: Enzyme-producing strains are easily inhibited by carbon and nitrogen sources, and the control of dissolved oxygen and pH during fermentation is complex, resulting in unstable yields after large-scale expansion; (3) High downstream purification costs: The crude enzyme solution contains a large amount of impurities and requires purification through multiple steps of chromatography (such as ion exchange and affinity chromatography), with yields often below 50%. In addition, the shelf life of enzyme preparations is short, and freeze-drying or immobilization technology further increases costs.

[0007] Natural chitinases suffer from low yields, difficult extraction, and unstable enzyme activity. Existing recombinant expression systems (such as Escherichia coli and Saccharomyces cerevisiae) also suffer from low secretion efficiency, complex products, and high purification costs. Traditional fermentation processes struggle to achieve high-density culture and large-scale production.

[0008] The methanol-inducible expression system of Pichia pastoris has significant limitations: its expression strictly relies on methanol as an inducer, which is toxic, increasing production safety risks and limiting its application in the pharmaceutical / food fields. In addition, the system requires staged cultivation (such as glycerol growth followed by methanol induction), the operation steps are cumbersome, and the dissolved oxygen, pH, and methanol gradient must be precisely controlled, making industrial scale-up difficult. Methanol metabolism requires the participation of peroxisomes, resulting in stagnant bacterial growth during the induction phase, with more energy used for protein expression rather than biomass accumulation, thus limiting fermentation efficiency.

[0009] In contrast, the Pichia pastoris constitutive expression system demonstrates significant potential for industrial application by eliminating methanol dependence, simplifying process flow, and improving metabolic compatibility. This system utilizes constitutive promoters (such as the GAP promoter) to continuously express target genes under conventional carbon sources such as glycerol / glucose, without the need for methanol induction, thereby significantly reducing safety risks. The fermentation process is completed in a single stage (with a consistent carbon source throughout), simplifying the operational process, shortening the production cycle, and making it more suitable for large-scale production. With the continued optimization of promoter engineering, host modification, and fermentation strategies, the expression efficiency of the Pichia pastoris constitutive expression system is expected to be comparable to that of the methanol-inducible system, making it an ideal platform for the safe and efficient production of recombinant proteins.

[0010] Trametes versicolor is a white-rot fungus. Using colloidal chitin as the sole carbon source, the inventors used transcriptomics to sequence the growing mycelium, discovering a highly abundant chitinase gene. This enzyme is hypothesized to play a key role in chitin hydrolysis. Currently, there is no mature solution for the constitutive, efficient secretory expression and industrial production of this chitinase using the Pichia pastoris expression system. Summary of the Invention

[0011] In view of this, one of the objectives of the present invention is to provide a highly expressed Trametes versicolor chitinase gene based on an artificially optimized cDNA. The nucleotide sequence of this gene is shown in SEQ ID NO. 1, which consists of 1029 deoxynucleotides. This sequence includes the native secretion signal peptide of the Trametes versicolor chitinase gene, the full-length reading frame of the mature protein, and a stop codon, encoding a mature protein consisting of 316 amino acid residues as shown in SEQ ID No. 2. The protein encoded by SEQ ID No. 1 includes the N-terminal secretion signal peptide totaling 26 amino acid residues, which is removed in the mature protein sequence of SEQ ID No. 2. In addition, the stop codon does not encode any amino acids.

[0012] The second object of the present invention is to provide a recombinant Trametes versicolor chitinase encoded by the above gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The third object of the present invention is to provide a biological material containing the above-mentioned Trametes versicolor chitinase gene, including a recombinant expression vector, an expression cassette, a recombinant bacterium, etc.

[0014] Furthermore, the recombinant expression vector is composed of an empty vector and the above-mentioned Trametes versicolor chitinase gene inserted into the empty vector, and the empty vector is a constitutive expression vector.

[0015] In particular, the constitutive expression vector is pGAPZB. Preferably, the target gene is inserted between the Xho I and Xba I restriction enzyme sites of the vector.

[0016] Furthermore, the recombinant vector is specifically obtained by inserting the above gene between the Xho I and Xba I restriction sites of the empty expression vector pGAPZB to obtain a recombinant vector expressing the above protein.

