DNA molecule for expressing alkaline protease, promoter and application

Abstract

The invention provides a DNA molecule for expressing alkaline protease, a promoter and application. Wherein the DNA molecule contains a promoter and a gene for expressing alkaline protease, and the sequence of the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 2. The method can solve the problem of low expression level of microbial alkaline protease in the prior art, and is suitable for the technical field of biology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a DNA molecule for expressing alkaline protease, a promoter and application. BACKGROUND

[0002] Alkaline protease is a kind of enzyme that can hydrolyze protein peptide bonds under alkaline conditions. Its optimum pH is generally 9-11. The alkaline protease produced by Bacillus licheniformis is an endoprotease, and the catalytic site is serine. Alkaline protease is widely used in detergent, food, medical, brewing, silk, leather and other industries. Alkaline protease is a popular washing additive on the market, which can greatly improve the washing and decontamination ability, especially for protein stains such as blood stains, sweat stains, milk stains and oil stains. It has unique washing effect and is the largest enzyme in industrial enzymes. With the rapid development of detergent, food, medicine and other industries, the demand for alkaline protease is increasing, but the existing alkaline protease-producing strains have the problem of low expression level of alkaline protease, which cannot meet the market demand.

[0003] The alkaline protease currently used is a protein hydrolytic enzyme obtained by deep fermentation, extraction and purification of alkaline protease derived from Bacillus licheniformis 2709 through mutagenesis method. Chinese patent application CN108570477A mentions a construction method of alkaline protease gene and recombinant Bacillus subtilis strain. The patent expresses the alkaline protease Bmp-opt gene with high enzyme activity under the control of different promoter signal peptides in the recombinant Bacillus subtilis strain to obtain the recombinant strain. In addition, Chinese patent application CN117448361A mentions a recombinant strain producing heterologous alkaline protease, which contains a combined DNA fragment containing two tandem promoters. The optimized double promoters are used to regulate and express the foreign genes for producing alkaline protease. However, the current technology mainly focuses on alkaline proteases from other sources, and does not involve research on alkaline protease expression from Bacillus licheniformis. The expression level of alkaline protease still cannot meet the demand of industrial production. SUMMARY

[0004] The main purpose of the present application is to provide a host cell for producing alkaline protease to solve the problem of low expression level of microbial alkaline protease in the prior art.

[0005] In order to achieve the above-mentioned purpose, according to the first aspect of the present application, a DNA molecule for expressing alkaline protease is provided, which contains a promoter and a gene for expressing alkaline protease, and the sequence of the promoter comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0006] Further, the DNA molecule further comprises any one or more of the following: a gene expressing a signal peptide, a gene expressing a chaperone protein.

[0007] Further, the promoter is located in the 5' direction of the alkaline protease gene.

[0008] To achieve the above object, according to a second aspect of the present application, a promoter is provided, the promoter comprising a nucleotide sequence as shown in SEQ ID NO: 2.

[0009] To achieve the above object, according to a third aspect of the present application, a recombinant plasmid is provided, the recombinant plasmid being linked with the DNA molecule or the promoter.

[0010] Further, the recombinant plasmid comprises one or more of PHYT or PHY300PLK.

[0011] To achieve the above object, according to a fourth aspect of the present application, a host cell is provided, the host cell comprising the DNA molecule, the promoter, or the recombinant plasmid.

[0012] Further, the host cell comprises Bacillus licheniformis or Escherichia coli.

[0013] Further, the host cell comprises an alkaline protease gene, the 5' direction of the alkaline protease gene comprising the promoter; the alkaline protease gene and the promoter being located in the genome of the host cell and / or being located on a free plasmid, the free plasmid being independently present in the host cell.

[0014] Further, the alkaline protease gene in the host cell has a copy number of ≥2.

[0015] To achieve the above object, according to a fifth aspect of the present application, a method for preparing alkaline protease is provided, the method comprising: culturing the host cell as claimed above, and collecting the alkaline protease.

[0016] Further, the culturing comprises: culturing the host cell in a fermentation medium.

[0017] Further, the fermentation medium comprises corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate.

[0018] Further, in the fermentation medium, the corn starch is 30-60 g / L, the soybean meal is 30-60 g / L, the disodium hydrogen phosphate is 1-3 g / L, and the sodium carbonate is 1.0-1.5 g / L; the pH of the fermentation medium is 7.2-7.6.

