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

Abstract

The invention discloses a chitin endonuclease gene for highly expressing trametes versicolor, a recombinant vector, recombinant bacteria, recombinase and a preparation method of the chitin endonuclease gene. The nucleotide sequence of the gene is shown as SEQ ID NO.1; according to the nucleotide sequence shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be achieved through a pichia pastoris inducible expression vector, and the total secretory expression level of the target protein can reach 4.1 g / L under the high-density fermentation condition; the recombinase is purified by nickel affinity chromatography, and the recombinase can efficiently hydrolyze colloidal chitin to generate chitin oligosaccharide. The recombinant trametes versicolor chitin endonuclease prepared by the invention has important application value and wide application prospect in the aspects of preparation of functional chitosan oligosaccharide, antibiosis, anti-tumor, environmental protection, agriculture and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to the construction of an artificially synthesized Trametes versicolor chitinase gene, the optimization of a Pichia pastoris expression system, high-level secretory expression under shake flask conditions, an efficient purification method, and enzymatic property analysis and application thereof. Background Art

[0002] The present invention relates to the field of bioengineering technology, and in particular to a method for efficiently producing Trametes versicolor endochitinase based on a Pichia pastoris methanol-inducible expression system and its application in biodegradation, agricultural disease resistance and pharmaceutical material development.

[0003] Chitin, a polymer of N-acetylglucosamine linked by β-1,4-glycosidic bonds, is the second-most abundant renewable resource in nature and is widely distributed in the shells of crustaceans, fungal cell walls, and insect exoskeletons. Its biodegradation relies on the specific hydrolase, endochitinase (EC 3.2.1.14), which cleaves the internal β-1,4-glycosidic bonds of chitin chains to produce low-polymerization oligochitosans. Research has shown that chitosans possess antibacterial, antioxidant, and immunomodulatory properties, and have important applications in agricultural antifungal preparations, medical healing materials, and food processing. Currently, natural strains such as Trichoderma spp. are commonly used in industry to produce endochitinases, but these methods face bottlenecks such as low enzyme yields (<1 g / L), complex extraction processes, and poor enzyme stability.

[0004] In the large-scale production of chitinase, current recombinant expression technology faces multiple technical obstacles. First, prokaryotic expression systems, such as Escherichia coli, are prone to forming inclusion bodies, resulting in a loss rate of enzyme activity as high as 60%-80% after renaturation, and lack the ability of eukaryotic post-translational modification, making the product activity lower than that of natural enzymes. Secondly, the constitutive Pichia pastoris expression system often has too low expression levels, which restricts its value in industrial applications. In addition, in traditional fermentation processes, enzyme-producing strains are easily inhibited by carbon and nitrogen sources, and the regulation of dissolved oxygen and pH is highly complex, resulting in enzyme yield fluctuations of more than 30% during large-scale production. Finally, the downstream purification cost is high, and the proportion of impurities in the crude enzyme solution exceeds 50%. It needs to go through multiple purification processes, with a yield of less than 40%, and the activity half-life of the enzyme preparation is short.

[0005] In response to the above technical bottlenecks, there is an urgent need in this field to develop a recombinant expression system that is efficient and suitable for industrial production. The Pichia pastoris methanol-inducible expression system is an efficient exogenous protein production platform with multiple significant advantages. First, it relies on the methanol-induced alcohol oxidase (AOX1) promoter, which can be strongly activated in the presence of methanol to drive high-level expression of the target protein, making it particularly suitable for large-scale industrial production. The regulation of this system is extremely strict. Under non-induction conditions (such as when glycerol or glucose is used as a carbon source), the promoter has almost no leakage expression, avoiding the metabolic burden or toxicity of premature synthesis of exogenous proteins on the host cells, thereby ensuring the stability of high-density culture. Pichia pastoris can grow to extremely high cell densities in cheap culture media. After methanol induction, the yield per unit volume is significantly improved, greatly reducing production costs. As a eukaryotic expression system, Pichia pastoris has post-translational modification capabilities, such as protein glycosylation, disulfide bond formation, etc., and can correctly fold complex eukaryotic proteins. It is particularly suitable for the production of pharmaceutical proteins or enzyme preparations that require specific modifications. In addition, the system supports secretory expression, guiding the secretion of the target protein to the extracellular space through signal peptides, simplifying the subsequent purification process and reducing host protein contamination. Methanol as an inducer is low-cost and easy to control, and combined with its adaptability to high-density fermentation, the system is both economical and scalable in industrial applications. Compared with mammalian cell culture, Pichia pastoris is easier to operate and has a shorter cycle; compared with prokaryotic systems such as Escherichia coli, the eukaryotic proteins it expresses are more active and have a wider range of applications. Therefore, this system has important value in the efficient and low-cost industrial production of chitinase. The present invention will provide a method for efficiently producing recombinant Trametes chlororaphis chitinase and the application of the recombinant protein.

