Novel protease with wide pH activity range and application thereof

Abstract

The invention provides a novel protease with a wide pH activity range and an application thereof, in particular relates to an application in vegetable protein raw materials, and particularly relates to an enzymatic property of the protease and an application thereof. The protease gene (AP) provided by the invention is derived from Ganoderma lucidum (Ganoderma Session), and gene sequence optimization design is carried out on the basis of a gene sequence as shown in SEQ ID NO: 1. The AP provided by the invention has the optimal catalysis temperature of 45 DEG C, the enzyme can maintain more than 60% of enzyme activity within the pH range of 6.0-9.0, and the residual enzyme activity is more than 60% after the AP is treated at 40-50 DEG C for 30 minutes; the AP disclosed by the invention has resistance to most metal ions; and the AP can be used for carrying out enzymolysis on vegetable protein raw materials to generate polypeptides, so that the content of antinutritional factors is reduced, the nutritional value is improved, and the AP has a wide application prospect in the field of feeds.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, in particular to a novel protease with a wide pH activity range and application thereof. Background Art

[0002] my country's high-quality protein feed raw materials are highly dependent on imports. In addition to animal protein feed, the import demand for plant protein raw materials is also large. At present, reducing soybean imports is an urgent problem that the feed industry needs to solve. It is necessary to reduce dependence on imported soybeans and reduce costs through comprehensive measures such as "improving efficiency, opening up new sources, and adjusting structure." Improving the utilization rate of plant protein feed is an effective way to achieve soybean meal reduction and substitution, which can be achieved mainly by reducing the content of anti-nutritional factors and developing its own nutritional value.

[0003] The active peptides in plant protein raw materials have high nutritional value. However, the content of active peptides in raw materials without any treatment is very low. Generally, chemical methods, microbiological methods and enzymatic methods can be used to increase the peptide content in raw materials. Compared with chemical methods and microbiological methods, enzymatic methods have the advantages of high efficiency, mild reaction conditions, easy operation and high product safety. Protease is an important material for the preparation of polypeptides by enzymatic hydrolysis. Under appropriate conditions, it catalyzes the hydrolysis of protein peptide bonds to generate small molecule polypeptides and free amino acids, thereby improving the nutritional value of plant protein raw materials and the digestibility and utilization rate of animals.

[0004] As the enzymatic hydrolysis product of protease, bioactive peptides have the advantages of anti-cancer activity and low cholesterol properties. In addition, adding an appropriate amount of protease to feed can promote the degradation of protein in the gastrointestinal tract of animals, improve the digestion and absorption of nutrients such as low molecular weight peptides and free amino acids, and significantly improve feed utilization and reduce the irritation of the gastrointestinal tract of young animals.

[0005] Protease is a general term for a class of enzymes that can catalyze protein hydrolysis, and there are many types of proteases. According to the differences in the position of the enzymatic reaction, proteases can be divided into endoproteases and exoproteases. According to the differences in the source, proteases can be divided into plant proteases, animal proteases, microbial proteases and other proteases. According to the differences in structure and active sites, proteases can be divided into serine proteases, aspartic acid proteases, cysteine ​​proteases and metalloproteases. In addition to being used to prepare bioactive peptides, proteases are widely used in industries such as washing, leather making, food and feed. In the washing industry, proteases can decompose stains such as sweat, blood, food residues, and have resistance to the denaturing effects of surfactants, oxidants, and chelating agents in detergents. In the food industry, proteases are often used in processes such as cheese making, meat tenderization, juice clarification, food baking and beer fermentation. In the leather industry, proteases can decompose macromolecular compounds in leather, reduce the toughness and strength of leather, and improve the softness and gloss of leather.

[0006] Studies have shown that proteases can effectively degrade β-conglobulin and globulin in soy protein isolate and produce a large number of small molecular oligopeptides. Therefore, protease-catalyzed enzymatic hydrolysis of plant protein raw materials can significantly improve their nutritional value and raw material utilization, which is of great significance to the feed industry and animal production. Summary of the invention

[0007] The purpose of the present invention is to provide a novel protease with a wide pH activity range and application thereof.

[0008] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a kind of Ganoderma Sessile ) protease, its optimum pH is 6.5, and its pH activity range is wide, its optimum temperature is 45°C, it has good metal ion resistance, and it still has more than 50% activity after incubation at 50°C for 1 hour. These characteristics are conducive to its application in the food and feed industry, and it has certain application prospects.

[0009] The protease is: i) the amino acid sequence shown in SEQ ID NO: 2; or ii) an amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of i); or iii) A protease having the same function obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence of i) or ii).