[0017] A fourth object of the present invention is to provide a method for preparing a recombinant Trametes versicolor chitinase, comprising the following steps:

[0018] 1) constructing the above-mentioned Trametes versicolor chitinase gene into a constitutive expression vector to obtain a recombinant expression vector;

[0019] 2) transforming the recombinant expression vector obtained in step 1) into Pichia pastoris host cells to obtain recombinant bacteria;

[0020] 3) Screening the recombinant bacteria from step 2) to obtain transformants expressing high levels of expression; screening for highly resistant transformants, and determining, by DNS method, the transformant with the strongest ability to hydrolyze colloidal chitin from the highly resistant transformants, i.e., the transformant with the largest absorbance value obtained by screening, as the transformant expressing the Trametes versicolor chitinase at the highest level;

[0021] 4) Fermenting the transformant obtained in step 3) to obtain a supernatant containing the recombinant Trametes versicolor chitinase.

[0022] Furthermore, in step 4), the fermentation includes continuing to culture at 28° C. for 72-96 hours and automatically adding 50% glycerol as a carbon source, the glycerol addition rate is connected in series with the dissolved oxygen to maintain the dissolved oxygen content of the fermentation at 30%; and using concentrated ammonia water to adjust the pH, setting the fermentation pH to 5.0.

[0023] Furthermore, the above preparation method also includes a protein purification step: precipitating the target protein with an ammonium sulfate solution with a relative saturation of 80%, dialyzing with 10mM Tris-HCl at pH 8 to remove salt ions, first balancing the DEAE chromatography column with 10mM Tris-HCl buffer at pH 8, then passing the dialysate supernatant treated by centrifugation and filtration through the column, rinsing the column with 10 times the column volume of 10mM Tris-HCl buffer at pH 6.8 containing 20mM NaCl, and then eluting the target protein with 20mM PBS at pH 6 containing 100mM NaCl to obtain high-purity recombinant Trametes versicolor chitinase.

[0024] Furthermore, the above preparation method further comprises a protein preservation step: the purified recombinant Trametes versicolor chitinase protein is quickly frozen at -80°C and then freeze-dried.

[0025] Particularly, the constitutive expression vector in the above preparation method is pGAPZB; the Pichia pastoris host strain is X33 strain.

[0026] A fifth object of the present invention is to provide the recombinant enzyme obtained by the above preparation method and the application of the recombinant enzyme in the preparation of functional oligochitosan, medicine, agriculture, biological control, environmental protection or industrial catalysis.

[0027] Beneficial effects: First, the expression method described in this technical solution can secrete, express and purify the recombinant chitinase with biological activity, and at the same time effectively prevent the host bacteria from degrading the expression product, reduce the metabolic load of the host cell and the toxic effect of the expression product on the host; second, the gene secretion expression of the target protein is guided by the self-signal peptide on the yeast vector pGAPZB-chitinase of the target protein, and the target protein will be secreted into the culture medium in large quantities and can form an accurate spatial structure, thereby maintaining the chitinase of the target protein. The present invention provides a novel method for expressing the chitinase from Trametes versicolor using the eukaryotic host Pichia pastoris and a method for rapidly and efficiently purifying the chitinase from Trametes versicolor, thereby reducing costs and enabling mass production. The invention also provides a novel method for rapidly purifying the chitinase from Trametes versicolor using a two-step method of ammonium sulfate precipitation and DEAE chromatography, and the purified protein exhibits strong biological activity in hydrolyzing colloidal chitin and generating chitin oligosaccharides. The chitinase gene and recombinant enzyme of the present invention have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the construction of the expression vector pGAPZB-Trametes versicolor chitinase in an embodiment of the present invention;

[0029] Figure 2 This is a diagram showing the SDS-PAGE detection results of the culture supernatant of the high Zeocin resistance (2 mg / mL) yeast transformant in the embodiment of the present invention;

[0030] Figure 3 Graph showing the SDS-PAGE detection results of the target protein expression at different time points under shake flask culture conditions in an embodiment of the present invention;

[0031] Figure 4 Graph showing the SDS-PAGE results of target protein expression at different time points under high-density fermentation culture conditions in an embodiment of the present invention;

[0032] Figure 5 This is a diagram showing the results of SDS-PAGE detection of the recombinant Trametes versicolor chitinase protein purified in an embodiment of the present invention;

[0033] Figure 6 This is a diagram showing the SDS-PAGE detection result of the culture supernatant of the gene sequence transformant in the comparative example of the present invention;

[0034] Figure 7 This is a graph showing the HPLC results of the recombinant Trametes versicolor chitinase hydrolyzing colloidal chitin to produce chitin oligosaccharides in an example of the present invention. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the examples, which are merely illustrative 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, and any modifications and variations made without violating the spirit of the present invention are intended to be included within the scope of the present invention. The experimental materials or reagents used in the following examples are commercially available unless otherwise specified.

