A highly efficient protease and a method for preparing the same

Abstract

This invention relates to a highly efficient protease and its preparation method, wherein the amino acid sequence of the protein includes the sequence shown in SEQ ID No. 1. This invention discovers a protein that possesses protease function, expanding the range of proteases and enabling the further development of highly efficient proteases. The nucleic acid sequence of the aforementioned protease is combined with an expression vector to construct a recombinant plasmid, obtaining a recombinant strain with protease hydrolysis activity. This recombinant strain has a simple process, low production cost, and stable and enhanced protease activity, making it suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of protease technology, and in particular to a highly efficient protease and its preparation method. Background Technology

[0002] Proteases participate in the degradation of proteins both inside and outside cells, hydrolyzing long-chain polypeptides or proteins into free amino acids or smaller peptides. Compared to traditional chemical methods, proteolytic protein hydrolysis is more environmentally friendly, with milder reaction conditions. Traditional thermochemical methods require harsh conditions and can cause the degradation of unstable amino acids such as serine, arginine, and threonine; furthermore, some specific chemicals may cause environmental problems. Therefore, proteolytic methods are increasingly favored.

[0003] In addition, proteases, as biocatalysts, not only have high selectivity and specificity, but also greatly reduce the generation of harmful byproducts. Therefore, they have significant advantages in terms of environmental protection and sustainability. Due to their mildness and high efficiency, protease catalysis technology is widely used in industries such as detergents, textiles, leather, feed and food, pharmaceuticals, and bioenergy.

[0004] However, low protease activity is a key issue limiting the application of proteases and hindering their development. Utilizing molecular biology techniques to clone and heterologously express protease genes, thereby improving expression efficiency and secretion activity, can effectively enhance protease production and performance.

[0005] To enhance protease activity, existing technologies often involve adding active ingredients. For example, CN108795908 describes adding dextran to improve protease activity. CN115698246 describes adding saponins to enhance protease activity in detergents. However, these methods of adding active ingredients are only applicable to specific use cases and cannot broaden the application environment. To address these issues, obtaining a highly active protease is the best solution.

[0006] Therefore, how to obtain proteases with high enzyme activity has become an urgent problem to be solved. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a highly efficient protease and its preparation method. The protease has an enzyme activity as high as 625 U / mL, and the preparation process is simple, with low production costs, making it suitable for large-scale industrial production.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the use of a protein in the preparation of a protease, said protein having an amino acid sequence comprising the sequence shown in SEQ ID No. 1.

[0010] In this invention, a protein was discovered that possesses protease function, expanding the range of proteases and enabling the further development of highly efficient proteases.

[0011] SEQ ID No. 1:

[0012] MNYTTKKCLITFIIVLFSMSFLFPSVSSARNHTSSAASTASVPSGQYYRSQEVALHSHVQKASLYYTTDGSQPTTQSQLYKKPIHIETDTTLKVSAYKNRHRLATSTYNYRFVTREDIASSFLSFEYQGMPYRLYIPKNHKRGKAYPLVLFLHGGGERGTDNQKQLLANDGAVLWASPE VQKKHPSYVLAPQARNAVDGGFALTRNVQNEIDLTNVFQFSPDLHKAYGVLQHVLTSYQIDQKRIYATGLSQGGFGTYQLNITYPRLFAAMIPIAGGADPRKAGILASKPIWAFHAEDDSIIPISYARNTIQAIQKAGGQPLYTEYATKYGYDHASWTPAYETPGLTDWLFAQRRSFR.

[0013] Secondly, the present invention provides a highly efficient protease, the nucleic acid sequence of which includes the sequence shown in SEQ ID No. 1.

[0014] Thirdly, the present invention provides an expression vector containing the nucleic acid sequence of the highly efficient protease described in the first aspect; and the expression vector, after transfection, transduction or transformation of host cells, enables the host cells to express the highly efficient protease described in the first aspect.

