High-temperature resistant protease AbTp and application thereof

Abstract

This invention relates to the field of biotechnology and its applications, specifically to a thermostable protease. Away Tp and its applications. The protease Away Tp contains at least one or more amino acid fragments of TNNHVVEGA, PFTDLAVIRAE, and GGALVNAAGQVI. The thermostable protease provided by this invention... Away Tp has an optimal temperature of 70℃ and an optimal pH of 6.0. At 70℃, its half-life can reach 267 minutes, and its specific activity can reach 1877 U·mg. ‑1 Protease Away When Tp is applied to the degradation of shrimp shell proteins, the protein degradation rate can reach 61%, which is higher than that of porcine trypsin and porcine pepsin commonly used in previous biodegradation methods.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and its applications, specifically to a thermostable protease AbTp and its applications. Background Technology

[0002] The main components of shrimp shells are calcium carbonate, protein, and chitin. Among them, the protein content accounts for 20% to 40%. By hydrolyzing shrimp shell protein with proteases, polypeptides and amino acids can be obtained, which can be used as feed and plant nutrient solutions.

[0003] Traditional proteases are mainly obtained from animal viscera, but this is limited by factors such as the animal's growth cycle, the source of the proteases, and the characteristics of the enzymes. Animal-derived proteases first require removing the fat and connective tissue from the fresh animal pancreas, crushing and filtering the contents, precipitating with ammonium sulfate to obtain crude protease, which is then activated with trypsin to obtain crude protease. This method results in low purity and low yield of animal-derived proteases. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a thermostable protease and its applications. The protease of this invention is expressed in large quantities in *E. coli* through genetic engineering and has a short growth cycle, making it more suitable for industrial applications.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The protease AbTp was obtained from the high-temperature compost metatranscriptome database. This database belongs to the research group of Liu Dongyang, College of Resources and Environmental Protection, Nanjing Agricultural University.

[0007] In a first aspect, the present invention protects a protease AbTp, said protease AbTp comprising at least one or more of the amino acid fragments TNNHVVEGA, PFTDLAVIRAE, and GGALVNAAGQVI.

[0008] Preferably, the amino acid sequence of the protease AbTp is selected from the following (A1) or (A2):

[0009] (A1) The amino acid sequence is shown in SEQ ID NO:1;

[0010] (A2) A protein having the same function as the protein shown in (A1) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in SEQ ID NO: 1.

[0011] Preferably, the protease AbTp is derived from the actinomycete Actinobacteria bacterium.

[0012] Secondly, the present invention also provides a biomaterial, which may be any one of the following (B1)-(B4):

[0013] (B1) DNA molecule encoding the protease AbTp;

[0014] (B2) An expression cassette containing the DNA molecule described in (B1);

[0015] (B3) A recombinant vector containing the DNA molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2);

[0016] (B4) A recombinant microorganism containing the DNA molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B3).

[0017] Preferably, the DNA molecule (B1) is selected from the following (C1) or (C2):

[0018] (C1) Nucleotide sequence is shown in SEQ ID NO:2;

[0019] (C2) A DNA molecule that has at least 80%, 85%, 90%, 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein with the same function.

[0020] The DNA molecule shown in SEQ ID NO:2 encodes the protease with the amino acid sequence SEQ ID NO:1.

[0021] (B1) The DNA molecule described also includes a DNA molecule obtained by codon optimization based on the nucleotide sequence shown in SEQ ID NO: 2.

[0022] This invention searches for genes of proteases with high transcriptional levels during the high-temperature period of composting from a metatranscriptome database. After codon optimization in E. coli, the target gene is synthesized and constructed into the expression vector pColdⅡ. The vector containing the target gene is extracted and transformed into E. coli expression strain BL 21 for induced expression of the target protein.

[0023] The purification steps of the target protein in this invention are as follows:

[0024] Weigh the cells, add cell lysis buffer to control the cell concentration between 10% and 15%, and stir the cell solution with a glass rod. This process should be carried out under ice bath conditions.

[0025] When the cell solution is mixed until there are no lumps, the cells are disrupted using a high-pressure homogenizer. The pressure is slowly increased to 600 bar, and the cell solution is disrupted until it is clear and transparent. The solution is then centrifuged at 15,000 rpm for 50 minutes at 4°C, and the supernatant is filtered through a 0.45 μm filter.

