High-activity calf abomasum chymosin suitable for dairy product processing, gene, strain and application of high-activity calf abomasum chymosin
By performing site-directed mutation of calf chermatis rennet, the temperature-sensitive rennet BtCHY-sup was designed, which solved the problem that existing rennet was difficult to terminate the procoagulant reaction in industrial cheese production, and achieved the improvement of the suitability and taste of cheese milk blocks.
Patent Information
- Application Number
- CN202411781209.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-04-25
AI Technical Summary
Existing rennet is difficult to effectively terminate the procoagulant reaction in industrial cheese production, resulting in the cheese milk block being too hard and may produce a bitter taste.
By performing site-directed mutation of calf chermatis rennet, a high-activity, temperature-sensitive rennet BtCHY-sup and its recombinant strain were designed to ensure that the enzyme remains highly active at lower temperatures and is rapidly inactivated at high temperatures.
The optimized rennet BtCHY-sup maintains high enzyme activity at 32°C and is completely inactivated after being left at 70°C for 30 minutes. It is suitable for termination of the procoagulant reaction and avoiding the hardness and bitterness of the cheese milk block.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-activity calf rennet suitable for dairy processing, a gene, a strain and an application thereof. Background Art
[0002] Calf rennet is an aspartic protease found in the stomach of unweaned calves. It can specifically cut the peptide bond between Phe105-Met106 of K-casein in milk, destroy casein micelles and coagulate milk. The coagulation ability and proteolytic ability of rennet make it a key enzyme for forming texture and special flavor in dairy processing, especially cheese production, and it is widely used in the production of cheese and yogurt. Common sources of rennet include animal, plant and microbial sources. Among them, animal-derived rennet is expensive and has low yield; plant-derived and microbial-derived rennet generally has strong proteolytic ability, weak specificity, and non-specific cutting of casein. The products produced have a strong bitter taste and poor taste, which is not the best way to produce rennet. Therefore, the production of calf rennet by recombinant strains is the current mainstream method.
[0003] The coagulation mechanism of rennet is as follows: milk usually coagulates in the stomach (under acidic conditions), which can be divided into two main stages: the first stage is called the enzymatic process, in which the rennet in the stomach cuts the peptide bond between Phe105-Met106 of K-casein under an acidic environment, releasing a large number of charged fragments and releasing α-casein and β-casein. The charged fragments aggregate and flocculate, making the original milk colloidal solution lose stability, and then enter the second stage in Ca 2+ Under the action of the protein, the proteins condense together to form clots.
[0004] Rennet is widely used. Application in dairy products: Ordinary fermented milk relies on weak non-covalent bonds and is kept at 4°C to maintain its micellar system, but due to various factors during transportation, the gel system is easily destroyed, the appearance changes, and the taste becomes worse. The appropriate addition of rennet to cow's milk can increase the degree of gelation and ensure the taste during transportation. Application in casein preparation: Casein is a phosphorus-containing protein polymer generated by milk in an acidic environment or under the action of proteases. It can be used in food production, textile building materials, leather craft manufacturing and other industries. One of the current mainstream preparation methods, "enzymatic preparation of casein", is to directly add rennet to skim milk to coagulate it, and then dehydrate and dry it to obtain the casein product. Casein prepared by rennet can be used as a protein additive in the food processing industry.
[0005] Temperature is an important factor affecting enzyme activity and is crucial in cheese production. In industry, considering the optimum temperature of lactic acid bacteria and the control of the coagulation process, the milk temperature is usually maintained at 30-33°C. At the same time, in order to prevent the milk curd from being too hard and avoid bitterness in industrial production, the temperature is quickly raised after the whey is discharged to inactivate the enzyme to terminate the pro-coagulation reaction. Therefore, the sensitivity of rennet to higher temperatures is crucial for the processing of dairy products. Currently, all rennet on the cheese market is heat-resistant, and heat-resistant rennet is more difficult to terminate the pro-coagulation reaction. The temperature-sensitive rennet provided by the present invention solves this problem to a certain extent. Compared with traditional rennet, new properties are accurately and efficiently given to the enzyme, thereby better meeting the needs of industrial production. Summary of the invention
[0006] The purpose of the present invention is to provide an optimized high-activity, temperature-sensitive chymosin BtCHY-sup and a recombinant strain, which are intended to be applied to the production of dairy products, especially cheese.