[0019] To achieve the above object, according to a sixth aspect of the present application, there is provided an application of the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease in the preparation of detergent, food or medicine.

[0020] To achieve the above object, according to a seventh aspect of the present application, there is provided an application of the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease in the detergent industry, the leather industry, the textile industry, the food industry, the feed industry or the field of alkaline wastewater treatment.

[0021] By using the technical solution of the present application, the above-mentioned DNA molecule for expressing alkaline protease contains a promoter and a gene for expressing alkaline protease. With the help of the promoter, the gene for expressing alkaline protease can be expressed in large quantities to obtain alkaline protease, thereby efficiently preparing alkaline protease. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The illustrations are shown to explain the present application and are not intended to limit the present application unduly. In the drawings:

[0023] Figure 1 A technical route schematic diagram for constructing a high-expression alkaline protease S8 recombinant strain according to Example 1 of the present application is shown.

[0024] Figure 2 A plasmid vector structure schematic diagram for expressing alkaline protease S8 according to Example 1 of the present application is shown.

[0025] Figure 3 A plasmid vector promoter optimization result diagram for constructing a high-expression alkaline protease S8 recombinant strain according to Example 2 of the present application is shown.

[0026] Figure 4 A plasmid vector signal peptide optimization result diagram for constructing a high-expression alkaline protease S8 recombinant strain according to Example 3 of the present application is shown.

[0027] Figure 5 A plasmid vector chaperone optimization result diagram for constructing a high-expression alkaline protease S8 recombinant strain according to Example 4 of the present application is shown. DETAILED DESCRIPTION

[0028] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0029] As mentioned in the background, there is a problem of low expression efficiency of microbial alkaline protease in the prior art. Therefore, in the present application, the inventors attempt to develop a host cell for producing alkaline protease, which can efficiently prepare the target product alkaline protease by optimizing the copy number of alkaline protease gene, signal peptide, promoter and chaperone, etc., and can be well applied to industrial scale-up production, thus a series of protection schemes of the present application are proposed.

[0030] In the first typical embodiment of the present application, a DNA molecule for expressing alkaline protease is provided, which contains a promoter and a gene for expressing alkaline protease, and the sequence of the promoter comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0031] In the present application, the inventors found that by analyzing and comparing the original ParprE promoter sequence SEQ ID NO: 1, the transcriptional negative regulator binding site sequence was obtained according to gene sequence analysis, and the sequence at this position was deleted to obtain the modified PaprE' promoter sequence SEQ ID NO: 2, which can significantly enhance the expression level of alkaline protease in Bacillus licheniformis E417. The above-mentioned DNA molecule significantly improves the expression amount of alkaline protease in the host cell under the condition of containing the optimized promoter sequence PaprE' and expressing alkaline protease. In the present application, by loading the optimized promoter PaprE' and expressing alkaline protease on the DNA molecule, the starting efficiency of transcription is enhanced, and the technical effect of promoting the efficient expression of alkaline protease is realized. In the actual production process of alkaline protease, the production cost can be significantly reduced, and the yield of alkaline protease can be improved.

[0032] SEQ ID NO: 1:

[0033] atctttcacccgtttctgtatgcgatatattgcatattttaatagatgatcgacaaggccgcaacctccttcggcaaaaaatgatctcataaaataaatgaatagtattttcataaaatgaatcagatggagcaatctcctgtcattcgcggccctcgggacctctttccctgccaggctgaagcggtctactaaaacagttattaataaccaaaaaattttaaattggtcctccaaaaaaataggcctaccatataattcattttttttctataataaattaacagaataattggaatagattatattatccttctatttaaattattctgaataaagaggaggagagtgagta.

[0034] SEQ ID NO: 2:

[0035] atctttcacccgtttctgtatgcgatatattgcatattttaatagatgatcgacaaggccgcaacctccttcggcaaaaaatgatctcataaaataaatgaatagtattttcataaaatgaatcagatggagcaatctcctgtcattcgcggccctcgggacctctttccctgccaggctgaagcggtctactaaaacagttattaataaccaaaaaattttaaattggtcctccaaaaaaataggcctaccatataattcattttttttctataataaattaacagaataattggaatagattatattatccttctatttaaattattctgaataaagaggaggagagtgagta.

[0036] In a preferred embodiment, the alkaline protease described above comprises alkaline protease S8.