[0026] The anion exchange column was pre-equilibrated for 5 column volumes. The filtered supernatant was then slowly loaded onto the column to allow the enzyme to fully bind to the column. Then, 3 to 5 column volumes of washing buffer were used to remove impurities. Finally, the target protein was eluted with 100 mM NaCl solution for subsequent biochemical characterization studies.

[0027] Thirdly, the present invention also provides a method for hydrolyzing proteins in shrimp shells, the method comprising adding the aforementioned protease to a shrimp shell buffer solution.

[0028] Fourthly, the present invention also protects the use of the aforementioned protease or the aforementioned biological material in the hydrolysis of proteins in shrimp shells.

[0029] Preferably, shrimp shells are dispersed in a pH 6.0 buffer solution, and protease AbTp is added, with a mass ratio of shrimp shells to protease AbTp of 5g:5mg.

[0030] More preferably, the shrimp shell is a crayfish shell, which is heated at 120°C for 30 minutes to inactivate the enzymes contained in the shrimp shell. The dried shrimp shell is then crushed using a grinder and used as experimental material for later use.

[0031] In a further preferred embodiment, 5g of crayfish shells were added to a pH 6.0 buffer solution, followed by 5mg of proteinase AbTp, and the volume was adjusted to 100mL. The reaction temperature was set at 60℃, the shaker speed at 200rpm, and the reaction was carried out for 13h.

[0032] Beneficial effects

[0033] (1) The heat-resistant protease AbTp provided by this invention has an optimal temperature of 70°C, an optimal pH of 6.0, a half-life of up to 267 minutes at 70°C, and a specific activity of up to 1877 U·mg. -1 .

[0034] (2) When the protease AbTp is applied to the degradation of shrimp shell protein, the protein degradation rate can reach 61%, which is higher than that of the porcine trypsin and porcine pepsin commonly used in previous biodegradation methods.

[0035] (3) The protease AbTp is expressed in large quantities in Escherichia coli through genetic engineering and has a short growth cycle, making it more suitable for industrial applications. Attached Figure Description

[0036] Figure 1SDS-PAGE gel electrophoresis image shows high-purity protease AbTp; among them, the target protease AbTp is located at 25kDa and 35kDa in lane 1.

[0037] Figure 2 The relative activity of the protease AbTp at different pH values;

[0038] Figure 3 Relative activity of protease AbTp at different temperatures;

[0039] Figure 4 The half-life of the protease AbTp at 70℃ and 80℃ is 267 minutes at 70℃ and 60 minutes at 80℃.

[0040] Figure 5 The effect of feed-to-liquid ratio on the degradation of shrimp shell protein;

[0041] Figure 6 Infrared spectrum of shrimp shell protein hydrolyzed by protease AbTp;

[0042] Figure 7 Scanning electron microscope image of untreated shrimp shells;

[0043] Figure 8 Scanning electron microscope image of shrimp shell protein after hydrolysis by protease AbTp. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are all conventional methods known to those skilled in the art:

[0045] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field.

[0046] The examples described in this invention are merely some embodiments of this invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0047] This invention discovers a heat-resistant protease AbTp (amino acid sequence shown in SEQ ID NO:1), optimizes the gene for protease AbTp (nucleotide sequence shown in SEQ ID NO:2), constructs it into the vector pCold II, and then transforms it into the Escherichia coli expression strain Escherichia coli BL21. TMThe expression of the enzyme was induced in (DE3); bacterial culture containing the target protein was collected, the cells were broken up, and the target protein was purified using an anion exchange column; enzymatic properties were studied to determine the relevant properties of the protease, such as temperature, pH and stability; the purified protease AbTp was applied to the study of hydrolysis of shrimp shell protein, and a degradation process for shrimp shell protein was designed based on the good thermal stability of the protease.

[0048] The composition of each 1L culture medium of the present invention is as follows:

[0049] Liquid LB medium: Tryptone 10g, Yeast extract 5g, NaCl 10g;

[0050] Solid LB medium: Tryptone 10g, Yeast extract 5g, NaCl 10g, 1.5% Agar.

[0051] Example 1: Expression and purification of protease AbTp

[0052] 1. Transform recombinant plasmids containing the target gene into competent expression cells.