[0015] Preferably, the nucleic acid sequence of the high-efficiency protease includes the sequence shown in SEQ ID No. 2.

[0016] This invention provides a nucleic acid sequence of a protease, which, when translated, forms a protein with protease function. This protease is highly reactive and stable, making it suitable for large-scale industrial production.

[0017] SEQ ID No. 2:

[0018]

[0019] Preferably, the expression vector comprises a plasmid and the nucleic acid sequence of the highly efficient protease.

[0020] Preferably, the plasmid comprises pHT43.

[0021] Preferably, the host cell comprises Basillus sp WB800N.

[0022] Preferably, the nucleic acid sequence of the expression vector includes the sequence shown in SEQ ID No. 3.

[0023] SEQ ID No. 3:

[0024]

[0025] Fourthly, the present invention provides an engineered bacterium that efficiently expresses a protease, wherein the engineered bacterium expresses the efficient protease described in the second aspect.

[0026] Fifthly, the present invention provides a method for constructing an engineered bacterium that efficiently expresses a protease according to the fourth aspect, characterized in that the construction method includes the steps of obtaining the nucleic acid sequence of the efficient protease according to the second aspect by PCR amplification, digesting it with a plasmid with enzymes, ligating it to construct the expression vector according to the third aspect, and transferring the expression vector into competent cells.

[0027] Preferably, the primers for the PCR include the sequences shown in SEQ ID No. 4 and SEQ ID No. 5.

[0028] SEQ ID No.4:

[0029] GGATCCATGCGAAATCATACGTCATCTGCTGCGTCAAC.

[0030] SEQ ID No. 5:

[0031] CCCGGGTTAGTGATGGTGATGGTGATGTCGAAAGCTTC.

[0032] Preferably, the restriction endonuclease used for digestion includes BamHI and / or SmaI.

[0033] Preferably, the ligase used for ligation includes T4 ligase.

[0034] Preferably, the method for preparing the competent cells includes: seeding the cells in GM medium, culturing them, incubating them in an ice bath, centrifuging them for the first time, and rinsing them to obtain the cells.

[0035] In this invention, self-made competent cells are used, which significantly improves the transformation success rate and provides a material basis for the subsequent production of proteases.

[0036] Preferably, the GM culture medium comprises 8-12 g / L peptone, 3-7 g / L yeast extract, 8-12 g / L NaCl, and 0.3-0.7 M sorbitol. The 8-12 g / L concentration can be, for example, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L. The 3-7 g / L concentration can be, for example, 3 g / L, 4 g / L, 5 g / L, 6 g / L, or 7 g / L. The 0.3-0.7 M concentration can be, for example, 0.3 M, 0.4 M, 0.5 M, 0.6 M, or 0.7 M.

[0037] Preferably, the amount of GM culture medium added is 1-5 mL, for example, it can be 1 mL, 2 mL, 3 mL, 4 mL or 5 mL, etc.

[0038] Preferably, the endpoint of the culture is determined by OD. 600 It is between 0.85 and 0.95, for example, it can be 0.85, 0.86, 0.88, 0.90, 0.92, 0.94 or 0.95, etc.

[0039] Preferably, the ice bath incubation temperature is 0-4℃ and the time is 5-15 min. The 0-4℃ can be, for example, 0℃, 1℃, 2℃, 3℃, or 4℃. The 5-15 min can be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min.

[0040] Preferably, the first centrifugation is performed at a speed of 4000-6000 rpm for 6-10 minutes at a temperature of 2-6°C. The 4000-6000 rpm can be, for example, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, or 6000 rpm. The 6-10 minutes can be, for example, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. The 2-6°C can be, for example, 2°C, 3°C, 4°C, 5°C, or 6°C.

[0041] Preferably, the rinsing step includes resuspending the bacterial cells in ETM medium followed by centrifugation.