[0037] In a preferred embodiment, the DNA molecule described above further comprises any one or more of the following: a gene expressing a signal peptide, a gene expressing a chaperone protein, or a gene expressing an alkaline protease.

[0038] In the present application, the inventors found that when the above-mentioned DNA molecule further comprises, but is not limited to, a gene expressing a signal peptide, a gene expressing a chaperone protein, or a gene expressing alkaline protease, the technical effect of improving the secretion efficiency, activity, and yield of alkaline protease can be achieved, the production cost of alkaline protease is reduced, and a good foundation is laid for further application of alkaline protease in industrial production.

[0039] In a preferred embodiment, the above-mentioned signal peptide is SacC, YurI, YoaW, Bli0820, Bli00338, or YbdN, preferably, the above-mentioned signal peptide is SacC or YurI.

[0040] In the present application, the inventors found that when the DNA molecule contains the optimized promoter PaprE', and the DNA molecule further contains, but is not limited to, SacC, YurI, YoaW, Bli0820, Bli00338, or YbdN, preferably, a gene expressing a signal peptide SacC or a gene expressing a signal peptide YurI, the activity of alkaline protease can be further improved, which indicates that the technical effect of improving the expression level of alkaline protease can be achieved by optimizing the signal peptide sequence in the DNA molecule.

[0041] In a preferred embodiment, the above-mentioned chaperone protein is PrsA.

[0042] The PrsA molecular chaperone protein can promote the correct folding of enzymes during protein folding, reduce misfolding and protein aggregation. In the present application, the inventors found that when the above-mentioned DNA molecule contains the nucleotide sequence of the molecular chaperone PrsA as shown in SEQ ID NO: 3, and the DNA molecule contains the optimized promoter PaprE' and the signal peptide gene SacC, the expression level of alkaline protease can be further improved, and the secretion efficiency and activity stability of alkaline protease can be improved.

[0043] SEQ ID NO: 3:

[0044]

[0045] In a preferred embodiment, the sequence of the alkaline protease comprises the amino acid sequence shown as SEQ ID NO: 4.

[0046] In the present application, the inventors found that when the sequence of the alkaline protease comprises the amino acid sequence shown as SEQ ID NO: 4 or the alkaline protease nucleotide sequence shown as SEQ ID NO: 5 is contained in the above-mentioned DNA molecule, the technical effect of increasing the yield of alkaline protease in host cells can be achieved by increasing the copy number of alkaline protease, in combination with the optimization of promoters, signal peptides and chaperones.

[0047] SEQ ID NO: 4:

[0048] MKKMLMLAFTFLLALTIHVGEASAMRKKSFWLGMLTALMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ.

[0049] SEQ ID NO: 5:

[0050]

[0051] In a preferred embodiment, in the above-mentioned DNA molecule, the above-mentioned promoter is located in the 5' direction of the above-mentioned alkaline protease gene.

[0052] In the present application, the inventors found that, in the above-mentioned DNA molecule, the promoter is located in the 5' direction of the alkaline protease gene, and the DNA molecule sequentially comprises, from 5' to 3', a promoter, a gene expressing a signal peptide, a gene expressing alkaline protease, a gene expressing chaperone PrsA, and a terminator, which can improve the expression level of alkaline protease in host cells, and improve the secretion efficiency and activity stability of alkaline protease.

[0053] In a second typical embodiment of the present application, a promoter is provided, which comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0054] In the present application, the inventors effectively solved the problems of low copy number of alkaline protease gene from Bacillus licheniformis and poor expression efficiency of the promoter by using the nucleotide sequence shown in SEQ ID NO: 2 as a promoter. By analyzing and comparing the original ParprE promoter sequence SEQ ID NO: 1, the sequence of the transcriptional negative regulator binding site was obtained according to gene sequence analysis, and the sequence at this position was deleted to obtain the modified PaprE' promoter sequence SEQ ID NO: 2, which can significantly enhance the expression level of alkaline protease in Bacillus licheniformis E417. Through the promoter optimization strategy of the present embodiment, not only the yield of alkaline protease is improved, but also the production cost is reduced, which shows the potential value of alkaline protease in industrial application, and provides strong technical support to meet the increasing demand for alkaline protease in detergent, food, medicine and other industries.

[0055] In a third typical embodiment of the present application, a recombinant plasmid is provided, which is connected with the above-mentioned DNA molecule or the above-mentioned promoter.