[0006] Trametes versicolor is a white rot fungus. The inventors used transcriptomic sequencing to discover a high-abundance chitinase, whose sequence was similar to that of Trametes versicolor FP-101664.

[0007] The SS1 strain is completely consistent. Currently, there is no mature solution to achieve efficient secretory expression of chitinase through the Pichia pastoris expression system. Summary of the Invention

[0008] In view of this, one of the objectives of the present invention is to provide a chitinase gene from Trametes versicolor, the nucleotide sequence of which is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2. The reading frame of SEQ ID No. 1 comprises 969 nucleotides, including a tag consisting of 6 histidine residues and a stop codon, TAA, that does not encode any amino acids. SEQ ID No. 2 consists of 322 amino acid residues.

[0009] The second object of the present invention is to provide a biological material containing the above-mentioned Trametes versicolor chitinase gene, and the biological material includes a recombinant expression vector, an expression cassette or a recombinant bacterium.

[0010] 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 secretory expression vector.

[0011] Furthermore, the secretory expression vector is any one of pPICZαA, pPIC9K and pHIL-S1.

[0012] Particularly, the recombinant vector is obtained by inserting the target gene sequence shown in SEQ ID NO. 1 into the pPICZαA secretion expression vector.

[0013] A third object of the present invention is to provide a method for preparing a recombinant Trametes versicolor chitinase, comprising the following steps:

[0014] 1) constructing the above-mentioned Trametes versicolor chitinase gene into a secretory expression vector to obtain a recombinant expression vector;

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

[0016] 3) Screening the recombinant bacteria from step 2) to obtain transformants with high-level expression;

[0017] 4) Fermenting the transformant obtained in step 3) to obtain a supernatant containing the recombinant Trametes versicolor chitinase.

[0018] Furthermore, the fermentation in step 4) includes continuing to culture at 28° C. for 72-96 hours, and occasionally adding methanol as a carbon source, the methanol addition rate is connected in series with the dissolved oxygen, and the dissolved oxygen in the fermentation is set to 30%; concentrated ammonia water is used to adjust the pH, and the pH of the fermentation is set to 5.0.

[0019] Furthermore, the method further includes purifying the supernatant obtained in step 4) using a nickel affinity chromatography column, comprising first equilibrating the chromatography column with an equilibration buffer, passing the supernatant through the column, rinsing the column with a buffer solution containing 10 mM Tris-HCl and 10 mM imidazole at pH 6.8, and then eluting the fusion protein with a pH 6.8 buffer solution containing 50-100 mM imidazole to obtain purified recombinant Trametes versicolor chitinase.

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

[0021] Particularly, the secretory expression vector is pPICZαA, the Pichia pastoris host strain is strain GS115, and high-density fermentation is carried out in a fermenter to obtain a large amount of recombinant Trametes versicolor chitinase.

[0022] A fourth objective of the present invention is to provide a recombinant enzyme obtained by the preparation method of the recombinant Trametes versicolor chitinase, and its use in the preparation of functional chitosan oligosaccharides, antibacterial and antitumor activities, environmental protection, or agricultural applications. The recombinant enzyme exhibits high activity and is capable of efficiently hydrolyzing colloidal chitin to produce chitosan oligosaccharides, possessing important applications in environmental protection and agriculture. The recombinant enzyme is recommended for use at pH 5 and 50°C for optimal results.