[0053] The target gene was derived from a high-temperature composting metatranscriptome database. After codon optimization in *E. coli*, a 6XHis tag was added to the C-terminus, and the gene was cloned into the pCold II vector. After plasmid extraction, it was transformed into an expression strain for protein expression.

[0054] 2. The conversion steps are as follows:

[0055] (1) Take 50 μL of competent E.coli BL 21 cells and place them on ice to thaw;

[0056] (2) After thawing, take 2 μL of recombinant plasmid and slowly add it to E.coli BL 21 competent cells, and place it on ice for 30 minutes;

[0057] (3) Heat shock in a 42℃ water bath for 45 seconds, then quickly place on ice to cool for 2 minutes;

[0058] (4) Add 500 μL of non-resistant LB solution and incubate for 1 h in a constant temperature shaker at 37 °C and 200 rpm;

[0059] (5) Take an appropriate amount of bacterial suspension and spread it onto a container containing 1 μL·mL⁻¹ -1 Ampicillin was incubated in an LB solid culture dish, inverted, at 37°C for 12 hours.

[0060] 3. The process of inducing the expression of the target protein is as follows:

[0061] (1) Pick a single colony from the above LB solid culture dish and inoculate it into a sterile shake flask containing 50 mL of LB culture medium (ampicillin concentration 1 μL·mL). -1 Incubate at 37℃ and 200rpm in a shaker until the OD600 value reaches 0.6–0.8;

[0062] (2) Take 10 mL of culture medium and inoculate it into 1 L of basic culture medium (ampicillin concentration is 1 μL·mL). -1 In the culture medium, the samples were incubated at 37°C and 200 rpm until the OD600 value reached 0.6–0.8.

[0063] (3) Place the shake flask containing cell culture medium in an ice-water bath, cool it to below 16°C, add 0.5 mM IPTG inducer, and incubate at 16°C and 200 rpm for 20 h.

[0064] (4) After 20 hours of induction, centrifuge at 4°C and 7000 rpm for 15 minutes, discard the supernatant, collect the cell pellet and weigh it. The cells can be broken up for further purification, or the cells can be frozen in a -80°C freezer.

[0065] 4. The purification procedure for the target protein is as follows:

[0066] (1) Weigh the cells, add cell lysis buffer to make the cell concentration between 10% and 15%, and stir the cell solution with a glass rod. This process should be completed under ice-water bath conditions.

[0067] (2) When the cell solution is mixed and there are no lumps, the cells are disrupted using a high-pressure homogenizer. During the process, the pressure is slowly increased to 600 bar and the cell solution is disrupted until it is clear and transparent. Centrifuge at 15,000 rpm for 50 minutes at 4°C. Filter the supernatant with a 0.45 μm filter and collect it.

[0068] (3) Pre-equilibrate the anion exchange column to create a favorable loading environment for the target protein. After equilibrating for 5 column volumes, slowly load the filtered supernatant to allow the protein to fully bind to the column. Use 3 to 5 column volumes of washing solution to remove impurities. Finally, use a gradient elution with a high-concentration salt solution to obtain the target protein for subsequent biochemical characterization studies.

[0069] Example 2: Biochemical Characteristics Study of Protease AbTp

[0070] (1) Detection of AbTp activity

[0071] In addition to hydrolyzing peptide bonds formed by basic amino acids and other amino acids, proteases can also hydrolyze ester bonds formed by basic amino acids, exhibiting highly specific catalytic activity. The activity of proteases can be measured using artificially synthesized N-benzoyl-L-arginine ethyl ester (BAEE) as a substrate: the absorbance change of the product benzoylarginine catalyzed by the protease in BAEE is measured at 253 nm, and the rate of change in absorbance reflects the activity of the protease.

[0072] The specific enzyme activity testing process is as follows:

[0073] Prepare a 50 mM BAEE solution (1 L of buffer solution to dissolve 85.705 mg of BAEE substrate). Measure the change in absorbance ΔA at 253 nm within 1 minute in a 1 cm reaction chamber between 200 μL of enzyme solution and 3 mL of BAEE substrate solution. 253nm .

[0074] Activity definition: At 25°C, ΔA 253nm An increase of 0.001 represents one esterase hydrolysis unit of a protease.

[0075] The formula for calculating enzyme activity is BAEE units (U / mg) = ΔA 253nm ×k / (0.001×c×0.2)

[0076] k is the dilution factor, c is the enzyme concentration (1 mg / mL), and 0.2 is the enzyme volume (mL).