[0007] In order to achieve the above object, the first aspect of the present invention provides a highly active calf chymosin BtCHY-sup suitable for dairy processing, the amino acid sequence of which is shown in SEQ ID NO:1.
[0008] The second aspect of the present invention provides a calf abomasum chymosin gene btchy-sup, which is used to encode the above-mentioned chymosin, and the nucleotide sequence of the calf abomasum chymosin gene btchy-sup has more than 95% identity with the nucleotide sequence shown in SEQ ID NO: 2. Preferably, the nucleotide sequence of the calf abomasum chymosin gene btchy-sup is as shown in SEQID NO: 2.
[0009] The third aspect of the present invention provides a method for obtaining the above-mentioned chymosin, comprising the following steps: mutating the proline at position 84 of the original calf abomasum chymosin to alanine (P84A), and mutating the glutamine at position 85 to glutamic acid (Q85E) by site-directed mutagenesis; the amino acid sequence of the original calf abomasum chymosin is shown in SEQ ID NO: 3.
[0010] The fourth aspect of the present invention provides a constitutive expression vector, comprising the above-mentioned calf chymosin gene btchy-sup.
[0011] The constitutive expression vector of the present invention adopts a constitutive TDH3 promoter, which simplifies the process and reduces production costs in the later fermentation. The TDH3 promoter is obtained by extracting the yeast genome through a kit and using it as a template to amplify the TDH3 promoter fragment through PCR technology.
[0012] The fifth aspect of the present invention provides a method for constructing the above-mentioned constitutive expression vector, firstly preparing the vector back-extended fragment and the TDH3 promoter fragment respectively, and constructing the constitutive expression vector pPTDH3-btchy through the homologous sequence of the vector and the TDH3 promoter fragment by T5 enzyme connection, and the homologous recombination method used is well known in the art; then using pPTDH3-btchy as a template, introducing a mutation site through PCR specific primers, and then constructing the constitutive pPTDH3-btchy-sup with the mutation site through seamless cloning technology. Specifically comprising the following steps:
[0013] (1) The original chymosin pPIC-btchy plasmid was reversely amplified by PCR using pPTDH3-btchy-F and pPTDH3-btchy-R to obtain a vector reverse amplification fragment, and the desired TDH3 promoter fragment was amplified by primers TDH3-F and TDH3-R using the X-33 genome as a template; the vector reverse amplification fragment and the TDH3 promoter fragment were purified separately, and then connected by T5 enzyme to construct a constitutive expression vector pPTDH3-btchy; the nucleotide sequences of pPTDH3-btchy-F and pPTDH3-btchy-R are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the nucleotide sequences of TDH3-F and TDH3-R are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
[0014] (2) Using the constitutive expression vector pPTDH3-btchy containing the original chymosin as a template, PCR was performed using the specific primer CHY P84A-Q85E -F and CHY P84A-Q85E -R introduced the mutation site into the constitutive expression vector pPTDH3-btchy, and then constructed the constitutive expression vector pPTDH3-btchy-sup with the mutation site by seamless cloning technology; PCR specific primer CHY P84A-Q85E -F and CHY P84A-Q85E The nucleotide sequences of -R are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
[0015] The sixth aspect of the present invention provides a recombinant expression strain, comprising the above-mentioned constitutive expression vector; the host cell of the recombinant expression strain is yeast.