[0042] Preferably, the ETM culture medium comprises 0.3-0.7M sorbitol, 0.3-0.7M mannitol, and 5-15% glycerol. The 0.3-0.7M can be, for example, 0.3M, 0.4M, 0.5M, 0.6M, or 0.7M. The 5-15% can be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0043] Preferably, the amount of ETM culture medium added is 40-60 mL, for example, 40 mL, 45 mL, 50 mL, 55 mL or 60 mL.

[0044] Preferably, the centrifugation speed is 4000-6000 rpm, the time is 6-10 min, and the temperature is 2-6℃. The 4000-6000 rpm can be, for example, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, or 6000 rpm. The 6-10 min can be, for example, 6 min, 7 min, 8 min, 9 min, or 10 min. The 2-6℃ can be, for example, 2℃, 3℃, 4℃, 5℃, or 6℃.

[0045] Preferably, the rinsing is performed 2-6 times. For example, it can be 2, 3, 4, 5, or 6 times.

[0046] Preferably, the method of transfer includes electroconvulsive conversion.

[0047] Preferably, the electroporation transformation step includes adding plasmids to competent cells, subjecting them to an ice bath followed by electroporation, adding RM medium and then shaking the culture, and then screening for antibiotics.

[0048] Preferably, the volume ratio of the plasmid to competent cells is 1:(8-14), where (8-14) can be, for example, 8, 9, 10, 11, 12, 13, or 14.

[0049] Preferably, the temperature of the ice bath is 0-4℃, and the time is 1-5 minutes. The 0-4℃ can be, for example, 0℃, 1℃, 2℃, 3℃, or 4℃. The 1-5 minutes can be, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes.

[0050] Preferably, the voltage of the electric shock is 1.5-2.5 kV / cm. The 1.5-2.5 kV / cm can be, for example, 1.5 kV / cm, 1.6 kV / cm, 1.8 kV / cm, 2.0 kV / cm, 2.2 kV / cm, 2.4 kV / cm, or 2.5 kV / cm, etc.

[0051] Preferably, the RM culture medium comprises 5-15 g / L peptone, 3-7 g / L yeast extract, 5-15 g / L NaCl, 0.3-0.7 M sorbitol, and 0.1-0.5 M mannitol. The 5-15 g / L concentration can be, for example, 5 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, 14 g / L, or 15 g / L. The 3-7 g / L concentration can be, for example, 3 g / L, 4 g / L, 5 g / L, 6 g / L, or 7 g / L. The 0.3-0.7 M concentration can be, for example, 0.3 M, 0.4 M, 0.5 M, 0.6 M, or 0.7 M. The 0.1-0.5 M concentration can be, for example, 0.1 M, 0.2 M, 0.3 M, 0.4 M, or 0.5 M.

[0052] Preferably, the shaking incubation temperature is 35-40℃, and the time is 1-5 hours. The 35-40℃ can be, for example, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. The 1-5 hours can be, for example, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0053] Preferably, the shaking incubation speed is 100-300 rpm, for example, it can be 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm.

[0054] Preferably, the antibiotics selected for screening include any one or a combination of at least two of ampicillin, chloramphenicol, or kanamycin.

[0055] In a sixth aspect, the present invention provides a method for preparing the highly efficient protease described in the second aspect, the method comprising inducing expression of the engineered bacteria that express the highly efficient protease described in the fourth aspect.

[0056] Preferably, the inducer for induced expression includes IPTG.

[0057] Compared with the prior art, the present invention has at least the following beneficial effects:

[0058] This invention discovers a highly efficient protease and transforms the sequence of this protease into Bacillus subtilis to obtain a recombinant strain with protease hydrolysis activity. The protease synthesized by this recombinant strain has high stability and an enzyme activity of up to 625 U / mL. Moreover, the process of this recombinant strain is simple, the production cost is low, and the activity of the protease produced is stable and improved, making it suitable for large-scale industrial production. Detailed Implementation

[0059] The technical solution of the present invention will be further illustrated through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0060] Example 1

[0061] This embodiment constructs recombinant plasmids.