[0023] In summary, the highly expressed Trametes versicolor chitinase gene of the present invention, such as the nucleotide sequence shown in SEQ ID NO. 1, can be used to achieve high-level recombinant secretory expression of the target protein in the Pichia pastoris host strain using a Pichia pastoris inducible expression vector. Under high-density fermentation conditions, the total secretory expression level of the recombinant Trametes versicolor chitinase can reach 4.1 g / L. The recombinant enzyme was purified using nickel affinity chromatography and can efficiently hydrolyze colloidal chitin to produce chitin oligosaccharides. The recombinant Trametes versicolor chitinase prepared by the present invention has important application value and broad application prospects in the preparation of functional oligochitosaccharides, antibacterial and anti-tumor activities, environmental protection, and agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the construction of the expression vector pPICZαA-Trametes versicolor chitinase in an embodiment of the present invention.

[0025] Figure 2 This is a diagram showing the results of SDS-PAGE detection of the culture supernatant of the yeast transformant expressing the high Zeocin-resistant Trametes versicolor chitinase gene in the embodiment of the present invention;

[0026] 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;

[0027] Figure 4 Graph showing the SDS-PAGE detection results of the target protein expression under high-density fermentation culture conditions in an embodiment of the present invention;

[0028] Figure 5This is a diagram showing the SDS-PAGE detection results of the target protein after nickel affinity purification in an embodiment of the present invention;

[0029] Figure 6 This is a diagram showing the SDS-PAGE detection results of the target protein after ultrafiltration and concentration in an embodiment of the present invention;

[0030] Figure 7 This is the SDS-PAGE detection result of the supernatant protein of the Pichia pastoris transformant induced by methanol;

[0031] Figure 8 This is a diagram showing the SDS-PAGE detection results of the supernatant protein of the Pichia pastoris transformant induced by methanol in Example 3;

[0032] Figure 9 TLC results of recombinant Trametes versicolor endochitinase hydrolyzing colloidal chitin to produce chitin oligosaccharides. DETAILED DESCRIPTION

[0033] 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.

[0034] The Pichia pastoris strain and secretory expression plasmid used in the present invention were purchased from Invitrogen Corporation of the United States.

[0035] The culture medium formula used is as follows:

[0036] 1) Yeast growth medium (BMGY)

[0037] Completely dissolve 10 g yeast extract and 20 g peptone and adjust the volume to 800 mL. Sterilize by autoclaving at 121°C for 15-20 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.

[0038] 2) Yeast induction medium (BMMY)

[0039] Completely dissolve 10g yeast extract and 20g peptone and adjust the volume to 800mL. Sterilize by autoclaving at 121°C for 15-20min. Cool to room temperature and add 100mL 1M potassium phosphate solution, 100mL YNB, 2mL 500X biotin, and 10mL methanol.

[0040] 3) YPD medium

[0041] Completely dissolve 10g of yeast extract and 20g of peptone, dilute to 900mL, and sterilize by autoclaving at 121°C for 15-20 minutes. Cool to approximately 70°C, then add 100mL of 20% sterile glucose solution. Add 1.6-1.8% agar to prepare YPD solid medium.

[0042] 4) YPG medium

[0043] Completely dissolve 10 g yeast extract and 20 g peptone, dilute to 900 mL, sterilize by steam autoclave at 121°C for 15-20 min, cool to about 70°C, and then add 100 mL of 20% sterilized glycerol solution.

[0044] 5) Yeast inorganic salt culture medium

[0045] Each liter of culture medium contains 6 grams of potassium sulfate, 5 grams of magnesium sulfate, 1 gram of potassium hydroxide, 9 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 5.5. After wet heat sterilization and cooling to room temperature, 5 milliliters of trace element solution are added to each liter of culture medium during inoculation.

[0046] 6) Trace element solution

[0047] 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, 0.1g KI, 0.05gH3BO3 and 5.0mL concentrated H2SO4. After filtering with a 0.22μm bacterial filter, store in a refrigerator at 4℃ until use.

[0048] Example 1

[0049] This example provides an optimized, artificially synthesized Trametes versicolor chitinase gene, the nucleotide sequence of which is shown in SEQ ID No. 1 and consists of 969 deoxynucleotides. This sequence includes the stable, mature protein full-length reading frame and stop codon of Trametes versicolor chitinase and encodes a protein having the amino acid residue sequence shown in SEQ ID No. 2.