[0077] (2) The optimal reaction temperature of the protease AbTp:

[0078] Prepare a buffer solution for the optimal pH of the protease. Dissolve 85.705 mg of BAEE substrate in 1 L of a 50 mM buffer solution. To investigate the optimal temperature for the protease, add temperature gradients as needed for the experiment. Take 3 mL of the BAEE substrate solution, preheat it in a water bath at various reaction temperatures for 2 minutes, and add 1 mg / mL of the solution. -1 After adding 200 μL of enzyme solution and reacting for 10 minutes, the absorbance at 253 nm was measured within 1 minute in a 1 cm reaction cell.

[0079] (3) Determine the optimal reaction pH of the protease AbTp:

[0080] Prepare a 50 mM buffer solution with a pH range of 2–3 (Glycine-HCl), a 50 mM phosphate buffer solution with a pH range of 5–8 (phosphate), and a 50 mM Glycine-NaOH buffer solution with a pH range of 9–13 (Glycine-NaOH). Based on preliminary experiments, explore the optimal pH of the protease AbTp at the optimal temperature in (2).

[0081] (4) Determine the thermal stability of the protein AbTp

[0082] Protein stability is related to its activity, referring to the time it retains its activity during irreversible inactivation, including the inactivation half-life t of the protease AbTp of this invention. 1 / 2 Its optimal reaction temperature T opt The thermal stability was determined at a temperature 10°C higher than the optimal reaction temperature.

[0083] The results are as follows:

[0084] (1) The purity of the purified target protein was determined. The theoretical molecular weight of the protease AbTp is 31.3 kDa. Purity analysis was performed using SDS-PAGE (results are shown below). Figure 1 (As shown).

[0085] (2) According to the protein marker, the protein in lane 1 between 25kDa and 35kDa is the target protein AbTp, and the purified protein is relatively pure.

[0086] (3) The optimal temperature for determining the protein AbTp is 70℃, and the optimal pH is 6.0. Figure 2 and Figure 3 As temperature increases, enzyme activity first increases and then decreases. AbTp activity is highest at 70℃, while enzyme activity at 60℃ and 80℃ is only 70% and 65% of that at the optimum temperature of 70℃, respectively.

[0087] (4) Based on the actual measured percentage of enzyme activity, a linear fit was performed with the natural logarithm of the residual enzyme activity as the ordinate and the measurement time t as the abscissa. Figure 4 Its slope is the inactivation constant k, and its half-life can be calculated using the following formula.

[0088] Half-life calculation formula: t 1 / 2 =ln2 / k

[0089] The protease AbTp exhibits good thermal stability at its optimal reaction temperature of 70°C.

[0090] (5) According to the first-order inactivation constant, the half-life of the protein AbTp at 70℃ is 267 minutes and the half-life at 80℃ is 60 minutes.

[0091] Example 3: Application of the thermostable protease AbTp to the hydrolysis of shrimp shell protein

[0092] (1) Pre-treatment of shrimp shells

[0093] Add water to the raw shrimp shells and heat at 120°C for 30 minutes to inactivate the enzymes contained in the shrimp shells. The dried shrimp shells are then ground into powder using a grinder and used as experimental material.

[0094] (2) The effect of feed-to-liquid ratio on product

[0095] Weigh out 5g, 10g, 15g, 20g, and 25g of shrimp shell powder and add them to ultrapure water. Add 5mg of purified proteinase AbTp and bring the volume to 100mL. Set the shaker speed to 200rpm and the temperature to 40℃, and react for 13h. After the reaction, remove the inactivated protein from the supernatant, leaving a red precipitate. Remove the water from the precipitate using a vacuum filter and dry it overnight in an oven at 60℃ for later use.

[0096] (3) Determination of total nitrogen content of the product

[0097] Weigh 0.0500 g of the dried product and place it in a microwave digestion tube. Add 5 mL of supercritical concentrated sulfuric acid and soak for 12 hours, then add 5 mL of 30% hydrogen peroxide. Turn on the microwave digester and set the gradient temperature program as follows: heat to 180°C for 15 minutes, hold for 20 minutes; then heat to 220°C for 15 minutes and hold for 10 minutes, then cool to room temperature and remove. Finally, place it in an electric furnace and heat at 180°C for 30 minutes.