[0016] The seventh aspect of the present invention provides a method for preparing the above-mentioned recombinant expression strain, comprising the following steps:
[0017] The constitutive expression vector pPTDH3-btchy-sup was linearized by enzyme digestion; the linearized recombinant expression vector was electroporated into the host cell, incubated at 28° C. for 2 hours, and then coated on a YPD plate to screen positive clones to obtain a recombinant expression strain.
[0018] The eighth aspect of the present invention provides a method for producing the above-mentioned chymosin, comprising the following steps:
[0019] The recombinant expression strain is fermented, and rennet is extracted from the supernatant obtained by fermentation.
[0020] The chymosin BtCHY-sup of the present invention has the characteristic of high-temperature inactivation. After being placed in a 70°C environment for 30 minutes, the enzyme activity was measured every 30 minutes and found to be completely inactivated. This temperature-sensitive strain is suitable for application scenarios where the coagulation reaction needs to be terminated during cheese production.
[0021] The ninth aspect of the present invention provides the use of the rennet in dairy processing, especially in the production of cheese. Specifically, the use method comprises: contacting the rennet with a milk-containing material.
[0022] The present invention designs a new high-activity calf rennet BtCHY-sup, which is based on the original enzyme through the rational design of enzyme molecules. It can ensure high enzyme activity at 32°C and become extremely sensitive to temperature after the temperature rises. Specifically, the optimized calf rennet BtCHY-sup has an enzyme activity that is 79% higher than that of the unoptimized one. At high temperatures, the temperature tolerance of the optimized calf rennet BtCHY-sup decreases by more than 90%. In dairy processing, it will be quickly inactivated during post-treatment to prevent the cheese block from being too hard and avoid producing bitterness, and is more suitable for the production of dairy products, especially cheese. The temperature-sensitive strain constructed by the present invention can be applied to a variety of cheese-producing industries.
[0023] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.
[0025] Figure 1 This is a design diagram of the chymosin mutation site in Example 1 of the present invention.
[0026] Figure 2 A is a construction diagram of the constitutive expression vector pPTDH3-btchy; B is a construction diagram of the constitutive expression vector pPTDH3-btchy-sup.
[0027] Figure 3 This is the sequencing map of the mutant gene btchy-sup.
[0028] Figure 4 These are the SDS-PAGE test results of the fermentation supernatant of the constitutive expression strain pPTDH3-btchy and the mutant expression strain pPTDH3-btchy-sup constructed in the examples of the present invention after shake flask fermentation.
[0029] Figure 5 This is a bar graph of enzyme activity in the fermentation supernatant of the pPTDH3-btchy expression strain and the pPTDH3-btchy-sup expression strain obtained in the examples of the present invention after fermentation in a shake flask.
[0030] Figure 6 The enzymatic activity of the pPTDH3-btchy expression strain and the pPTDH3-btchy-sup expression strain obtained in the examples of the present invention at different temperature gradients is measured, as well as a graph showing the change in enzyme activity over time at 70° C. within 4 hours.
[0031] Figure 7 This is a diagram of the actual flocculation reaction between the fermentation supernatant (chymosin) of the pPTDH3-btchy-sup expression strain and the milk-containing material (serial number 1 represents the control group, and serial numbers 2, 3, and 4 are parallel experimental groups for flocculation reactions). DETAILED DESCRIPTION
[0032] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.
[0033] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.
[0034] The primer sequences used to construct the pPTDH3-btchy and pPTDH3-btchy-sup expression vectors in the examples are shown in Table 1.
[0035] Table 1
[0036]
[0037] Example 1
[0038] This example is used for the design and modification process of the chymosin BtCHY-sup of the present invention.