[0050] The DNA sequence of SEQ ID No. 1 was directly ligated into the Pichia pastoris secretory expression vector pPICZαA to obtain a recombinant vector, which was then transformed into the Pichia pastoris host strain GS115 using the lithium chloride method. After transformation, the cells were screened using YPD plates containing 100 μg / mL Zeocin antibiotic. The Pichia pastoris transformants grown on the YPD plate with 100 μg / mL Zeocin antibiotic were washed with sterile water and inoculated onto the YPD plate with a final concentration of 500 μg / mL Zeocin antibiotic; the Pichia pastoris transformants grown on the YPD plate with 500 μg / mL Zeocin antibiotic were washed with sterile water and inoculated onto the YPD plate with a final concentration of 1000 μg / mL Zeocin antibiotic; the Pichia pastoris transformants grown on the YPD plate with 1000 μg / mL Zeocin antibiotic were washed with sterile water and inoculated onto the YPD plate with a final concentration of 1500 μg / mL Zeocin antibiotic, and finally the highly resistant Pichia pastoris transformants that could grow normally on the YPD plate with 1500 μg / mL Zeocin antibiotic were obtained. The highly Zeocin-resistant transformants screened were cultured in a 50-ml centrifuge tube containing 6 mL of BMGY culture medium at 28°C and 250 rpm until the OD 600 =10~15, collect the bacteria by centrifugation and add 1.5mL BMMY medium, and induce culture at 28 ℃ and 250rpm for 3 days. Add 20μL of methanol to the centrifuge tube approximately every 24 hours after adding BMMY, and add methanol twice in total. When the induction is completed, centrifuge and take the supernatant. Take 20μL of the supernatant for SDS-PAGE detection to analyze the expression of the target protein. The expression of the target protein was observed.

[0051] Example 2

[0052] This embodiment provides a method for preparing chitinase protein, which specifically comprises the following steps:

[0053] S1: Construction of recombinant expression vector: The DNA shown in SEQ ID No. 1 of Example 1 was connected to the Pichia pastoris inducible secretory expression vector pPICZαA to obtain the recombinant vector pPICZαA-Trametes versicolor chitinase. Figure 1 Schematic diagram of the construction of the pPICZαA-Trametes versicolor chitinase recombinant expression vector. The main steps of vector construction are preferably as follows:

[0054] (1) The artificially synthesized 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. The reaction system was as follows (the endonucleases and buffer used were purchased from Biyuntian Biotechnology): 12 μL of the plasmid containing the synthetic Trametes versicolor chitinase gene, 4 μL of 10×M buffer, 4 U of Xho I, 4 U of Xba I, and sterile water was added to 40 μL;

[0055] (2) pPICZαA was double-digested with Xho I and Xba I to obtain the vector fragment. The reaction system was as follows: 12 μL of plasmid pPICZαA, 4 μL of 10× M buffer, 4 U of Xho I, 4 U of Xba I, and sterile water to 40 μL;

[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). The target gene was accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor. The reaction system was as follows: 2 μL of the vector pPICZαA fragment, 6 μL of the target fragment, 2 μL of 10× buffer, 1 μL of T4 ligase, and sterile water was added to 20 μL.

[0058] S2: Transformation of the recombinant plasmid: The recombinant vector pPICZαA-Trametes versicolor chitinase was linearized using PmeI and transformed into the Pichia pastoris host strain (GS115 in this example) using the lithium chloride method. After transformation, the cells were screened using YPD plates containing 100 μg / mL Zeocin. The Pichia pastoris transformants grown on YPD plates with 100 μg / mL Zeocin antibiotic were washed with sterile water and spread on YPD plates with a final concentration of 500 μg / mL Zeocin antibiotic. The transformants grown on the resistant plates were then spread on YPD plates with 1000 μg / mL Zeocin antibiotic. The Pichia pastoris transformants grown on YPD plates with 1000 μg / mL Zeocin antibiotic were washed with sterile water and spread on YPD plates with a final concentration of 1500 μg / mL Zeocin antibiotic. Finally, highly resistant Pichia pastoris transformants that could grow normally on YPD plates with 1500 μg / mL Zeocin antibiotic were obtained.