[0010] In a second aspect, the present invention provides a gene encoding the protease, the nucleotide sequence of which is: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 1 is replaced, deleted and / or one or more nucleotides are added and the nucleotide sequence expresses a protease with the same function; iii) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protease with the same function, wherein the stringent conditions are hybridization in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS at 65°C, and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protease.

[0011] In a third aspect, the present invention provides biological materials containing the gene, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0012] In a fourth aspect, the present invention provides a method for preparing the protease, wherein the gene encoding the protease is optimized according to the preferred codons of Pichia pastoris, and then introduced into Pichia pastoris via a plasmid to express the target protein; Furthermore, the optimized gene sequence is shown in SEQ ID NO:3.

[0013] In a fifth aspect, the present invention provides application of the protease or the biomaterial in the field of feed.

[0014] In a sixth aspect, the present invention provides the use of the protease or the biomaterial in improving the degradation efficiency of plant protein raw materials.

[0015] In a seventh aspect, the present invention provides a method for degrading plant protein, using the protease to degrade the plant protein.

[0016] Furthermore, the reaction system used to degrade the plant protein contains a phosphate solution.

[0017] Furthermore, the reaction conditions are: 40-50°C, pH 4.0-10.0.

[0018] Preferably, the reaction conditions are: 45°C, pH 6.0-9.0.

[0019] More preferably, the reaction conditions are: 45°C, pH 6.5.

[0020] Preferably, the plant protein includes but is not limited to soybean meal and rapeseed meal.

[0021] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) The recombinant protease provided by the present invention has an optimum pH of 6.5 and good pH stability, maintaining a high relative activity in the pH range of 4.0-10.0.

[0022] (ii) The protease provided by the present invention has an optimum catalytic temperature of 45°C and still maintains more than 50% activity after incubation at 50°C for 1 hour.

[0023] (III) The recombinant protease provided by the present invention has a certain resistance to most metal ions, including 10 mM 2+ The enzyme activity after treatment was about 135.67%.

[0024] (IV) The recombinant protease provided by the present invention can catalyze the hydrolysis of protein in soybean meal to generate small molecule polypeptides, thereby improving its nutritional value and reducing its antigenic protein content. It has good enzymatic hydrolysis effect and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the electrophoresis diagram of SDS-PAGE analysis of the fermentation supernatant in Example 2 of the present invention.

[0026] Figure 2 This is the effect of pH on the activity of the recombinant protease in Example 3 of the present invention.

[0027] Figure 3 It is the pH stability of the recombinant protease in Example 3 of the present invention.

[0028] Figure 4 This is the effect of temperature on the activity of the recombinant protease in Example 3 of the present invention.

[0029] Figure 5-Figure 6 This is the temperature stability of the recombinant protease in Example 3 of the present invention.

[0030] Figure 7 This is a protein SDS-PAGE image of soybean meal degraded by the recombinant protease in Example 4 of the present invention. M: protein marker; 1: soybean meal; 2: enzymatic soybean meal control group (only buffer was added); 3: enzymatic soybean meal treatment group (buffer and protease were added). DETAILED DESCRIPTION

[0031] The present invention aims to provide a new protease. Specifically, the nucleotide sequence encoding the protease is shown in SEQ ID NO:1.

[0032] The amino acid sequence of the protease provided by the present invention is shown in SEQ ID NO:2.

[0033] The optimum pH of the novel protease provided by the present invention is 6.0-9.0.

[0034] The present invention also provides biological materials containing the above-mentioned protease, wherein the biological materials are expression boxes, vectors, and engineering bacteria.

[0035] The present invention further provides the use of the above-mentioned protease or the above-mentioned biomaterial in the feed industry (feed or feed additives), and the use of the above-mentioned protease or the above-mentioned biomaterial in the degradation efficiency of plant protein raw materials.

[0036] In the application, protease is used to degrade the protein of the plant protein raw material.

[0037] Furthermore, the reaction system used to degrade the plant protein contains a phosphate solution.

[0038] In the application, the plant protein raw materials include but are not limited to soybean meal and rapeseed meal.

[0039] The present invention also provides a feed or a feed additive, wherein the feed or the feed additive contains the above-mentioned protease or the above-mentioned biological material.

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0041] The Pichia pastoris expression vector pPpinkα-HC and strains used in the following examples Pichia PinkStrain 4 Purchased from Invitrogen.

[0042] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0043] Example 1 Optimization and synthesis of protease genes The gene sequence optimization design was performed based on the protease gene sequence shown in SEQ ID NO: 1 (excluding the signal peptide sequence). The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0044] Without changing the amino acid sequence, the codons with low usage frequency were replaced with the codons with high usage frequency of Pichia pastoris, while eliminating the continuous pyrimidine base sequence and reducing the stability of the secondary structure of mRNA 5'. After codon optimization, the G+C content of the gene was reduced from 59.88% to 51.77%, which is closer to the GC content of the Pichia pastoris genome. The optimized sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (SEQ ID NO: 3).