[0098] The total nitrogen content of the product was determined using a flow analyzer. After diluting the product to 100 mL, 10 mL of the mixed product was taken to determine the total nitrogen content. The efficiency of the protease AbTp in hydrolyzing shrimp shell protein was calculated. Figure 5 (As shown).

[0099] The protein hydrolysis rate is calculated using the following formula:

[0100] Deproteinization (%) = [(P o ×O)-(P R ×R)] / (P o ×O)×100%

[0101] P o This refers to the protein content (g / g) before treatment, P R This refers to the protein content (g / g) after processing. O refers to the weight of the untreated original sample, and R refers to the weight of the processed and dried sample.

[0102] A 5% material-to-liquid ratio is optimal. Figure 5 As shown.

[0103] (4) Qualitative analysis of shrimp shell protein hydrolysates

[0104] Fourier transform infrared spectroscopy (FT-IR) is one of the classic methods for chitin identification. Infrared spectroscopy can analyze amide bonds and amino groups, making it a classic method for chitin analysis. The dried product after the above reaction and dried KBr were ground, compressed into a tablet, and placed in the detector of a Fourier transform infrared spectrometer. The sample was measured at 4000–400 cm⁻¹. -1 The chitin exposed after the degradation of shrimp shell protein was analyzed.

[0105] Fourier transform infrared spectroscopy can determine molecular groups and molecular structure by analyzing the number and intensity of spectral peaks. The infrared spectrum of chitin is located at 2447 cm⁻¹. -1 The vicinity shows an absorption peak of amino stretching vibration in the chitin functional region, at 1655 cm⁻¹. -1 The vicinity contains amide band absorption peaks (amide I and amide II bands). The sample treated with the protease of this invention (shown by the blue line) exhibits a significantly different change in peak intensity at the corresponding wavelength compared to the untreated shrimp shell sample (shown by the black line). Figure 6 (As shown). The changes in absorption peak and absorption intensity indicate that after AbTp hydrolysis, the chitin-coated protein is degraded, exposing the chitin.

[0106] In the qualitative detection of chitin, in addition to the common infrared spectroscopy method, scanning electron microscopy (SEM) can directly analyze the surface morphology and physical state of the product.

[0107] Scanning electron microscopy (SEM) uses an electron probe to scan and illuminate the surface of a material to observe its surface morphology. SEM results show that untreated shrimp shells exhibit a flocculent structure (e.g., ...). Figure 7 (As shown). After treatment with AbTp protease, the scanning electron microscope images of the shrimp shell samples showed the unique microfibrils and porous structure of chitin (as shown). Figure 8 As shown in the image, this indicates that the proteins in the shrimp shell have been hydrolyzed, exposing the chitin.

[0108] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A protease Ab Tp, the protease Ab The amino acid sequence of Tp is shown in SEQ ID NO:

1.

2. The protease according to claim 1 Ab Tp, characterized in that The protease Ab Tp originates from actinomycetes Actinobacteria bacterium .

3. A biomaterial, wherein the biomaterial may be any one of the following (B1)-(B4): (B1) encodes a protease Ab Tp's DNA molecule; (B2) An expression cassette containing the DNA molecule described in (B1); (B3) A recombinant vector containing the DNA molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2); (B4) A recombinant microorganism containing the DNA molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B3); in, (B1) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

2.

4. A method for hydrolyzing proteins in shrimp shells, characterized in that, The method comprises adding the protease described in claim 1 to a shrimp shell buffer solution for hydrolysis.

5. The application of the protease of claim 1 and the biomaterial of claim 3 in the hydrolysis of proteins in shrimp shells.

6. The application according to claim 5, characterized in that, Disperse shrimp shells in a pH 6.0 buffer solution and add protease. Ab Tp, shrimp shell and protease Ab The mass ratio of Tp is 5g:5mg.

7. The application according to claim 5, characterized in that, The shrimp shells mentioned are crayfish shells. They are heated at 120°C for 30 minutes to inactivate the enzymes contained in the shrimp shells, and then the dried shrimp shells are crushed using a grinder.

8. The application according to claim 5, characterized in that, Add 5g of crayfish shells to a pH 6.0 buffer solution, then add 5mg of protease. Ab The volume was adjusted to 100 mL, the reaction temperature was set at 60℃, the shaking speed was set at 200 rpm, and the reaction was carried out for 13 h.

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