[0039] The chymosin BtCHY-sup provided by the present invention is obtained by performing site-directed mutagenesis on the original calf abomasum chymosin BtCHY through artificial rational design. The amino acid sequence of the original calf abomasum chymosin BtCHY is cited from NCBI GenBank: AAA30446.1, and the sequence is shown in SEQ ID NO: 3. The specific transformation process includes: mutating the 84th proline to alanine (P84A) by a site-directed mutagenesis method, introducing a hydrophilic amino acid at this position, which can enhance the hydrophilicity of the enzyme molecule and eliminate the flexibility of proline in the chymosin structure. Secondly, mutating the 85th glutamine to glutamic acid (Q85E), because the 85th glutamine has a larger side chain functional group, it is replaced by glutamic acid with a relatively small side chain group, which weakens the stability between enzyme molecules. While ensuring high enzyme activity, the heat resistance of the enzyme is also reduced.
[0040] Thus, the modified high-activity, temperature-sensitive chymosin BtCHY-sup is obtained, and its amino acid sequence is shown in SEQ ID NO: 1. According to the amino acid sequence of the chymosin BtCHY-sup, the nucleotide sequence of the chymosin gene is artificially redesigned according to the codon preference of yeast, and high-frequency codons are selected to reduce the complexity and minimum free energy of the secondary structure of the mRNA encoded by the gene, balance the content and distribution of GC in the gene, remove the repetitive sequence and cis-acting unit in the gene, and thus increase the expression of chymosin in the recombinant strain. Thus, the gene sequence btchy-sup corresponding to the high-activity, temperature-sensitive chymosin is designed, and its nucleotide sequence is shown in SEQ ID NO: 2.
[0041] After determining the mutation site, the present invention predicts and generates a three-dimensional structural model of the mutant chymosin with the help of SWISS-MODEL, such as Figure 1 The constitutive expression vector pPTDH3-btchy containing the original chymosin gene btchy was used as a template, the mutation site was introduced into the chymosin gene by PCR, and then a constitutive plasmid with the mutant gene was constructed by seamless cloning technology. The nucleotide sequence of the original calf abomasum chymosin gene btchy is shown in SEQ ID NO: 4.
[0042] Example 2
[0043] This example is used to illustrate the design and construction of the constitutive expression vector pPTDH3-btchy.
[0044] Based on the original thermostable chymosin expression vector pPIC-btchy, the required TDH3 promoter was added, such as Figure 2As shown in A, the upstream vector reverse amplification primer pPTDH3-btchy-F and the downstream vector reverse amplification primer pPTDH3-btchy-R were designed, and the promoter fragment primers TDH3-F and TDH3-R were designed upstream and downstream of the TDH3 promoter.
[0045] The original chymosin pPIC-btchy plasmid was reversely amplified by PCR using pPTDH3-btchy-F and pPTDH3-btchy-R to obtain a vector reverse amplification fragment, and the required TDH3 promoter fragment was amplified using primers TDH3-F and TDH3-R with the X-33 genome as a template. After the vector reverse amplification fragment was purified by Dpn I template elimination and the TDH3 promoter fragment was gel-recovered and purified, the homologous sequences of the vector and the fragment were connected by T5 enzyme and then transformed into E.coli DH5α and sequenced for verification, thereby constructing a recombinant expression vector pPTDH3-btchy with a constitutive promoter sequence.
[0046] The PCR reaction system and PCR reaction procedure in Example 2 are mentioned in Example 3 below and will not be described in detail here.
[0047] Example 3
[0048] This example is used to illustrate the construction of the mutant expression vector pPTDH3-btchy-sup.
[0049] The constitutive expression vector pPTDH3-btchy was used as a template to construct this mutant, and upstream and downstream reverse amplification primers CHY were designed at the 68th and 69th mutation sites. P84A-Q85E -F and CHY P84A-Q85E -R, such as Figure 2 As shown in B.
