Chitinase chi-6690 and application thereof

Abstract

The invention provides chitinase chi-6690 and application thereof, the amino acid sequence of the chitinase chi-6690 is as shown in SEQ ID NO: 2, the chitinase chi-6690 has the highest activity at 50 DEG C (improved by more than 20% compared with conventional chitinase), the stability is optimal when the pH is 6.0, and the application blank of the chitinase in a medium-temperature region is filled. Through a recombinant expression technology, the enzyme can be efficiently expressed in escherichia coli. The enzyme can efficiently degrade chitin in the cell wall of penicillium, an efficient and pollution-free bio-enzyme preparation is provided for disease prevention and control of picked fruits and vegetables and food industry preservation, and the enzyme has important industrialization value.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to a chitinase chi-6690 and an application thereof. Background Art

[0002] Stropharia rugoso-annulata, also known as red pine mushroom, belongs to the genus Stropharia in the subphylum Basidiomycetes and is one of the top ten edible mushrooms recommended by the Food and Agriculture Organization of the United Nations. The optimal temperature for mycelial growth is 22-25°C, while the optimal temperature for fruiting body formation is 16-21°C. Its fruiting body has a brown cap and thick flesh, rich in nutrients such as protein, 18 amino acids, polysaccharides, vitamins, and minerals. It possesses antioxidant, immune-enhancing, and potential anti-tumor activities. As a straw-rot edible fungus, Stropharia rugoso-annulata can be cultivated using agricultural and forestry waste such as rice straw and stalks. Returning the residue to the field improves soil quality. Its genome is rich in glycoside hydrolase genes, including the chitinase GH18 family. These genes degrade chitin in the fungal cell wall, disrupting the cell structure and inhibiting mycelial growth and spore germination.

[0003] Chitinase chi-6690, a chitinase gene cloned from the genome of Stropharia rugosodium, hydrolyzes β-1,4-glycosidic bonds, gradually degrading chitin into chito-oligosaccharides (such as chitobiose and chitotriose), ultimately producing N-acetylglucosamine monomers. Some broad-spectrum chitinases may also have some degradative effect on structurally similar polysaccharides (such as deacetylated chitosan), but their primary substrate remains chitin.

[0004] Compared with traditional chemical fungicides, chitinase-mediated biological control strategies have the advantages of strong mechanism specificity, high environmental compatibility, and no chemical residues. They have important theoretical research value and industrial application prospects in the fields of agricultural product preservation, food industry antiseptic treatment, and green control of plant diseases, and provide a green and sustainable technical path for solving the hazards of Penicillium fungi.

[0005] Therefore, it is necessary to develop a fungus that can inhibit Penicillium. Summary of the Invention

[0006] The present invention aims to provide a chitinase chi-6690 and its application. The chitinase chi-6690 can inhibit Penicillium fungi, has good stability and an optimum temperature of 50°C, filling the gap in the existing technology in the field of healthy antibacterial and antiseptic applications.

[0007] The present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a chitinase chi-6690 is provided. The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0009] In a second aspect of the present invention, a chitinase chi-6690 is provided. The amino acid sequence of the chitinase chi-6690 is shown in SEQ ID NO: 2.

[0010] In the third aspect of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector is capable of expressing the chitinase chi-6690.

[0011] Furthermore, the recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0012] In the fourth aspect of the present invention, a recombinant bacterium or an engineered cell line comprising the recombinant expression vector is provided.

[0013] Furthermore, the host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, and an insect cell.

[0014] In a fifth aspect of the present invention, provided is the use of the gene, the chitinase chi-6690, the recombinant expression vector, the recombinant bacteria or the engineered host cell line in the preparation of a product for fermented steamed cake.

[0015] In a sixth aspect of the present invention, a method for preparing chitinase chi-6690 is provided, the method comprising:

[0016] Total RNA was extracted from Stropharia rugosa and reverse transcribed into cDNA;

[0017] Using the cDNA as a template, PCR amplification was performed with the primer pair shown in SEQ ID NO.3-SEQ ID NO.4 to obtain the chi-6690 gene;

[0018] The chi-6690 gene was inserted between the EcoRI and XhoI restriction sites of the expression vector pET-28a to construct the Escherichia coli recombinant expression vector pET-28a-chi-6690;

[0019] The recombinant expression vector is transformed into a host cell and induced to express under the condition of 18-25° C., and low-temperature xylanase is obtained through purification.