[0062] The sequence described in SEQ ID No. 1 was modified by removing the signal peptide and adding a histidine tag. Upstream and downstream primers SEQ ID No. 4 and SEQ ID No. 5 were designed, respectively. Then, PCR amplification was performed using the genome of Basillus sp. 8A6 as a template.

[0063] The PCR amplification product and plasmid pHT43 were double-digested with restriction endonucleases BamHI and SmaI. The PCR amplification product and plasmid pHT43 were then ligated using T4 ligase to obtain a recombinant plasmid containing a highly efficient protease gene.

[0064] Example 2

[0065] This embodiment constructs recombinant engineered bacteria.

[0066] (1) Preparation of competent cells

[0067] 2 mL of Bacillus subtilis sp. WB800N was inoculated into 50 mL of GM medium. GM medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 0.5 M sorbitol. The culture was carried out until OD200 reached. 600 When the saturation value was 0.9, the cells were placed in an ice bath for 10 minutes at 0°C. After centrifugation at 5000 rpm for 6 minutes at 4°C, the cells were collected.

[0068] After centrifugation, the cells were rinsed and resuspended in 50 mL of pre-cooled ETM medium containing 0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol. The rinsing was repeated four times. The resulting cells were then resuspended in 1 mL of ETM medium, mixed thoroughly, and aliquoted into 60 μL tubes into Eppendorf tubes to obtain competent Bacillus subtilis cells.

[0069] (2) Transformation

[0070] 5 μL of the recombinant plasmid prepared in Example 1 was added to 60 μL of Bacillus subtilis competent cells and incubated at 0°C on ice for 2 min. The mixture was then placed in a 0.1 cm electroporation cuvette and electroporated once at 2 kV / cm. 1 mL of RM medium was added to the electroporation cuvette and mixed well. The RM medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 0.5 M sorbitol, and 0.38 M mannitol. The mixture was cultured at 37°C with shaking at 200 rpm for 3 h. Afterward, the mixture was plated on LB solid medium containing chloramphenicol and cultured overnight. Positive clones were screened to obtain the highly efficient protease-expressing engineered strain.

[0071] Example 3

[0072] This embodiment involves inducing the expression of recombinant engineered bacteria.

[0073] The selected positive transformants were inoculated into 5 mL of liquid LB medium and cultured at 37°C and 200 rpm until OD500 was reached. 600IPTG was added at a concentration of 0.9 to induce expression. The final IPTG concentration was 1 mM. After induction overnight, the sample was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected, which was the protease.

[0074] Comparative Example 1:

[0075] In this comparative example, the nucleic acid sequence encoding the protease in Example 1 was replaced with the nucleic acid sequence shown in SEQ ID NO. 6. The expression vector was obtained according to the preparation process provided in Example 1, and the protease was obtained by induction according to Examples 2 and 3. SEQ ID NO. 6 was derived from Bacillus 8A6.

[0076] SEQ ID NO.6:

[0077] ATGGAAAAGGAAAACAAACGTGAACAACTGCTCAGTCTGTTAGGAGAAATGCCAGAACGTCATCGCGTGGAGGCTTATACGTTAAAAATGGAAGAACGTGAATCATATGTAGTGGAAACACTCATTCTCTCCATGAATGGTGTAGAGGAAGTCCCTGCTTATTTCGTGAAACCGAAGGATACAGTGAAAAAAAGACCCGTTGTGCTGTTCCAGCACTCTCATGGCGGGAATTACGTGAATGGGAAGGACGAGCTGTTAAAAGGTGCTCATTATTTGCAAACGCCTTCATATGCAAAAGAGTTTACGTCAAAAGGATATAGTGTACTGGCCATTGATCATGCAGGGTTCGGCGAGAGAAGAGGAAGAACAGAAAGTGAAATTTTTAAGGAAATGCTTTTAACAGGAAAAGTGATGTGGGGCATGATGCTGTATGAAAGTATGTGTGCGATTGATTATTTGCTGTCTCGATCTGATGTGCCGGCGGACAGGTTAGCTGTCTTTGGGATGTCAATGGGCGGTCTTCTTTCCTGGTGGACGGCTGCACTTGATGAGAGGGTCAGTGTGTGTATTGATCTTTGTGCACAGGTGGACCATCATACATTAATTGAAACGAACAATTTGGACAGACATGGCTTTTATTACTATGTACCGAGCTTGGCGAAGCATTTTACAGCAGCGGACATTCAAGAAATGATTTTTCCAAGACCGCATTTAAGCTTAGTTGGAAAACTTGATCAGCTCACGCCTGCTGAAGGCGTTGGAAGGATTCAAAAGGTATTGAGTCAAACCTATGAAGCTGCTTCATTGAAAGAACGATATCAGCTTACCCGTCTTCATGCAGGTCATTTTGAAACAGCCGCTATGCGTCACGAGGCGACACGATTTTTGAAAAAGTGGCTGTAA。

[0078] Comparative Example 2:

[0079] In this comparative example, the nucleic acid sequence encoding the protease in Example 1 was replaced with the nucleic acid sequence shown in SEQ ID NO.7. The expression vector was obtained according to the preparation process provided in Example 1, and the protease was obtained by induction according to Examples 2 and 3. SEQ ID NO.7 was derived from Bacillus 8A6.

[0080] SEQ ID NO.7:

[0081]

[0082] Test Example 1

[0083] This test example measures the enzyme activity of the proteases induced and expressed in Example 3 and Comparative Examples 1-2. The test method is as follows:

[0084] Using Ac-Leu-pNA as the substrate, the reaction system was 1 mL, the buffer system was 50 mM phosphate buffer (pH 8.0), the final substrate concentration was 0.2 mM, and 40 μL of enzyme was added. The absorbance at 405 nm was measured using a UV spectrophotometer. Enzyme activity unit definition: 1 unit of enzyme activity is the amount of substrate hydrolyzed to produce 1 μmol of Ac-Leu-pNA per minute (ε = 0.016 L / μmol). - 1 The amount of enzyme required (cm).

[0085] Vitality calculation method:

[0086] Vitality unit / L = △OD 405 / 0.016×V / V E ;

[0087] OD 405 △OD per minute 405 Changes;

[0088] V: Total volume of the reaction system (mL);

[0089] V E Volume of enzyme in the reaction system (mL).

[0090] The enzyme activity of the proteases induced in Examples 3 and 1-2 was determined using the method described above, and the test results are shown in Table 1.

[0091] Table 1

[0092] Group Enzyme activity (U / mL) Example 3 625 Comparative Example 1 389 Comparative Example 2 573

[0093] As shown in Table 1, this gene can efficiently express proteases in a specific expression system. Compared to the other two genes, the protease obtained by the gene specified in this invention in this system has significantly higher activity. The discovery of a protein with protease function expands the range of proteases and allows for further development of highly efficient proteases. This recombinant strain has a simple process, low production cost, and produces stable and enhanced protease activity, making it suitable for large-scale industrial production.

[0094] In summary, this invention constructs a recombinant plasmid by combining the nucleic acid sequence of the aforementioned protease with an expression vector, thereby obtaining a recombinant strain with protease digestion activity. This recombinant strain has a simple process, low production cost, and provides stable and enhanced protease activity, making it suitable for large-scale industrial production.

[0095] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of protein in the preparation of proteases, characterized in that, The amino acid sequence of the protein includes the sequence shown in SEQ ID No.

1.

2. A highly efficient protease, characterized in that, The amino acid sequence of the protease includes the sequence shown in SEQ ID No.

1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid sequence of the highly efficient protease as described in claim 2; and after transfection, transduction, or transformation of host cells, the expression vector enables the host cells to express the highly efficient protease as described in claim 1.