[0036] The present invention uses the Pichia pastoris strain and the integrative expression plasmid pGAPZB, both of which are purchased from Invitrogen Corporation of the United States.

[0037] The culture medium formula used is as follows:

[0038] 1) YPD medium

[0039] Completely dissolve 10g yeast extract and 20g peptone, dilute to 900mL, sterilize by autoclaving at 121°C for 15-20min, cool to approximately 70°C, and add 100mL of 20% sterile glucose solution. Add 1.8% agar to prepare YPD solid medium.

[0040] 2) YPG medium

[0041] Completely dissolve 10 g yeast extract, 20 g peptone, and 20 g glycerol, adjust the volume to 1000 mL, and sterilize by steam autoclaving at 121°C for 15-20 min.

[0042] 3) Inorganic salt culture medium

[0043] Each liter of culture medium contains 6 grams of potassium sulfate, 5 grams of magnesium sulfate, 1 gram of potassium hydroxide, 7 milliliters of concentrated phosphoric acid, 30 grams of glycerol, an appropriate amount of defoaming agent and 0.3 grams of calcium sulfate. Ammonia water is used to adjust the pH to 4.5. After wet heat sterilization and cooling to the room temperature, 5 milliliters of trace element solution are added to each liter of culture medium during inoculation.

[0044] 4) Trace element solution

[0045] Each liter of trace element solution contains: 65g FeSO4·7H2O, 24g MoNa2O4·2H2O, 20g ZnCl2, 6gCuSO4·5H2O, 3g MnSO4·H2O, 0.5g CoCl2, 0.2g biotin, 0.09g KI, 0.02g H3BO3 and 5.0mL concentrated H2SO4. After filtering with a 0.22μm bacterial filter, store in a refrigerator at 4℃ until use.

[0046] Example 1

[0047] This embodiment provides an optimized artificially synthesized Trametes versicolor chitinase gene, the specific sequence of which is shown in SEQ ID No. 1, and the protein sequence corresponding to the gene is shown in SEQ ID No. 2.

[0048] The DNA sequence synthesized according to the sequence characteristics of the chitin endonuclease gene of Trametes versicolor and the yeast codon preference is ligated to the Pichia pastoris expression vector pGAPZB to obtain a recombinant vector. The recombinant vector is then transformed into the Pichia pastoris host strain X-33 using the lithium chloride transformation method provided in the Invitrogen operating manual. After transformation, the recombinant vector is screened using a YPD plate containing 100 μg / mL Zeocin antibiotic. The Pichia pastoris transformants grown on the YPD plate containing 100 μg / mL Zeocin antibiotic are washed with sterile water and plated onto a YPD plate containing a final concentration of 250 μg / mL Zeocin antibiotic. The transformants grown on the resistant plate are then plated onto a YPD plate containing 500 μg / mL Zeocin antibiotic. The Zeocin concentration is then doubled to obtain highly resistant Pichia pastoris transformants that can grow normally on a 2 mg / mL Zeocin YPD plate. Transformants with high Zeocin resistance were screened and cultured in a 100 mL Erlenmeyer flask containing 5 mL YPG culture medium at 28°C and 250 rpm until OD 600 = 10, centrifuge, remove the supernatant, and add 50 μL of the supernatant to 100 μL of colloidal chitin (5 mg / mL) and 100 μL of 50 mM citric acid-phosphate buffer, pH 5.0. Incubate at 50°C for 60 minutes, cool, and centrifuge (12,000 g, 5 minutes) to remove unhydrolyzed particles. Add 0.25 mL of DNS reagent to terminate the reaction, develop color in a boiling water bath, and measure absorbance at 540 nm. The transformant with the highest absorbance value is the transformant expressing the highest level of Trametes versicolor endochitinase.