[0020] In a seventh aspect of the present invention, there is provided a use of the chitinase chi-6690 in preparing a product for inhibiting Penicillium fungi.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] 1. The chitinase chi-6690 of the present application has highly effective antibacterial properties: in the inhibition experiment on Penicillium fungi, the growth rate of the plaque treated with 50 μL of enzyme solution was significantly slowed by >50% (compared with the Buffer B group and the blank group), confirming that it directly destroys the structure of pathogens by degrading cell wall chitin.

[0023] 2. High thermal stability and activity advantages: Orthogonal experiments confirmed that the enzyme has the highest activity at 50°C (more than 20% higher than conventional chitinase) and the best stability at pH 6.0, filling the application gap of chitinase in the medium temperature zone.

[0024] 3. Expression system suitable for industrial production: Successfully expressed in E. coli using pET-28a vector ( Figure 3 ), a 44kDa high-purity protein was obtained after low-temperature induction (18℃ for 20h) and nickel column purification, with a yield of 0.8mg / mL, meeting the needs of large-scale applications.

[0025] 4. Green and environmentally friendly characteristics: Compared with chemical fungicides, this enzyme has a specific mechanism of action (Km=0.6539mg / mL) and no residual pollution, providing a sustainable biological control solution for agricultural product preservation and food antiseptic. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the gel electrophoresis diagram after PCR.

[0028] Figure 2 The gel electrophoresis diagram is for double enzyme digestion verification.

[0029] Figure 3 This is the SDS-page image of the purified protein. The target protein is 44kDa.

[0030] Figure 4 The optimal pH was determined by orthogonal experiments.

[0031] Figure 5The optimum temperature was obtained through orthogonal experiments.

[0032] Figure 6 Kinetic parameters were calculated for the reaction rates based on different substrate concentrations.

[0033] Figure 7 This is the initial stage of culture, and the differences in colony growth among the enzyme treatment group, Buffer B control group and blank group were compared.

[0034] Figure 8 In the late stage of culture, the differences in colony growth among the enzyme treatment group, Buffer B control group and blank group were compared. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0036] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0038] The chitinase chi-6690 and its applications are described in detail below, using examples and experimental data. The pET28a (SUMO) vector used in this application is a commercial vector, available from biotechnology companies such as Addgene, catalog number MLCC1025. Escherichia coli Rosetta 2 (DE3) is also commercially available. In this application, there are no specific requirements for the type of expression vector or the strain of E. coli used.

[0039] Example 1. Cloning and expression of xylanase chi-6690 gene

[0040] (1) Chitinase chi-6690 gene cloning

[0041] The RNA genome of Stropharia rugosa was extracted from the mycelium of Stropharia rugosa, and the cDNA group of Stropharia rugosa was obtained by reverse transcriptase. Primers were designed and the target gene chi-6690 was cloned by PCR using cDNA as a template. The PCR program was set as denaturation temperature at 94℃, 5min per cycle, annealing temperature at 52℃, 30s per cycle, extension temperature at 72℃, 2min per cycle, and the number of cycles was 34. The PCR results are as follows: Figure 1 .

[0042] Depend on Figure 1 It can be seen that 1% agarose gel electrophoresis showed a specific band of about 1.3 kb.

[0043] (2) Construction and expression of recombinant expression plasmid

[0044] The amplified target gene chi6690 was ligated to the expression vector PET-28a to construct the Escherichia coli recombinant expression vector PET-28a-chi6690. The culture was fermented at 37°C for 4 h using ZYM-5052 lactose induction medium until the OD600 reached 0.6-0.8, and then transferred to 18°C ​​for fermentation for 20 h. The bacteria were harvested by centrifugation and then ultrasonically disrupted to obtain the crude enzyme solution. The purified pure enzyme solution was obtained by nickel column purification, and the protein size was verified by SDS-page.

[0045] Depend on Figure 3 It can be seen that a single target band with a molecular weight of 44 kDa was obtained, which is xylanase chi-6690.