[0056] The above-mentioned plasmid, when connected with the above-mentioned promoter or the above-mentioned DNA molecule, can effectively transcribe and translate the carried DNA molecule in host cells to obtain a large amount of target product alkaline protease. The problems of low yield and high production cost of alkaline protease from Bacillus licheniformis in the prior art are effectively solved, which provides technical support for large-scale production and application of alkaline protease.

[0057] In a preferred embodiment, the above-mentioned recombinant plasmid comprises one or more of pHYT or PHY300PLK.

[0058] PHY300PLK is an E. coli-Bacillus shuttle expression plasmid, the nucleotide sequence of which is shown as SEQ ID NO: 6, which is mainly used in Bacillus system to realize intracellular or secretory expression of proteins. In the present application, the inventors further optimize PHY300PLK to obtain a recombinant vector PHYT shown as SEQ ID NO: 7. PHYT vector can be used as an intermediate cloning vector for prokaryotes such as E. coli, and has the characteristics of high copy number of target product gene. Preferably, the recombinant vector PHYT contains a P43-SigP fragment shown as SEQ ID NO: 8. In the present application, the inventors preferably select PHYT as the expression vector of the recombinant plasmid, which can significantly improve the expression level of alkaline protease gene in host cells, thereby achieving the technical effect of improving the yield of alkaline protease.

[0059] SEQ ID NO: 6:

[0060]

[0061] SEQ ID NO: 7:

[0062]

[0063] SEQ ID NO: 8:

[0064]

[0065] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell comprises the DNA molecule, the promoter or the recombinant plasmid.

[0066] With the host cell, the replication of the recombinant plasmid in the host cell can be carried out, and the DNA molecule carried by the recombinant plasmid can be transcribed and translated to obtain a large amount of the target product alkaline protease. With the prior art, the host cell can be broken to purify the protein, the broken crude enzyme can be catalyzed, or other methods can be used to obtain the alkaline protease. The host cell is a non-plant-derived host cell.

[0067] In a preferred embodiment, the host cell comprises Bacillus licheniformis or Escherichia coli.

[0068] In the present application, the inventors found that the host cell, including but not limited to Bacillus licheniformis or Escherichia coli, has a natural adaptive and expression system, which can effectively carry and express the target product alkaline protease, especially under the driving of the promoter mentioned above, which can significantly improve the expression amount of the alkaline protease. By using the promoter PaprE' in the host cell, not only the gene copy number of the alkaline protease is increased, but also the expression regulation mechanism is optimized, thereby realizing a substantial increase in the yield of the alkaline protease. In the present application, the inventors preferably select Bacillus licheniformis E417 as the host cell, which can efficiently and stably prepare the alkaline protease, and is suitable for large-scale fermentation to prepare high-activity alkaline protease, meeting the application demands of the detergent, food, pharmaceutical and other industries.

[0069] In a preferred embodiment, the host cell comprises the alkaline protease gene, and the 5' direction of the alkaline protease gene comprises the promoter; the alkaline protease gene and the promoter are located in the genome of the host cell and / or on a free plasmid, and the free plasmid exists independently in the host cell.

[0070] In the present application, the inventors found that the host cell comprises the alkaline protease gene, and the 5' direction of the alkaline protease gene comprises the promoter, wherein the alkaline protease gene is located in the genome of the host cell and / or on a free plasmid, and the free plasmid exists independently in the host cell, and the constructed host cell can significantly improve the yield and activity of the alkaline protease, reduce the production cost of the alkaline protease, and meet the market demand for high-enzyme-activity alkaline protease.

[0071] In a preferred embodiment, the copy number of the alkaline protease gene in the host cell is ≥2.

[0072] In the present application, the inventors found that by increasing the copy number of alkaline protease gene in the host cell, wherein the copy number is ≥ 2 (including but not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10), the expression level of the target product alkaline protease can be effectively promoted, the production cost of alkaline protease is reduced, and the market demand for alkaline protease is met.

[0073] In a fifth typical embodiment of the present application, a method for preparing alkaline protease is provided, which comprises culturing the host cell or the host cell obtained by the construction method, and collecting the alkaline protease.

[0074] In the present application, the inventors provide a method for preparing alkaline protease by culturing the host cell or the host cell obtained by the construction method, and collecting the alkaline protease. In addition, the inventors found that the host cell containing the genes including but not limited to the optimized promoter PaprE' gene, the signal peptide SacC gene and the YurI gene, the chaperone PrsA gene or the alkaline protease gene can be better applied to the preparation process of alkaline protease, and can meet the market demand for alkaline protease.