[0059] S3: Screening of high-level secretory expression yeast transformants and small-scale expression: The transformants with high Zeocin resistance verified by PCR were screened for expression: 6 mL of BMGY culture medium was used in a 50 mL centrifuge tube at 28°C and 250 rpm to culture until the OD 600 = 15, centrifuged at 2000g for 10 minutes, collected the cells and added 1.5mL of BMMY medium, and induced at 28°C and 250rpm for 3 days. 20μL of methanol was added to the centrifuge tube approximately every 24 hours (a total of 2 additions were required). After the induction was completed, 20μL of the supernatant was centrifuged and used for SDS-PAGE analysis. Several transformants with high-level secretory expression of the recombinant Trametes versicolor chitinase were screened. The Pichia pastoris transformant with the highest expression level was shaken for small-scale expression: a 1L Erlenmeyer flask containing 200mL of BMGY culture medium was cultured at 28°C and 250rpm until the OD 600 = 15, centrifuge at 2000g for 10 minutes, collect the cells, add 40mL of BMMY medium to resuspend the cells, and transfer them to a 250mL Erlenmeyer flask. Induce the culture at 28°C and 250rpm for 4 days. Add 500μL of methanol to the flask approximately every 24 hours (a total of 3 additions). Sample 500μL every day (24 hours) and centrifuge to obtain the supernatant. After induction, 20μL of the supernatant from each sample was used for SDS-PAGE analysis to determine the relationship between the secretory expression level of the target protein and the methanol induction time under shake flask conditions.

[0060] S4: Mass expression of recombinant enzyme under high-density fermentation conditions: The transformant with the highest expression of Trametes versicolor chitinase screened in S3 was cultured in YPG medium until OD 600 The method comprises the following steps: using 15 as seed bacteria, inoculating the seed bacteria into a bioreactor containing an inorganic salt culture medium at a volume ratio of 1:5-10 for fermentation; continuing to culture at 28°C and adding 50% glycerol as a carbon source; the glycerol addition rate is connected in series with the dissolved oxygen to maintain the dissolved oxygen at 30% during fermentation; adjusting the pH to 5.0 with concentrated ammonia water until the wet weight of the bacteria reaches more than 200g / L; then, adding methanol to the fermentation liquid for inducing expression; setting the methanol addition rate to be linked with the dissolved oxygen; automatically adding methanol when the dissolved oxygen is higher than 30% and always keeping the dissolved oxygen at about 30%; and after 4 days of induction culture, a supernatant obtained from the fermentation contains a large amount of recombinant Trametes versicolor chitinase.

[0061] It should be noted that the transformants with high Zeocin resistance were screened and expressed in BMMY medium. After SDS-PAGE analysis, several yeast transformants that stably and highly secreted Trametes versicolor chitinase were obtained from the transformants with high resistance (resistance level of at least 1500 μg / mL Zeocin). The transformants from YPD plates with resistance level below 200 μg / mL Zeocin had significantly lower secretion ability of the target protein than the transformants with high resistance, and the enzyme activity test showed the same results. The SDS-PAGE results of the target protein secretion from some high Zeocin resistance transformants are shown in Figure 2. Figure 2 shown.

[0062] It should also be noted that for small-scale expression in shake flasks, the total amount of protein obtained by induction culture for 1 to 4 days and the addition of methanol can be higher. The specific total amount of supernatant protein is shown in Table 1 below.

[0063] Table 1 Total protein content

[0064] Induction time 1 day 2 days 3 days 4 days Total protein concentration (mg / L) 150 370 450 470

[0065] SDS-PAGE was used to detect the expression of target proteins at different time points. Figure 3 The SDS-PAGE analysis of the supernatant from a small shake flask culture after methanol induction for 1 to 4 days shows clear expression of the target protein. Overall, the total amount of target protein produced increases with increasing induction time with methanol supplementation. Additionally, the proportion of target protein in the total supernatant protein is shown in Table 2 below.

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

[0067] Induction time 1 day 2 days 3 days 4 days Percentage content (%) 66 51 47 46

[0068] According to calculations, the target protein content can reach about 210 mg / L after 3 days of induction.