[0046] (3) Plasmid double enzyme digestion verification

[0047] Double enzyme digestion verification uses two restriction endonucleases, EcorI and XhoI, for reaction. First, prepare a 20 μL reaction system in a sterile centrifuge tube, add the DNA template to be digested, EorI and XhoI restriction endonucleases, the corresponding 10× buffer and sterile water in sequence, mix thoroughly and centrifuge briefly. Place the reaction system in a 37°C constant temperature metal bath and incubate for 1 hour to allow the two endonucleases to fully exert their effects and specifically cut the DNA sequence. After the reaction is completed, add 4 μL 10× loading buffer to the system, mix thoroughly, and transfer all 24 μL of the reaction product to the sample well of the nucleic acid gel. Subsequently, place the gel in an electrophoresis tank containing TAE buffer, set a suitable voltage (such as 130 V), perform agarose gel electrophoresis separation, and after staining with nucleic acid dyes (such as EB or SYBR Green), observe the enzyme-digested fragment bands under the gel imaging system, and compare the number and size of the bands with the expected results to verify the enzyme digestion effect and the integrity of the DNA sample, such as Figure 2 The recombinant plasmid was digested with enzymes to release the 1.3 kb target fragment.

[0048] Example 2: Determination of the optimum temperature and pH of the enzyme

[0049] Colloidal chitin was used as the substrate for the determination of xylanase activity. The chitinase activity was determined at pH 3, 4, 5, 6, 7, 8, 9, 10, and 11 and at temperatures of 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C by orthogonal assay.

[0050] Wherein, the preparation method of colloidal chitin is as follows:

[0051] To 0.25 g of chitin, add 5 mL of concentrated hydrochloric acid, stirring occasionally. Allow to swell overnight at 4°C. Then, add 50 mL of ice-cold 50% ethanol, stir thoroughly, and let stand at room temperature for 12 hours to allow the colloidal chitin to precipitate. Centrifuge at 8000 g for 30 minutes to collect the colloidal chitin precipitate. Wash the precipitate repeatedly with deionized water to a pH of approximately 7. Alternatively, adjust the pH to 7.0 with 1 M NaOH and then rinse with water to remove salts. Finally, resuspend the precipitate in deionized water and dilute to 25 mL to obtain 1% colloidal chitin.

[0052] Depend on Figure 4 It can be seen that chitinase chi-6690 reaches its maximum enzyme activity peak at 50℃.

[0053] Depend on Figure 5 It can be seen that the activity reached its maximum at pH 6.0, which was 27.5% and 34.1% higher than the enzyme activities at pH 5.0 and pH 7.0, respectively.

[0054] Example 3: Determination of enzyme kinetics

[0055] The standard curve of N-acetylglucosamine was calculated using the OD value at OD540 after the reaction of known N-acetylglucosamine concentration with DNS. The reaction system was prepared with different 1% colloidal chitin concentrations. After measuring the OD value, the amount of 1% colloidal chitin produced was calculated according to the standard curve, and a Lineweaver-Burk plot was drawn.

[0056] The results are as follows Figure 6 As shown, the chitinase kinetic parameters Km=0.6539 mg / mL and Vmax=0.5372 μmol / min were calculated.

[0057] Example 4: Application of chitinase chi-6690 in inhibiting Penicillium

[0058] 1. Material Preparation

[0059] 1. A cultured Penicillium plate (make sure the Penicillium is in good growth state and in the logarithmic growth phase or a suitable experimental stage); the Penicillium used in this application was purchased from Wuhan Huizao Biotechnology Co., Ltd. (Cat. No. HZB547526).

[0060] 2. Several sterile unused culture plates

[0061] 3. Freshly prepared PDA (potato dextrose agar) medium plates containing AMP (ampicillin) (prepare and sterilize in advance, cool and solidify before use, and the AMP concentration should be accurately prepared according to the experimental requirements)

[0062] 4. Enzyme solution (prepare in advance to ensure activity and store in an ice box)

[0063] 5. Buffer B solution (prepare according to the formula, sterilize after packaging, and dilute 100% before use as needed, for example, take 50 μL of the stock solution and add 50 μL of sterile water and mix well)

[0064] 6. 75% ethanol, sterile water

[0065] 2. Instruments and Consumables

[0066] 1. Clean bench (turn on the UV lamp for sterilization 30 minutes in advance and turn on the fan for 10-15 minutes before the experiment)

[0067] 2. Micropipette (10 - 100 μL range, paired with sterile pipette tips)

[0068] 3. Alcohol lamp, inoculation loop, tweezers (sterilized)

[0069] 4. Constant temperature incubator (set to a temperature suitable for the growth of Penicillium, such as 25-28°C)

[0070] 3. Specific experimental steps

[0071] 1. Sample Preparation

[0072] (1) Clean the clean bench, wipe the table with 75% ethanol, and light the alcohol lamp.