[0075] In a preferred embodiment, the culturing comprises culturing the host cell in a fermentation medium.

[0076] In a preferred embodiment, the fermentation medium comprises corn starch, soybean meal, disodium hydrogen phosphate or sodium carbonate.

[0077] In the present application, the inventors found that the fermentation medium contains corn starch, soybean meal, disodium hydrogen phosphate or sodium carbonate, and the host cell can achieve efficient expression and secretion of alkaline protease. The nutritional components of the fermentation medium improve the yield and activity of alkaline protease, and provide a more efficient and stable alkaline protease production scheme for industrial application.

[0078] In a preferred embodiment, the total amount of the fermentation medium, the corn starch is 30-60 g / L, the soybean meal is 30-60 g / L, the disodium hydrogen phosphate is 1-3 g / L, and the sodium carbonate is 1.0-1.5 g / L; the pH of the fermentation medium is 7.2-7.6.

[0079] In the present application, the inventors further found that the content of corn starch in the fermentation medium is, for example, 30-60 g / L (including but not limited to 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L or 60 g / L), the content of soybean meal is, for example, 30-60 g / L (including but not limited to 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L or 60 g / L), the content of disodium hydrogen phosphate is, for example, 1-3 g / L (including but not limited to 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L or 3 g / L), the content of sodium carbonate is, for example, 1.0-1.5 g / L (including but not limited to 1.05 g / L, 1.1 g / L, 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L or 1.5 g / L); under the condition that the pH of the fermentation medium is, for example, 7.2-7.6 (including but not limited to 7.2, 7.3, 7.4, 7.5 or 7.6), the optimal nutritional environment required for the growth of host cells and the synthesis of alkaline protease can be provided. Corn starch and soybean meal are rich in various vitamins and trace elements, which are helpful for microbial metabolism; disodium hydrogen phosphate and sodium carbonate are essential inorganic salts in the process of microbial growth and enzyme synthesis, which maintain the osmotic pressure and pH stability of the medium, promote the nutrient absorption and metabolic balance of microorganisms, and thus improve the yield of alkaline protease.

[0080] In the sixth typical embodiment of the present application, the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease is used in the preparation of detergent, food or medicine.

[0081] In the present application, the inventors find that the expression level and enzyme activity of the alkaline protease prepared by using the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease are both good, and the alkaline protease can be well used for preparing detergent, food or pharmaceutical products. Specifically, in the process of preparing detergent products, the above-mentioned alkaline protease can be used in enzyme-added laundry liquid or laundry powder to improve the decontamination ability, especially the decontamination effect of protein stains (including but not limited to blood stains or milk stains) is remarkable; in the process of preparing food products, the above-mentioned alkaline protease can be used for hydrolyzing animal and plant proteins to prepare products such as yeast extract, proteose peptone or amino acid; in the process of preparing pharmaceutical products, the above-mentioned alkaline protease can be used for nucleic acid purification or anti-inflammatory and detumescence drugs, and has the characteristics of high specificity, low toxicity, suitable for biopharmaceuticals and laboratory research.

[0082] In the seventh typical embodiment of the present application, the application of the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease in the fields of washing industry, leather industry, textile industry, food industry, feed industry or alkaline wastewater treatment is provided.

[0083] In the present application, the inventors find that the expression level and enzyme activity of the alkaline protease prepared by using the above-mentioned DNA molecule, the above-mentioned promoter, the above-mentioned recombinant plasmid, the above-mentioned host cell or the above-mentioned method for preparing alkaline protease are both good, and the above-mentioned alkaline protease has good application value in the fields of washing industry, leather industry, textile industry, food industry, feed industry or alkaline wastewater treatment. Specifically, in the washing industry, the above-mentioned alkaline protease can be used in enzyme-added laundry liquid or laundry powder to improve the decontamination ability, especially the decontamination effect of protein stains (including but not limited to blood stains or milk stains) is remarkable; in the leather industry, the above-mentioned alkaline protease can be used for unhairing and softening leather, decomposing non-collagen proteins in leather, and has the advantages of reducing pollution and improving leather quality compared with traditional sulfide unhairing; in the textile industry, the above-mentioned alkaline protease can be used for wool anti-felting treatment, silk degumming or removing protein impurities, and has the advantages of improving fabric hand and luster and improving fiber processing performance; in the food industry, the above-mentioned alkaline protease can be used for hydrolyzing animal and plant proteins to prepare products such as yeast extract, proteose peptone or amino acid; in the feed industry, the above-mentioned alkaline protease can be used for decomposing plant proteins such as soybean meal to improve the digestion and absorption rate of animals to proteins, thereby improving the feed conversion rate and reducing the breeding cost; in the field of alkaline wastewater treatment, the above-mentioned alkaline protease can be used for degrading organic nitrogen pollutants, for sewage treatment and bioremediation, and has the advantages of being green and environmentally friendly, and being suitable for alkaline wastewater environment.