4. The expression vector according to claim 3, characterized in that, The nucleic acid sequence of the highly efficient protease includes the sequence shown in SEQ ID No. 2; Preferably, the expression vector comprises a plasmid and the nucleic acid sequence of the highly efficient protease; Preferably, the plasmid includes pHT43; Preferably, the host cell comprises Basillus sp. WB800N; Preferably, the nucleic acid sequence of the expression vector includes the sequence shown in SEQ ID No.

3.

5. An engineered bacterium that efficiently expresses a protease, characterized in that, The engineered bacteria express the highly efficient protease described in claim 2.

6. A method for constructing an engineered bacterium that efficiently expresses a protease according to claim 5, characterized in that, The construction method includes obtaining the nucleic acid sequence of the high-efficiency protease described in claim 2 by PCR amplification, digesting it with a plasmid and ligating it to construct the expression vector described in claim 3 or 4, and then transferring the expression vector into competent cells.

7. The method for constructing an engineered bacterium that efficiently expresses protease according to claim 6, characterized in that, The primers for the PCR include the sequences shown in SEQ ID No. 4 and SEQ ID No. 5; Preferably, the restriction endonuclease used for digestion includes BamHI and / or SmaI; Preferably, the ligase used for ligation includes T4 ligase; Preferably, the method for preparing the competent cells includes: seeding the cells in GM medium, culturing them, incubating them in an ice bath, centrifuging them for the first time, and rinsing them to obtain the cells; Preferably, the GM culture medium comprises 8-12 g / L peptone, 3-7 g / L yeast extract, 8-12 g / L NaCl, and 0.3-0.7 M sorbitol; Preferably, the amount of cells added is 1-5 mL; Preferably, the amount of GM culture medium added is 50-100 mL; Preferably, the endpoint of the culture is determined by OD. 600 It is 0.85-0.95; Preferably, the ice bath incubation temperature is 0-4℃ and the time is 5-15 min; Preferably, the first centrifugation is performed at a speed of 4000-6000 rpm for 6-10 minutes at a temperature of 2-6°C. Preferably, the rinsing includes resuspending the bacterial cells in ETM medium followed by centrifugation; Preferably, the ETM culture medium comprises 0.3-0.7M sorbitol, 0.3-0.7M mannitol and 5-15% glycerol; Preferably, the amount of ETM culture medium added is 40-60 mL; Preferably, the centrifugation speed is 4000-6000 rpm, the time is 6-10 min, and the temperature is 2-6℃; Preferably, the rinsing is performed 2-6 times.

8. The method for constructing an engineered bacterium that efficiently expresses protease according to claim 6 or 7, characterized in that, The method of transfer includes electroconvulsive conversion; Preferably, the electroporation transformation step includes adding plasmids to competent cells, performing electroporation after an ice bath, adding RM medium and culturing with shaking, and then screening for antibiotics. Preferably, the volume ratio of the plasmid to competent cells is 1:(8-14); Preferably, the temperature of the ice bath is 0-4℃ and the duration is 1-5 minutes; Preferably, the voltage of the electric shock is 1.5-2.5 kV / cm; Preferably, the RM culture medium comprises 5-15 g / L peptone, 3-7 g / L yeast extract, 5-15 g / L NaCl, 0.3-0.7 M sorbitol, and 0.1-0.5 M mannitol; Preferably, the temperature for the shaking culture is 35-40℃, and the time is 1-5 hours; Preferably, the shaking incubation speed is 100-300 rpm; Preferably, the antibiotics screened include any one or a combination of at least two of chloramphenicol, ampicillin, chloramphenicol, or kanamycin.

9. A method for preparing the high-efficiency protease according to claim 2, characterized in that, The preparation method includes inducing the expression of the engineered bacteria that efficiently expresses the protease as described in claim 5.

10. The method for preparing a highly efficient protease according to claim 9, characterized in that, The inducer for induced expression includes IPTG.