[0049] Example 2

[0050] This embodiment provides a method for preparing Trametes versicolor chitinase protein, which specifically includes the following steps:

[0051] S1: Construction of recombinant expression vector: The DNA sequence shown in SEQ ID No. 1 synthesized in Example 1 according to the sequence characteristics of the gene itself and the yeast codon preference was ligated into the Pichia pastoris constitutive expression vector pGAPZB to obtain the recombinant vector pGAPZB-Trametes versicolor chitinase. Figure 1 Schematic diagram of the construction of the recombinant vector pGAPZB-Trametes versicolor chitinase. The main steps of vector construction are preferably as follows:

[0052] (1) The plasmid containing the synthetic Trametes versicolor chitinase gene (the plasmid contains the Trametes versicolor chitinase gene and the restriction sites of Xho I and Xba I at both ends of the gene) was double-digested with Xho I and Xba I to obtain the target fragment. The reaction system is as follows (the endonucleases and buffer used were purchased from Biyuntian Biotechnology):

[0053]

[0054] (2) pGAPZB was double-digested with Xho I and Xba I to obtain the vector fragment. The reaction system was as follows (the enzymes and buffer used were purchased from Biyuntian Biotechnology):

[0055]

[0056] (3) The target fragment and vector fragment obtained in steps (1) and (2) were recovered using a DNA gel recovery kit purchased from Biyuntian Biotechnology. The specific operation was carried out according to the kit instructions.

[0057] (4) The target fragment recovered in step (3) and the vector were ligated using T4 DNA ligase (purchased from Biyuntian Biotechnology) to accurately insert the target gene into the constitutive vector reading frame. The reaction system is as follows:

[0058]

[0059] S2: Transformation of the recombinant plasmid: The recombinant vector pGAPZB-Trametes versicolor chitinase was linearized using AvrII digestion and transformed into the Pichia pastoris host strain (strain X-33 was used in this example) using the lithium chloride transformation method provided in the Invitrogen manual. After transformation, the strains were screened using YPD plates containing 100 μg / mL Zeocin. Pichia pastoris transformants grown on YPD plates containing 100 μg / mL Zeocin were washed with sterile water and plated onto YPD plates containing a final concentration of 250 μg / mL Zeocin. Transformants grown on these resistant plates were then plated onto YPD plates containing 500 μg / mL Zeocin. Following the Zeocin concentration multiplication method, highly Zeocin-resistant Pichia pastoris transformants were obtained that could grow normally on YPD plates containing 2 mg / mL Zeocin.

[0060] S3: Screening of yeast transformants with high secretory expression and small-scale protein expression: Select the transformants with high Zeocin resistance and screen for expression: Culture in a 100 mL Erlenmeyer flask containing 5 mL YPG culture medium at 28°C and 250 rpm until the OD 600= 10, centrifuged and the supernatant was collected. 50 μL of the supernatant was added to 100 μL of colloidal chitin (5 mg / mL) and 100 μL of 50 mM citric acid-phosphate buffer (pH 5.0). After incubation at 50°C for 60 minutes, the supernatant was cooled and centrifuged (12000 g for 5 minutes) to remove unhydrolyzed particles. 0.25 mL of DNS reagent was added to terminate the reaction, and the color was developed in a boiling water bath. The absorbance was measured at 540 nm. The transformant with the highest absorbance value was the transformant expressing the highest level of Trametes versicolor endochitinase. The Pichia pastoris transformant with the highest expression level was shaken for small-scale expression: 1000 mL of YPG culture medium was cultured at 28°C and 250 rpm in a 1000 mL Erlenmeyer flask. Glycerol was added to a final concentration of 2% every 24 hours, and 1 mL of the culture supernatant was collected. The relationship between the target protein expression level and fermentation time was analyzed by SDS-PAGE.

[0061] It should be noted that the transformants with high Zeocin resistance were screened and expressed in YPG medium, and three yeast transformants that stably and highly secreted Trametes versicolor chitinase were obtained by DNS method. The transformants with resistance levels below 200 μg / mL on Zeocin YPD plates had significantly lower secretion and expression of the target protein than the transformants with high resistance, and the enzyme activity test showed the same results. The products of hydrolyzed colloidal chitin in the fermentation broth of the three high Zeocin resistance transformants had the maximum absorbance value after DNS determination. At the same time, the SDS-PAGE results of the target proteins in their YPG culture broth are as follows: Figure 2 As shown, the target protein was expressed at about 33 kDa. The transformant expressing the chitinase of Trametes versicolor was cultured in a small amount by shaking flask culture. After 1-4 days of culture, the electrophoresis results were as follows: Figure 3 The SDS-PAGE results of the target protein expression at different time points are shown. After 2 days of culture, the target protein is clearly expressed at around 33 kDa. After 3-4 days of culture, the total amount of target protein is even higher by continuing to add glycerol.