[0045] Example 2 Construction of a recombinant protease expression engineered strain and high-density fermentation The recombinant pPinkα-HC-AP plasmid was transformed into Pichia pastoris by electroporation Pink Strain 4 In competent cells, the recombinant protease AP Pichia yeast engineering strain was obtained by adenine auxotrophy screening and shake flask verification. The shake flask expression level of the recombinant protease was 6.6 U / mL (measured using casein as substrate), and the size on SDS-PAGE was about 35~40 kDa ( Figure 1 ).

[0046] According to the Invitrogen Pichia fermentation manual, the recombinant strain was fermented at a high density of 30 L. When the wet bacterial weight and enzyme activity reached a peak and remained stable or showed a downward trend, the fermentation was stopped, the supernatant was centrifuged and freeze-dried, and the enzyme activity was measured after freeze-drying, which was approximately 19,579.3 U / g.

[0047] Example 3 Analysis of Enzymatic Properties of Recombinant Protease Protease activity assay: Preheat 10 g / L casein (Sigma, USA) in a 40°C water bath for 5 min, then take 100 μL of the preheated enzyme solution and casein substrate solution diluted 1,000 times with pH 3.0 lactic acid-sodium lactate buffer, mix thoroughly, and place in a 40°C constant temperature water bath for 10 min. Add 200 μL of 0.4 M trichloroacetic acid solution to terminate the reaction and shake to mix. Take 100 μL of the static filtrate, add 500 μL of 0.4 M sodium carbonate solution and 100 μL of Folin working solution, and place in a 40°C constant temperature water bath for 20 min.

[0048] Measure the absorbance OD using an enzyme-labeled 680 OD was obtained based on the tyrosine standard curve. 680 The relationship between the tyrosine concentration of the substrate and the tyrosine concentration after protease degradation is calculated, and then the enzymatic activity of the protease is calculated.

[0049] The enzyme solution was diluted with buffer solutions of pH 2.0-11.0, and the substrate solution was prepared with buffer solutions of corresponding pH. The enzyme activity was measured at 40°C.

[0050] After the enzyme solution was appropriately diluted with a phosphate buffer of pH 6.5, the enzyme activity was measured and calculated at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ to determine its optimal temperature. The test results showed that the optimal pH of AP was 6.5, and the pH stability was good, maintaining a high relative activity between pH 4.0 and 9.0 ( Figure 2 and Figure 3 The optimum catalytic temperature of AP was determined to be 45°C under the condition of pH 6.5 ( Figure 4 ), and the activity remained above 50% after incubation at 50°C and 55°C for 60min and 30min respectively ( Figure 5 and Figure 6 ).

[0051] A 20 mM compound solution of different metal ions was prepared using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 6.5. The enzyme solution and metal ions were mixed in equal volumes to make the final concentration of metal ions 10 mM. After treatment at room temperature for 1 h, the enzyme activity was measured under the optimal conditions, and the enzyme solution without metal ions was used as a blank control. The results showed that AP had different tolerances to different metal ions at a final concentration of 10 mM and EDTA incubation at room temperature for 1 h. Fe 3+ , Cu 2+ 、Zn 2+ Metal ions such as Ca and EDTA will inhibit enzyme activity. Except for EDTA, which has a strong inhibitory effect, the relative activity of the enzyme after treatment with other ions is above 60%. 2+ , Mn 2+ Metal ions such as manganese sulfate can promote enzyme activity, and the enzyme activity was about 135.67% after treatment with manganese sulfate (Table 1). We also studied the tolerance of AP to protease inhibitors (Table 1). In the presence of 1 mM pepstatin A, AP lost more than 92% of its activity, and PMSF (phenylmethylsulfonyl fluoride) had little effect on AP activity.

[0052] Table 1 Effects of metal ions and protease inhibitors on the activity of protease AP

[0053] The enzymatic activity of AP was determined under the optimal conditions using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 6.5, with 10 g / L casein, bovine hemoglobin, skim milk powder and soy protein isolate substrates, respectively. AP showed a wide range of substrate specificity (Table 2). The results showed that the specific activity of AP to casein was the highest (100.0%), followed by skim milk powder (69.45%), bovine hemoglobin (31.18%) and soy protein isolate (26.61%).