[0084] The beneficial effects of the present application will be further explained in detail below in connection with specific embodiments.

[0085] Experimental materials and reagents:

[0086] 1. Strains and vectors:

[0087] The strain types used in the present application include Escherichia coli strain DH5a and Bacillus licheniformis E417 with preservation number CCTCC NO: M20232199, and the sequence of the expression vector PHYT is the nucleotide sequence shown in SEQ ID NO: 7.

[0088] 2. Enzymes and kits:

[0089] The homologous recombination enzyme was purchased from Nanjing Novozyme Company, ClonExpress Ultra One Step Cloning Kit, item number: C115-02; specification: 50rxns, the plasmid extraction and gel purification kit was purchased from Shanghai Sangon Company, and the restriction endonuclease was purchased from Takara Company.

[0090] 3. Culture medium:

[0091] LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0);

[0092] LB-Tc is LB medium with the addition of 15 μg / mL tetracycline;

[0093] Fermentation medium: corn starch (30 g / L), soybean meal (30 g / L), disodium hydrogen phosphate (2 g / L), sodium carbonate (1.25 g / L), pH 7.2-7.6.

[0094] Example 1

[0095] In the prokaryotic expression system, the gene copy number, promoter, signal peptide and protein folding efficiency have the most significant influence on gene expression. Since alkaline protease S8 is expressed by secretion in Bacillus licheniformis, the secretory signal peptide plays a decisive role in the protein secretion process. Therefore, the copy number, promoter, signal peptide and chaperone of alkaline protease S8 are optimized in this scheme to improve the fermentation expression level of alkaline protease S8 by Bacillus licheniformis E417. In the present application, the technical route diagram of constructing a recombinant strain with high expression of alkaline protease S8 is shown in Figure 1 The existing Bacillus licheniformis E417 strain expressing alkaline protease S8 is optimized in terms of gene copy number, promoter, signal peptide and chaperone to construct a recombinant strain with high fermentation expression level of alkaline protease S8.

[0096] In order to improve the gene copy number of alkaline protease S8 in Bacillus licheniformis E417, a plasmid vector for free expression of alkaline protease S8 was constructed, and the alkaline protease S8 gene expression frame (containing a promoter, a signal peptide and a terminator) was amplified by PCR using the genome of Bacillus licheniformis E417 as a template. P43-SigP was obtained by gene synthesis, and the two target fragments were cloned into pHYT by in vitro recombination method. The specific method includes: the amino acid fragment of P43-SigP shown in SEQ ID NO: 8 is amplified by using the primers F1 and R1 in Table 1, and the restriction enzymes BglII and SmaI are used to perform enzyme digestion reaction on the PHY300 vector at 37°C, and the double enzyme digestion of the vector is completed after 1 hour. The linearized vector is recovered by the product recovery kit. The two DNA fragments are assembled using an in vitro recombination kit, and then the cloned product is transformed into Escherichia coli DH5α, which is verified by colony PCR and sequencing to construct the plasmid vector pHYT-P43-SigP-S8. The structure of the plasmid vector is shown in Figure 2 The pHYT-P43-SigP-S8 is transformed into Bacillus licheniformis E417 to obtain the recombinant strain E417 (pHYT-P43-SigP-S8).

[0097] Table 1

[0098]

[0099] Example 2

[0100] On the basis of the plasmid vector for free expression of alkaline protease S8 obtained in Example 1, in order to screen the promoter for efficient expression of alkaline protease S8 in Bacillus licheniformis E417, on the one hand, the P43 promoter is replaced by a double-promoter expression system, including the stable phase expression promoter PYkzA, PGsiB and the constitutive expression promoter PserA; on the other hand, the original alkaline protease S8 promoter PaprE gene sequence in E417 is analyzed and compared, and the transcriptional negative regulator binding site sequence is obtained according to the gene sequence analysis. The sequence at this position is deleted to obtain the modified promoter sequence PaprE'.