[0062] S4: Mass expression of recombinant enzyme under high-density fermentation conditions: The transformant expressing the highest level of Trametes versicolor chitinase screened in S3 was cultured in YPG medium until OD 600 The seed bacteria are inoculated into a bioreactor containing an inorganic salt medium at a volume ratio of 1:10 for fermentation. The fermentation is continued at 28°C for 96 hours and 50% glycerol is automatically added as a carbon source. The glycerol addition rate is connected in series with the dissolved oxygen, and the dissolved oxygen is set to 30%. Concentrated ammonia is used to adjust the pH to 5.

[0063] It should be noted that the transformant highly expressing Trametes versicolor chitinase was fermented in a fermenter at high density, and the expression of the target protein at different times under fed-batch culture conditions in the fermenter was detected by SDS-PAGE. The electrophoresis results were as follows: Figure 4 As shown, after 24 hours of cultivation, the target protein was clearly expressed at around 33 kDa. As the fermentation time increased, the expression level of the target protein increased significantly. However, after 84 hours of cultivation, the expression of the target protein was close to the highest level. If the cultivation time was extended, the fermentation cost would be greatly increased, but the target protein did not increase significantly. This shows that under the conditions of the fermenter, the optimal expression can be achieved when the fermentation cycle is about 84 hours. In short, as the fermentation time increases, the total amount of protein is also higher. The specific results of the total protein concentration of each supernatant are shown in Table 1 below; the results of the proportion of the target protein to the total protein in the supernatant are shown in Table 2 below.

[0064] Table 1 Total protein content

[0065] Cultivation time (hours) 12 24 36 48 60 72 84 96 Total protein concentration (mg / L) 330 605 1235 1978 3254 4548 4935 5029

[0066] Table 2 The percentage of target protein in supernatant protein obtained at different induction times

[0067] Cultivation time (hours) 12 24 36 48 60 72 84 96 Target protein ratio (%) 79 73 68 65 59 60 63 61

[0068] After calculation, the target protein content is shown in Table 3 below, with the highest content at 84 hours.

[0069] Table 3 Total amount of target protein

[0070] Cultivation time (hours) 12 24 36 48 60 72 84 96 Target protein content (mg / L) 261 442 839 1285 1920 2729 3110 3068

[0071] The supernatant of the fermentation broth, cultured for 1 to 4 days, was collected by centrifugation and 50 μL of the supernatant was added to 100 μL of colloidal chitin (5 mg / mL) and 100 μL of 50 mM citric acid-phosphate buffer, pH 5.0. The mixture was incubated at 50°C for 60 minutes, cooled, and centrifuged (12,000 g for 5 minutes) to remove unhydrolyzed particles. The reaction was terminated by the addition of 0.25 mL of DNS reagent, and color was developed in a boiling water bath. The absorbance was measured at 540 nm. The resulting absorbance changes are shown in Table 4.

[0072] Table 4 Absorbance value change results

[0073] Cultivation time (hours) 12 24 36 48 60 72 84 96 Absorbance was measured at 540 nm 0.18 0.43 0.82 1.27 1.58 1.86 2.01 1.98

[0074] The absorbance value was converted to glucose concentration using a glucose standard curve. After 84 hours of fermentation, the enzyme activity of the Trametes versicolor chitinase in the supernatant reached its maximum value. If one enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μg of reducing sugar per hour, the enzyme activity in the fermentation broth was calculated to be approximately 9570 U / mL, and the specific activity of the enzyme was approximately 3120 U / mg. Preferably, after step S4, the following protein purification step is also included:

[0075] S5: The culture broth after the fermentation in S4 was centrifuged, and ammonium sulfate powder was slowly added to the supernatant while stirring in an ice-water bath until the relative saturation reached 80%. The mixture was allowed to stand at 4°C and centrifuged at a speed of 12,000 g or higher for 10 to 20 minutes. The obtained precipitate was added to a 10 mM pH 8 Tris-HCl buffer to dissolve the precipitate. The resulting solution was dialyzed against a 10 mM pH 8 Tris-HCl buffer using a 10 kDa dialysis bag and centrifuged at a speed of 12,000 g or higher for 30 to 60 minutes. The supernatant was collected and injected into a DEAE chromatography column equilibrated with 2 to 4 times the volume of the chromatography column and a pH 8 buffer containing 10 mM Tris-HCl, and then loaded onto the column.