[0054] Table 2 Effects of different substrates on the activity of protease AP

[0055] Example 4 Study on the degradation of soybean meal by protease The lyophilized protease was applied to the soybean meal substrate (ordinary soybean meal), which was previously crushed, passed through a 40-mesh sieve and treated with high temperature and high pressure (121°C for 20 min). The same system containing only the substrate without enzyme and treated in the same way was used as a blank control. The reaction system (5 ml) contained 0.5 g of substrate, 50 mM phosphate buffer (pH 6.5), and the amount of recombinant protease added was 2000U / g of substrate. It was incubated at 45°C for 12 h, dried and crushed for later use. Weigh an appropriate amount of sample, extract the protein therein, and quantitatively detect the content of antigen protein therein, and calculate the antigen protein degradation rate (Table 3). Pipette an appropriate amount of the obtained sample protein extract into a centrifuge tube containing a 10kDa retention membrane, and then centrifuge it on a centrifuge at 12,000 rpm for 5 minutes, leaving the submembrane solution, which is the sample polypeptide solution. The polypeptide content and protein content of the substrate were determined by the principle of biuret reaction, and the polypeptide conversion rate was calculated (Table 4). The calculation formula is polypeptide conversion rate = unit mass polypeptide content ÷ unit mass protein content.

[0056] The SDS-PAGE assay ( Figure 7 ) and mass spectrometry results (Table 3) show that the content of globulin and β-conglobulin is significantly reduced after protease hydrolysis. This time, when the protease degrades the substrate, it can catalyze the hydrolysis of the protein in it to produce small molecular peptides (Table 4). Recombinant protease AP can degrade antigenic proteins in soybean meal and improve the nutritional value of soybean meal. It has a good enzymatic hydrolysis effect and broad application prospects.

[0057] Table 3 Effects of protease hydrolysis on the content and degradation rate of globulin and β-conglobulin in soybean meal

[0058] Table 4 Effect of protease hydrolysis on the polypeptide content and conversion rate of soybean meal

[0059] Example 5 Study on the degradation of rapeseed meal by protease The lyophilized protease was applied to the rapeseed meal substrate, which was previously crushed, passed through a 40-mesh sieve, and treated with high temperature and high pressure (121°C for 20 min). The same system containing only the substrate without enzyme and treated in the same way was used as a blank control. The reaction system (5 ml) contained 0.5 g of substrate, 50 mM phosphate buffer (pH 6.5), and the amount of recombinant protease added was 2000 U / g of substrate. It was incubated at 45°C for 12 h, dried, and crushed for later use. Weigh an appropriate amount of sample and extract the protein therein. Pipette an appropriate amount of the obtained sample protein extract into a centrifuge tube containing a 10 kDa retention membrane, and then centrifuge it on a centrifuge at 12,000 rpm for 5 minutes, leaving the submembrane solution, which is the sample polypeptide solution. The polypeptide content and protein content of the substrate were determined using the principle of biuret reaction, and the polypeptide conversion rate was calculated (Table 5). The calculation formula is as follows: Peptide conversion rate = peptide content per unit mass ÷ protein content per unit mass Table 5 Effect of protease hydrolysis on the polypeptide content and conversion rate of rapeseed meal

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A protease, characterized in that It is: i) the amino acid sequence shown in SEQ ID NO: 2; or ii) an amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of i); or iii) A protease having the same function obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence of i) or ii).

2. A gene encoding the protease according to claim 1, characterized in that: Its nucleotide sequence is: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 1 is replaced, deleted and / or one or more nucleotides are added and the nucleotide sequence expresses a protease with the same function; iii) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protease with the same function, wherein the stringent conditions are hybridization in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS at 65°C, and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protease.

3. The biological material containing the gene according to claim 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

4. The method for preparing the protease according to claim 1, characterized in that: The gene encoding the protease of claim 1 is optimized according to the preferred codons of Pichia pastoris, and then introduced into Pichia pastoris via a plasmid to express the target protein; The optimized gene sequence is shown in SEQ ID NO:

3.

5. Use of the protease according to claim 1 or the biomaterial according to claim 3 in the field of feed.

6. Use of the protease according to claim 1 or the biomaterial according to claim 3 in improving the degradation efficiency of plant protein raw materials.

7. A method for degrading plant protein, characterized in that: The protease described in claim 1 is used to degrade plant protein.

8. The method according to claim 7, characterized in that The reaction system used to degrade the plant protein contains a phosphate solution.

9. The method according to claim 7 or 8, characterized in that: Reaction conditions: 40-50°C, pH 4.0-10.0; Preferably, the reaction conditions are: 45°C, pH 6.0-9.0; More preferably, the reaction conditions are: 45°C, pH 6.

5.

10. The method according to any one of claims 7 to 9, characterized in that: The plant protein is selected from soybean meal and rapeseed meal.

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