[0101] The newly constructed recombinant vectors PHYT-PaprE-SigP-S8, PHYT-PaprE'-SigP-S8, PHYT-PYkzA-PserA-SigP-S8, and PHYT-PGsiB-PserA-SigP-S8 were respectively transformed into Bacillus licheniformis E417 strain. The correct transformants were verified by streaking on LB plates, and the colonies were scraped and inoculated into fermentation medium for 12 h of shake flask culture. Then, 1% of the culture was transferred to fresh fermentation medium, and the fermentation culture was sampled at intervals to determine the alkaline protease activity in the fermentation supernatant.

[0102] The results are shown in Table 3. Figure 3 As shown in Table 3, the experimental results show that the shake flask fermentation enzyme activity of Bacillus licheniformis E417 strain containing the recombinant vector PHYT-PaprE'-SigP-S8 (named E417-PaprE'-S8) is about 50% higher than that of the wild-type Bacillus licheniformis E417.

[0103] Example 3

[0104] In the present application, we further optimized the alkaline protease S8 secretion signal peptide in Bacillus licheniformis E417. Since alkaline protease S8 is expressed in Bacillus licheniformis, the signal peptide plays a key role. Based on the high-efficiency alkaline protease S8 recombinant vector (PHYT-PaprE'-SigP-S8) obtained in Example 2, we further analyzed the effect of alkaline protease S8 secretion signal peptide on the enzyme activity of fermentation broth. Through relevant literature research (The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions.), we selected six strong signal peptides in Bacillus licheniformis, including Bli00338, YbdN, SacC, YurI, Yoaw, or Bli02820. The nucleotide sequences of the above six signal peptides are shown in Table 2.

[0105] The above signal peptides were replaced with the signal peptide SigP in PHYT-PaprE'-SigP-S8 to construct six recombinant expression vectors: PHYT-PaprE'-Bli00338-S8, PHYT-PaprE'-YbdN-S8, PHYT-PaprE'-SacC-S8, PHYT-PaprE'-YurI-S8, PHYT-PaprE'-Yoaw-S8, or PHYT-PaprE'-Bli02820-S8.

[0106] The above recombinant vectors were respectively transformed into Bacillus licheniformis E417 strain, and the correct transformants were verified by streaking on LB+Tet plates. The colonies were scraped and inoculated into fermentation medium, and shaken for 12 hours. Then, 1% of the culture was transferred into fresh fermentation medium, and the fermentation culture was sampled at intervals to determine the alkaline protease activity in the fermentation supernatant.

[0107] The results are shown in Figure 4 As shown in the experimental results, after replacing the S8 signal peptide in the E417 strain with the above six new secretion signal peptides, SacC and YurI signal peptides slightly improved the shake flask fermentation enzyme activity of alkaline protease S8, but the fermentation enzyme activity of the strains transformed with the remaining secretion signal peptides was reduced to some extent. Among them, the SacC signal peptide improved the shake flask fermentation enzyme activity of alkaline protease S8 the most compared with the remaining signal peptides. Therefore, the recombinant expression vector (pHYT-PaprE'-SacC-S8) was selected for subsequent experiments.

[0108] Table 2

[0109]

[0110] Example 4

[0111] In the present application, we further studied the influence of overexpression of molecular chaperones on the expression of alkaline protease S8 in Bacillus licheniformis E417. Since alkaline protease S8 is expressed by secretion in Bacillus licheniformis E417, the signal peptide is cut during the transmembrane process of the polypeptide chain after protein translation, and the correct protein is folded with the help of molecular chaperones.

[0112] In order to analyze the influence of extracellular molecular chaperone PrsA on the expression of alkaline protease S8 in Bacillus licheniformis E417 strain, we cloned the complete PrsA expression frame (including the original promoter and terminator) from Bacillus licheniformis E417 strain, and cloned it into the pHYT-PaprE'-SacC-S8 recombinant plasmid in Example 3 to obtain a recombinant expression plasmid containing the PrsA expression frame (pHYT-PaprE'-SacC-S8-PrsA). The correct recombinant vector was transformed into Bacillus licheniformis E417 strain to obtain a recombinant strain. The recombinant strain was streaked on LB+Tet plates, and the colonies were scraped and inoculated into fermentation medium. The culture was shaken for 12 hours, and then 1% of the culture was transferred into fresh fermentation medium. The fermentation culture was sampled at intervals to determine the alkaline protease activity in the fermentation supernatant.