[0076] S6: The DEAE column was rinsed with 10 column volumes of a buffer solution containing 10 mM Tris-HCl and 20 mM sodium chloride at pH 6.8, and then eluted with a buffer solution containing 20 mM PBS and 100 mM sodium chloride at pH 6. The resulting eluate was concentrated using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa. The SDS-PAGE results of the concentrated protein were as follows: Figure 5 As shown, the results showed that the concentrated protein had a higher purity.

[0077] The SEQ ID No. 1 sequence provided by the present invention uses pGAPZB as an expression vector and X33 as an expression strain. After high-density fermentation, about 200 mg of the target protein can be purified from every 100 mL of fermentation broth. The final recovery rate of the target protein can reach nearly 65% and the purity is above 90%. It can be seen that the sequence SEQ ID No. 1 provided by the present invention uses pGAPZB as an expression vector and X33 as an expression strain in the Pichia pastoris expression system. The target protein is expressed in a high amount with few impurities and is easy to purify. The relevant purification results are shown in Table 5.

[0078] Table 5 Fermentation supernatant protein purification results

[0079] Purification steps Total volume Endonuclease (mg) Purity of target protein (%) Recovery rate (%) Before purification 100 310 63 100 Thiamin deeply 32 262 >80 84.5 dialysis 35 209 >90 67.4 Ultrafiltration concentration 11 202 >90 65.1

[0080] Preferably, after step S6, the method further comprises the following steps of preserving the protein:

[0081] S7: The product obtained by ultrafiltration and concentration was quickly frozen at -80°C and then freeze-dried to obtain a lyophilized protein powder. The lyophilized powder was dissolved in physiological saline and centrifuged at 4°C and 12,000 g for 20 minutes. The supernatant was subjected to SDS-PAGE analysis, and the target protein was detected as a single band, indicating that this protein treatment method did not cause significant protein denaturation or degradation.

[0082] Since constitutive transformants do not require the use of flammable and hazardous substances such as methanol during bacterial growth, the key is to achieve constitutive secretory expression of the new Trametes versicolor chitinase gene in Pichia pastoris and establish a high-density fermentation culture system.

[0083] Comparative Example:

[0084] Trametes versicolor belongs to the Polyporaceae family and the genus Versicolor, and is an annual corky fungus. The inventors used transcriptome technology to analyze the expression profile of the chitinase gene of Trametes versicolor and discovered a chitinase gene with the highest abundance. Using the data obtained from the transcriptome, primers were designed, and the target gene was amplified by RT-PCR and ligated into a cloning vector. The amplified target gene native sequence is shown in SEQ ID NO.3 in the sequence listing. Similarly, several other optimized synthetic artificial DNA sequences were synthesized and ligated into the Pichia pastoris constitutive expression vector pGAPZB. The optimized synthetic artificial sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 (represented by these sequences). The vector construction, transformation, screening, and culture methods of Reference Example 2 were used to double-digest the above-mentioned Trametes versicolor chitinase gene sequence with Xho I and Xba I, and ligated into the pGAPZB expression vector that had also been double-digested with Xho I and Xba I. The recombinant vector pGAPZB-Trametes versicolor chitinase was linearized with AvrII and transformed into the Pichia pastoris host strain using the lithium chloride method. Transformants that could grow normally on YPD plates containing 2 mg / mL Zeocin were selected by Zeocin screening. Transformants with high Zeocin resistance were selected and cultured in 100 mL Erlenmeyer flasks containing 5 mL YPG culture medium at 28°C and 250 rpm until the OD 600= 10, centrifuge and take the supernatant, add 50 μL of the supernatant to 100 μL of colloidal chitin (5 mg / mL) and 100 μL of 50 mM citric acid-phosphate buffer at pH 5.0, incubate at 50°C for 60 minutes, cool and centrifuge (12000g, 5 minutes) to remove unhydrolyzed particles, add 0.25 mL of DNS reagent to terminate the reaction and develop color in a boiling water bath, and measure the absorbance at 540 nm. The results showed that almost no difference in the absorbance values of the above transformants was detected and almost all were 0. At the same time, the expression of the target protein in the supernatant protein was detected by SDS-PAGE. The results of SDS-PAGE are as follows: Figure 6 As shown, no target protein band was detected at the target location. This result indicates that only the sequence shown in SEQ ID NO. 1 in the DNA sequence table, synthesized according to the sequence characteristics of the Trametes versicolor endonuclease gene itself and the yeast codon preference, can achieve high-level secretory expression of the target protein when transformed into Pichia pastoris.