[0069] In addition, it should be noted that for the large-scale expression and preparation of recombinant Trametes versicolor chitinase under high-density fermentation conditions, the total amount of protein obtained by induction culture for 1 to 4 days and the addition of methanol is higher. The specific total amount of supernatant protein is shown in Table 3 below.

[0070] Table 3 Total protein content

[0071] Induction time 1 day 2 days 3 days 4 days Total protein concentration (g / L) 0.41 2.07 4.76 6.61

[0072] Take the supernatant of the fermentation broth after 1 to 4 days of induction culture, and perform SDS-PAGE detection under the condition that the electrophoresis sample buffer does not contain reducing agent. The detection results of SDS-PAGE are as follows: Figure 4As shown, there is significant expression of the target protein at around 35 kDa, and the total amount of target protein obtained by adding methanol is higher. Grayscale scanning results of the SDS-PAG gel show that the proportion of target protein is positively correlated with the incubation time. In addition, the proportion of target protein in the total protein in the supernatant is shown in Table 4 below.

[0073] Table 4 Results of percentage content of target protein in supernatant under high-density fermentation conditions

[0074] Induction time 1 day 2 days 3 days 4 days Percentage content (%) 19 43 55 62

[0075] After calculation, the target protein content results are shown in Table 5 below. After 4 days (96 hours) of induction, the total amount of the target protein can reach about 4.1 g / L.

[0076] Table 5 Total amount of target protein

[0077] Induction time 1 day 2 days 3 days 4 days Recombinant Trametes versicolor chitinase (g / L) 0.078 0.89 2.61 4.10

[0078] Preferably, after step S4, the method further comprises the following steps of purifying the protein:

[0079] S5: Centrifuging the culture broth after the fermentation in S4, taking the supernatant, adjusting the pH to 7.5 with Tris base, and centrifuging at a speed greater than or equal to 15,000 g for 10 to 20 minutes, adding the obtained supernatant to a nickel affinity chromatography column equilibrated with a pH 7.5 Tris-HCl buffer, and rinsing the nickel affinity chromatography column with 10 to 15 column volumes of a pH 6.8 buffer containing 10 mM Tris-HCl and 10 mM imidazole;

[0080] S6: The nickel affinity chromatography column was eluted with 10 mM Tris-HCl buffer at pH 6.8 containing 0-400 mM imidazole. The SDS-PAGE results of the eluted protein were as follows: Figure 5 As shown in the figure, it can be seen that when the sample eluted with 50-100mM imidazole buffer was collected, the target protein was specifically eluted and had a high purity. The eluate containing the target protein (50-100mM imidazole) was dialyzed in 10mM pH 6 PBS buffer using a 10kDa dialysis bag, and the recombinant protein was then concentrated by ultrafiltration. The SDS-PAGE results of the concentrated protein are shown in Figure 2. Figure 6 The results show that the concentrated protein also has a high purity. The protein purity and recovery rate of each purification step after high-density fermentation in the fermenter are shown in Table 6.

[0081] Table 6 Fermentation broth supernatant protein purification results

[0082]

[0083] SEQ ID No. 1 provided by the present invention uses pPICZαA as an expression vector and GS115 as an expression strain. After high-density fed-batch fermentation in a fermenter, 330 mg of the target protein can be purified per 100 mL of fermentation broth. The final recovery rate of the target protein is greater than 80% and the purity is greater than 90%. It can be seen that Pichia pastoris is used as an expression system, pPICZαA is used as an expression vector, and GS115 is used as an expression strain. The target protein has a high expression level and is easy to purify.

[0084] Comparative Example:

[0085] Using data from the NCBI database, primers were designed, and the target gene was amplified by RT-PCR and ligated into a cloning vector. The amplified native sequence of the target gene is shown in SEQ ID NO. 3. During the research process of the present invention, several other optimized artificial DNA sequences were also synthesized, two of which are shown in SEQ ID NO. 4 and SEQ ID NO. 5. The recombinant Trametes versicolor chitinase gene sequence was double-digested with Xho I and Xba I according to the vector construction, transformation, screening, and induction methods of Example 2 and ligated into the pPICZαA expression vector, which had also been double-digested with Xho I and Xba I. The recombinant vector pPICZαA-recombinant Trametes versicolor chitinase was linearized with PmeI and transformed into the Pichia pastoris host strain using the lithium chloride method. Positive clones were then screened with Zeocin. The highly resistant transformants of Pichia pastoris verified by PCR were streaked onto YPD plates containing 1500 μg / mL Zeocin. The transformants that could grow normally were cultured in 50 mL centrifuge tubes containing 6 mL BMGY culture medium at 28°C and 250 rpm until the OD 600 = 10-15, collect the cells by centrifugation and add 1.5 mL of BMMY medium, culture at 28°C and 250 rpm for 3 days, add 20 μL of methanol to the centrifuge tube every 24 hours, and after the induction is completed, centrifuge and take 20 μL of the supernatant and add the loading buffer. The SDS-PAGE detection results of the target protein are as follows Figure 7 As shown, no obvious target protein band was detected at the target position. The results show that only when the DNA sequence provided by the present invention, such as the sequence shown in SEQ ID NO. 1, is transformed into Pichia pastoris can high-level secretory expression of the target protein be achieved.

[0086] Example 3

[0087] This example shows the expression of SEQ ID NO. 1 in other Pichia pastoris secretory expression vectors and strains.

[0088] In addition to using pPICZαA as the expression vector and GS115 as the expression strain to achieve high-level expression of the sequence in SEQ ID NO.1 in Pichia pastoris, the sequence in SEQ ID NO.1 can also achieve high-level induced secretory expression in other Pichia pastoris secretory expression vectors such as pPIC9K, pHIL-S1, and other expression strains such as X33, KM71, SMD1168, etc. Since there are too many combinations to be presented one by one in the examples, the inducible secretory expression vector pPIC9K and the expression strains X33 and SMD1168 are used as examples for illustration. The steps are briefly as follows: the sequences in SEQ ID NO.1 are cloned into the inducible secretory expression vector pPIC9K respectively, and the recombinant vector pPIC9K-Trametes versicolor chitinase gene is transformed into Pichia pastoris competent cells X33 and SMD1168 respectively by electroporation according to the method of the Invitrogen expression instruction manual. Transformants with a resistance level of 1.5 mg / mL are selected by G418, and the transformants verified by PCR are cultured in a 50-ml centrifuge tube containing 6 mL of BMGY culture medium at 28°C and 250 rpm until the OD 600 =10-15, collect the cells by centrifugation and add 1.5 ml of BMMY medium, culture at 28°C and 250 rpm for 3 days, add 20 μL of methanol to the centrifuge tube every 24 hours, after the induction is completed, centrifuge and take 20 μL of the supernatant and add the loading buffer, the SDS-PAGE detection results of the target protein are as follows Figure 8 As shown, the sequence in SEQ ID NO. 1 also achieved high-level secretory expression in X33 and SMD1168. Therefore, the sequence in SEQ ID NO. 1 can also achieve high-level secretory expression in other secretory expression vectors and other P. pastoris as host bacteria.

[0089] Example 4

[0090] In this example, the activity of purified Trametes versicolor endochitinase was detected, and the specific steps and results are as follows:

[0091] The optimal pH of recombinant Trametes versicolor chitinase was detected by DNS method. 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 0.1M pH 3-8 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 concentration gradient of 0-1.0 mg / mL 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 in the blank group). After incubation at 50°C for 30 minutes, 1 mL was immediately added. 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 (12000 rpm, 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 7.

[0092] Table 7 Determination of optimal pH

[0093]

[0094]

[0095] The results in Table 7 indicate that the optimal pH of the recombinant enzyme is approximately 5.

[0096] 1) DNS method for detecting the optimal temperature of recombinant Trametes versicolor chitinase. Referring to the experimental methods and procedures in 1), chitin was dissolved in concentrated hydrochloric acid, stirred in an ice bath, and then centrifuged and washed until neutral. It was then ultrasonically dispersed in 0.1 M citric acid-phosphate buffer (pH 5). 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, centrifugation (12,000 rpm, 5 minutes) was performed to remove unhydrolyzed particles. The supernatant was collected and the absorbance at 540 nm was measured. 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 8.

[0097] Table 8 Determination of optimum temperature

[0098] Temperature (℃) 35 40 45 50 55 60 Relative enzyme activity (%) 45 67 88 100 84 17

[0099] The results in Table 8 indicate that the optimum temperature of the recombinase is approximately 50°C.