[0113] The experimental results are shown in Figure 5As shown, the experimental results show that the enzyme activity of the Bacillus licheniformis E417 strain containing the pHYT-PaprE'-SacC-S8-PrsA recombinant plasmid is increased by about 10% compared with the E417 strain containing the pHYT-PaprE'-SacC-S8 recombinant plasmid, indicating that the pHYT-PaprE'-SacC-S8-PrsA recombinant plasmid can further improve the enzyme activity of alkaline protease S8 after expression in the E4171 strain.

[0114] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects:

[0115] Through the research on the copy number of alkaline protease genes from Bacillus licheniformis and the influence of signal peptides and chaperones on the expression of alkaline protease, compared with other inventions and methods, the method provided by the present application starts from increasing the protein expression amount, combines the research on protein secretion and folding and transportation, and more systematically improves the expression efficiency of alkaline protease in Bacillus licheniformis. By increasing the copy number of alkaline protease S8 in the genome, combining signal peptide optimization and chaperone protein gene expression combination, a recombinant Bacillus licheniformis alkaline protease strain is constructed, and the expression amount of alkaline protease is increased by 10-50%.

[0116] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A DNA molecule for expressing an alkaline protease, characterized in that, The DNA molecule contains a promoter and a gene expressing an alkaline protease, the sequence of which includes the nucleotide sequence shown in SEQ ID NO:

2.

2. The DNA molecule according to claim 1, characterized in that, The DNA molecule also contains any one or more of the following: a gene expressing a signal peptide, or a gene expressing a chaperone protein.

3. The DNA molecule according to claim 2, characterized in that, In the DNA molecule, the promoter is located at the 5' direction of the alkaline protease gene.

4. A promoter, characterized in that, The promoter includes the nucleotide sequence shown in SEQ ID NO:

2.

5. A recombinant plasmid, characterized in that, The recombinant plasmid is linked to the DNA molecule of any one of claims 1-3 or the promoter of claim 4.

6. The recombinant plasmid according to claim 5, characterized in that, The recombinant plasmid includes one or more of pHYT or PHY300PLK.

7. A host cell, characterized in that, The host cell contains a DNA molecule as described in any one of claims 1-3, a promoter as described in claim 4, or a recombinant plasmid as described in claim 5 or 6.

8. The host cell according to claim 7, characterized in that, The host cells include Bacillus licheniformis or Escherichia coli.

9. The host cell according to claim 7 or 8, characterized in that, The host cell contains an alkaline protease gene, and the promoter of the alkaline protease gene is located at the 5' position. The alkaline protease gene and the promoter are located in the genome of the host cell and / or on a free plasmid that exists independently in the host cell.

10. The host cell according to claim 9, characterized in that, The copy number of the alkaline protease gene in the host cell is ≥2.

11. A method for preparing alkaline protease, characterized in that, The method comprises: culturing the host cell according to any one of claims 7-10, and collecting the alkaline protease.

12. The method according to claim 11, characterized in that, The culture includes culturing the host cells in a fermentation medium.

13. The method according to claim 12, characterized in that, The fermentation medium includes corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate.

14. The method according to claim 13, characterized in that, The fermentation medium, in total quantity, comprises 30-60 g / L corn starch, 30-60 g / L soybean meal, 1-3 g / L disodium hydrogen phosphate, and 1.0-1.5 g / L sodium carbonate; the pH of the fermentation medium is 7.2-7.

6.

15. The use of the DNA molecule of any one of claims 1-3, the promoter of claim 4, the recombinant plasmid of claim 5 or 6, the host cell of any one of claims 7-10, or the method for preparing alkaline protease of any one of claims 11-14 in the preparation of detergents, food, or medicine.

16. The application of the DNA molecule of any one of claims 1-3, the promoter of claim 4, the recombinant plasmid of claim 5 or 6, the host cell of any one of claims 7-10, or the method for preparing alkaline protease of any one of claims 11-14 in the detergent industry, leather industry, textile industry, food industry, feed industry, or alkaline wastewater treatment field.

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