[0085] Example 3

[0086] In this example, the activity of purified Trametes versicolor endochitinase was detected, and the specific steps and results are as follows:

[0087] 1) DNS method was used to detect the optimal pH of recombinant Trametes versicolor chitinase. First, colloidal chitin was prepared (chitin was dissolved in concentrated hydrochloric acid, stirred in an ice bath, centrifuged and washed until neutral, and ultrasonically dispersed in 50mM pH3-8 citric acid-phosphate buffer), and DNS reagent (containing 3,5-dinitrosalicylic acid, NaOH and potassium sodium tartrate) was prepared; when establishing a standard curve, glucose was used as the reducing sugar standard, and a 0-1.0 mg / mL concentration gradient was prepared. 1 mL of each was taken and mixed with an equal volume of DNS reagent. After boiling in a water bath for 5 minutes, the absorbance at 540 nm was measured and a curve was drawn; the enzyme reaction system contained 500 μL of colloidal chitin (5 mg / mL), 400 μL of buffer and 100 μL of enzyme solution (inactivated enzyme solution was used for the blank group). After incubation at 50°C for 30 minutes, 1 mL of The reaction was terminated with DNS reagent and color was developed in a boiling water bath; after cooling, the unhydrolyzed particles were removed by centrifugation (12000g, 5 minutes), and the supernatant was measured for absorbance at 540 nm. The absorbance was converted to glucose concentration using a standard curve, and the relative activity at different pH values was calculated. The results are shown in Table 6.

[0088] Table 6 Determination of optimal pH

[0089] pH 3 4 5 6 7 8 Relative enzyme activity (%) 25 82 100 43 18 —

[0090] The results in Table 6 indicate that the optimal pH of the recombinant enzyme is approximately 5.

[0091] The optimal temperature of recombinant Trametes versicolor chitinase was determined by the DNS method. Referring to the experimental methods and procedures in 1), the enzyme reaction system consisted of 500 μL of colloidal chitin (5 mg / mL), 400 μL of buffer, and 100 μL of enzyme solution (inactivated enzyme solution was used for the blank control). After incubation at 35-60°C for 30 minutes, 1 mL of DNS reagent was immediately added to terminate the reaction and color was developed in a boiling water bath. After cooling, the supernatant was centrifuged (12,000 g, 5 minutes) to remove unhydrolyzed particles. The supernatant was then measured for absorbance at 540 nm. The absorbance was converted to glucose concentration using a standard curve, and the relative activity at different temperatures was calculated. The results are shown in Table 7.

[0092] Table 7 Determination of optimum temperature

[0093] Temperature (℃) 35 40 45 50 55 60 Relative enzyme activity (%) 41 62 92 100 87 22

[0094] The results in Table 7 indicate that the optimum temperature of the recombinase is approximately 50°C.

[0095] 3) DNS assay for the specific activity of recombinant Trametes versicolor endochitinase. Using the same experimental methods and procedures as described in 1), the enzyme reaction system consisted of 500 μL of colloidal chitin (5 mg / mL), 400 μL of buffer, and 100 μL of enzyme solution (1 μg / μL). After incubation at 50°C for 30 minutes, 1 mL of DNS reagent was immediately added to terminate the reaction and color was developed in a boiling water bath. After cooling, the supernatant was centrifuged (12,000 g, 5 minutes) to remove unhydrolyzed particles. The absorbance at 540 nm was measured and converted to glucose concentration using a standard curve. One unit (U) of enzyme activity equals the amount of enzyme required to catalyze the production of 1 μg of reducing sugar per hour, from which the specific activity of the enzyme was calculated. The results showed that the specific activity of the recombinant enzyme was approximately 3200 U / mg.