[0073] (2) Place the plate with cultured Penicillium in a clean bench and use a medium-sized gun tip to evenly punch 9 round holes of the same size on the plate (try to avoid the edge of Penicillium and areas with dense or sparse growth, and keep the hole spacing appropriate to avoid mutual influence between samples).

[0074] (3) Use sterilized tweezers to carefully pick up the culture medium samples in the circular holes and place them in sterile unused culture plates in turn, marking them as samples 1 - 9.

[0075] 2. Group processing

[0076] (1) Enzyme solution treatment group:

[0077] A. Use a marker to mark three sterile plates as "enzyme solution treatment group."

[0078] B. Use a micropipette to draw up 50 μL of enzyme solution and add it to the circular well culture medium of three corresponding samples (such as samples 1, 2, and 3), ensuring that the enzyme solution completely covers the sample surface.

[0079] C. Let it soak for 30 minutes (start timing and keep the plate still during this time. You can place the plate in a humidified box to prevent water evaporation).

[0080] D. After 30 minutes, use sterile tweezers to inoculate the treated samples onto new PDA culture medium plates containing AMP, gently press the samples to ensure full contact with the culture medium, and mark them.

[0081] (2) Buffer B treatment group:

[0082] A. Label the other three sterile plates as "Buffer B Treatment Group."

[0083] B. Use a micropipette to draw up 50 μL of 1-fold diluted Buffer B solution and add it to the three round-hole culture media corresponding to the samples (e.g., samples 4, 5, and 6), ensuring that the solution covers the samples.

[0084] C. Soak for 30 minutes (the operation is the same as the static standing requirements for the enzyme solution treatment group).

[0085] D. After the time is up, inoculate the treated sample onto a new PDA medium plate containing AMP and mark it clearly.

[0086] (3) Control group:

[0087] A. Label the last 3 sterile plates as "Control Group."

[0088] B. Use sterile tweezers to directly inoculate the remaining three samples (such as samples 7, 8, and 9) onto a new PDA culture medium plate containing AMP without soaking in other solutions, and mark them.

[0089] 3. Cultivate observation

[0090] (1) Place all the inoculated PDA culture medium plates containing AMP upside down in a constant temperature incubator (inversion can prevent condensation water from dripping and affecting the growth of Penicillium), and set the temperature to 25-28℃ for incubation.

[0091] (2) Regularly observe and record the growth of Penicillium (e.g., observe at the same time every day, record the colony size, color, morphological changes, etc., and continue observing until the growth of Penicillium stabilizes).

[0092] like Figure 7 As shown in Figure 8, on the third day, the colony diameter of the enzyme treatment group (12±1mm) was significantly smaller than that of the Buffer B group (25±2mm) and the blank group (28±3mm).

[0093] In summary, the chitinase chi-6690 can inhibit Penicillium fungi, has good stability and an optimum temperature of 50°C, filling the gap in the existing technology in the field of healthy antibacterial and antiseptic applications.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

[0095] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A gene encoding chitinase chi-6690, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO: 1, chitinase chi-6690.

2. A chitinase chi-6690, characterized in that The amino acid sequence of the chitinase chi-6690 is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector is capable of expressing the chitinase chi-6690 according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

5. A recombinant bacterium or engineered host cell line comprising the recombinant expression vector according to any one of claims 3-4.

6. The recombinant bacteria or engineered host cell line according to claim 5, characterized in that The host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, and an insect cell.

7. Use of the gene according to claim 1, the chitinase chi-6690 according to claim 2, the recombinant expression vector according to any one of claims 3-4, and the recombinant bacteria or engineered host cell line according to any one of claims 5-6 in preparing a product for fermented steamed cake.

8. A method for preparing chitinase chi-6690, characterized in that: The method comprises: Total RNA was extracted from Stropharia rugosa and reverse transcribed into cDNA; Using the cDNA as a template, PCR amplification was performed with the primer pair shown in SEQ ID NO.3-SEQ ID NO.

4. Obtain the chi-6690 gene; The chi-6690 gene was inserted between the EcoRI and XhoI restriction sites of the expression vector pET-28a to construct the large intestine. E. coli recombinant expression vector pET-28a-chi-6690; The recombinant expression vector is transformed into host cells and induced to express under the condition of 18-25°C, and low-temperature xylan is obtained through purification. enzyme.

9. Use of the chitinase chi-6690 according to claim 2 in the preparation of a product for inhibiting Penicillium fungi.

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