[0100] 3) DNS assay for the specific activity of the recombinant Trametes versicolor chitinase. Using the experimental methods and procedures described in 1), chitin was dissolved in concentrated hydrochloric acid, stirred in an ice bath, and then centrifuged to neutralize. The chitin was then ultrasonically dispersed in 0.1 M citric acid-phosphate buffer (pH 5). 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). The reaction was incubated at 50°C for 30 minutes, immediately followed by the addition of 1 mL of DNS reagent to terminate the reaction, and color was developed in a boiling water bath. After cooling, the supernatant was centrifuged (12,000 rpm for 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 3,000 U / mg.

[0101] Example 5

[0102] In this example, the activity of purified recombinant Trametes versicolor chitinase in hydrolyzing colloidal chitin to produce chitin oligosaccharides was detected by thin layer chromatography. The specific steps and results are as follows:

[0103] The enzyme reaction system contains 500 μL of colloidal chitin (5 mg / mL), 400 μL of pH 5.0 buffer and 100 μL of enzyme solution. After incubation at 50°C for 0.5, 1, 2 and 4 hours, heat inactivate at 90°C for 10 minutes, and centrifuge (12000 rpm, 5 minutes) to remove unhydrolyzed particles after cooling. Activate the silica gel G plate in a 100°C oven and cool to room temperature. Use a capillary to spot 1 μL. The spotting position is 1.5 cm from the bottom of the silica gel plate and 2 to 3 cm on both sides. Each sample point is 1 to 1.5 cm apart. The developing agent (ethyl acetate: acetic acid: water = 2:1:1) is balanced in the chromatography cylinder. After a few minutes, the silica gel plate is placed in. When the developing agent moves to 2 to 3 cm from the top, take out the silica gel plate and blow dry it. Spray the color developer (25% sulfuric acid) evenly on it. After color development at 100°C for 10 to 15 minutes, take it out of the oven. The color development results are as follows. Figure 9 As shown in the figure, it can be seen that the colloidal chitin sample has no staining spots when no enzyme is added, and color spots appear after adding the endonuclease reaction for 0.5 hours, and the molecular weight of the spots is significantly larger than that of the glucose standard. Moreover, as the reaction time is prolonged, the spots are colored darker, indicating that the addition of the endonuclease hydrolyzes the colloidal chitin to produce a large amount of chitin oligosaccharides with different molecular weights, indicating that the recombinant Trametes versicolor chitin endonuclease produced by the present invention can efficiently hydrolyze colloidal chitin and produce chitin oligosaccharides.

[0104] 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 chitinase gene of Trametes versicolor, 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. 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.

3. The biomaterial according to claim 2, 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 secretory expression vector.

4. The biomaterial according to claim 3, wherein The secretory expression vector is any one of pPICZαA, pPIC9K and pHIL-S1.

5. 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 secretory 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 expression levels; 4) Fermenting the transformant obtained in step 3) to obtain a supernatant containing the recombinant Trametes versicolor chitinase.

6. The preparation method according to claim 5, wherein In step 4), the fermentation comprises continuing to culture at 28° C. for 72-96 hours, and occasionally adding methanol as a carbon source, wherein the methanol addition rate is connected in series with the dissolved oxygen, and the dissolved oxygen during fermentation is set to 30%; and using concentrated ammonia to adjust the pH, and the pH during fermentation is set to 5.

0.

7. The preparation method according to claim 5 or 6, characterized in that The method further includes purifying the supernatant obtained in step 4) using a nickel affinity chromatography column, comprising first equilibrating the chromatography column with an equilibration buffer, passing the supernatant through the column, rinsing the column with a buffer solution containing 10 mM Tris-HCl and 10 mM imidazole at pH 6.8, and then eluting the fusion protein with a pH 6.8 buffer solution containing 50-100 mM imidazole to obtain purified recombinant Trametes versicolor chitinase.

8. The preparation method according to any one of claims 5 to 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 GS115, X33, KM71 and SMD1168.

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

10. The recombinant enzyme obtained by the preparation method according to any one of claims 5 to 9, and its use in the preparation of functional chitosan oligosaccharides, antibacterial, antitumor, environmental protection or agricultural fields.

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