[0096] Example 4

[0097] In this example, high performance liquid chromatography was used to detect the activity of colloidal chitin hydrolyzed by recombinant Trametes versicolor chitinase to produce chitin oligosaccharides. The specific method was as follows: 500 μg of purified recombinant Trametes versicolor chitinase was added to 5 mL of colloidal chitin (5 mg / mL) at pH 5. The reaction was incubated at 50°C on a shaking table for 0, 0.5, 1, 2, and 4 hours. 1 mL of each sample was inactivated in a 100°C water bath for 10 minutes. After cooling, the mixture was centrifuged (12,000 g for 5 minutes) to remove unhydrolyzed particles. The supernatant was collected and filtered through a 0.22 μm microporous membrane into a sample bottle for liquid chromatography analysis. The liquid phase method is as follows: chromatographic column: SRT-C SEC-100 7.8*300mm 5μm column; mobile phase 0.1M ammonium acetate, flow rate: 0.6mL / min, injection volume: 10μL, column temperature: 30℃, detector: evaporative light detector. HPLC detection was performed using a Waters 2695 high performance liquid chromatograph, and chitin oligosaccharides 3-8 were used as standard. The detection results are shown in Table 8. It can be seen from Table 8 that no oligosaccharides were detected in the unreacted colloidal chitin. Trisaccharides, hexasaccharides and heptasaccharides were detected after 0.5 hours of reaction, among which trisaccharides were the most. However, the increase in chitin oligosaccharides slowed down significantly after 4 hours of reaction. The curve of the HPLC detection results of the sample after 4 hours of reaction is shown in the figure below. Figure 7 As shown, this indicates that the endonuclease does have a strong ability to hydrolyze colloidal chitin and generate chitin oligosaccharides.

[0098] Table 8 Types and contents of chitin oligosaccharides produced by hydrolysis of colloidal chitin

[0099]

[0100]

[0101] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art and will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple variations of the technical scheme of the present invention can be made, and these simple variations all fall within the scope of protection of the present invention.

Claims

1. A highly expressed chitinase gene of Trametes versicolor based on artificially optimized cDNA, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The recombinant Trametes versicolor endochitinase obtained by the gene encoding according to claim 1, characterized in that The amino acid sequence of the enzyme is shown in SEQ ID NO.

2.

3. The biological material containing the gene according to claim 1, characterized in that The biological material includes a recombinant expression vector, an expression cassette or a recombinant bacterium.

4. The biomaterial according to claim 3, wherein The recombinant expression vector consists of an empty vector and the gene according to claim 1 inserted into the empty vector, and the empty vector is a constitutive expression vector.

5. The biomaterial according to claim 4, wherein The constitutive expression vector is pGAPZB.

6. A method for preparing a recombinant Trametes versicolor chitinase, characterized in that: The following steps are involved: 1) constructing the gene according to claim 1 into a constitutive expression vector to obtain a recombinant expression vector; 2) transforming the recombinant expression vector obtained in step 1) into Pichia pastoris host cells to obtain recombinant bacteria; 3) Screening the recombinant bacteria from step 2) to obtain transformants with high-level expression; 4) Fermenting the transformant obtained in step 3) to obtain a supernatant containing the recombinant Trametes versicolor chitinase.

7. The preparation method according to claim 6, wherein In step 4), the fermentation includes continuing to culture at 28° C. for 72-96 hours and automatically adding 50% glycerol as a carbon source, the glycerol addition rate is connected in series with the dissolved oxygen to maintain the dissolved oxygen content of the fermentation at 30%; and using concentrated ammonia to adjust the pH, setting the fermentation pH to 5.

0.

8. The preparation method according to claim 6 or 7, characterized in that The method also includes a protein purification step: precipitating the target protein with an ammonium sulfate solution with a relative saturation of 80%, dialyzing with 10mM Tris-HCl at pH 8 to remove salt ions, first equilibrating the DEAE chromatography column with 10mM Tris-HCl buffer at pH 8, then passing the dialysate supernatant treated with centrifugation and filtration through the column, rinsing the column with 10 times the column volume of 10mM Tris-HCl buffer at pH 6.8 containing 20mM NaCl, and then eluting the target protein with 20mM PBS at pH 6 containing 100mM NaCl to obtain highly pure recombinant Trametes versicolor chitinase.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The constitutive expression vector is pGAPZB; the Pichia pastoris host strain is X33 strain.

10. The recombinant enzyme obtained by the preparation method according to any one of claims 6 to 9, and its use in the preparation of functional chitosan oligosaccharides, medicine, agriculture, biological control, environmental protection or industrial catalysis.

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