[0050] The mutation site was introduced into the constitutive expression vector pPTDH3-btchy by PCR, and then the recombinant plasmid pPTDH3-btchy-sup with the mutation site was constructed by seamless cloning technology. The specific method is as follows: the constitutive expression vector pPTDH3-btchy was diluted 20 times as a template, and the upstream and downstream primers CHY P84A-Q85E -F, CHY P84A-Q85E -R The constitutive expression vector pPTDH3-btchy was reversely amplified. The PCR amplification enzyme used in the present invention was 2×Vazyme LAmp Master Mix. The reaction system was the recombinant expression vector (diluted 20 times) pPIC-btchy as template 1 μL, 2×Vazyme LAmp Master Mix 25 μL, CHY N68A-Y69E -F 2 μL, CHYN68A-Y69E -R 2μL, ddH2O fills up to 50μL. The reaction program is 94℃5min, 94℃30s 55℃30s 72℃2min (the length of the fragment to be reverse-amplified is 5kb, 2×Vazyme LAmp Master Mix 30s / K), set 32 cycles, and extend thoroughly at 72℃ for 7min. After the PCR is completed, add 1μL of DpnI enzyme and 5μL of 10×T buffer to the reverse-amplification product and place it at 37℃ for 1h. The purpose is to eliminate the circular plasmid in the system after the PCR reaction is completed. Secondly, prepare 0.08% nucleic acid gel and use the gel recovery kit to recover the reverse-amplification fragment of the reverse-amplification product after template elimination. The purified fragment is connected with T5 enzyme according to the homologous sequence of the primer and then transformed into E.coli DH5α and sequenced for verification, such as Figure 3 As shown, the constitutive plasmid pPTDH3-Btchy with the mutation site was obtained.
[0051] Example 4
[0052] This example is used to illustrate the construction of the mutant expression strain pPTDH3-btchy-sup and the fermentation culture to produce chymosin.
[0053] The constitutive mutant expression vector pPTDH3-btchy-sup obtained in Example 3 was transformed into Pichia pastoris by electroporation. The electroporation method was as follows: (1) pPIC-btchy-sup was digested with restriction endonuclease Bgl II, and then precipitated with anhydrous ethanol at 37°C for two hours to convert it from a circular shape to a linear shape; (2) 10 μL of the linearized product of the mutant expression plasmid was mixed with 100 μL of fresh Pichia pastoris competent cells and placed in an ice bath for 5 min; (3) The electroporator was turned on and the electroporation parameters were adjusted to 2500 V and 200 Ω, and the mixture of the linearized product and competent cells in the ice bath was transferred to an electroporator precooled on ice. (4) After the electroporation transformation is completed, 1 mL of electroporation incubation solution (YPD: 1 M sorbitol = 1:1) preheated at 28°C is quickly added to the electroporation cup, and after pipetting and mixing, the mixture is transferred to a 1.5 mL sterile centrifuge tube and shaken at 28°C for 2 h; (5) 100 μL of the bacterial solution is spread on the YPD plate and cultured in a 28°C incubator for 48 h. After a single colony grows, the Pichia pastoris recombinant expression strain containing the chymosin gene btchy is obtained.
[0054] The original expression strain pPTDH3-btchy mentioned above was constructed in the same manner as in Example 4. No further details will be given here.
[0055] The obtained recombinant expression strain was inoculated into 5 mL of YPD medium containing bleomycin and cultured at 28°C with constant temperature shaking. When OD600 was about 3.0-6.0, it was inoculated into 25 mL of BMGY medium and continued to be cultured in a shake flask. 50% glycerol was added every 24 hours for fermentation and cultured for 96 hours. After the fermentation was completed, the protein in the fermentation broth was detected by SDS-PAGE, and its chymosin activity was detected.
[0056] The chymosin activity was determined by the Arima K method.
[0057] The fermentation supernatant (chymosin) was adjusted to pH = 2 with 1 mol / L H2SO4 and allowed to stand at room temperature for 2 h. The pH = 2 fermentation liquid was then adjusted to pH = 6 with 2 mol / L Tris for the following chymosin activity test.
[0058] Dissolve skim milk powder in 0.01 mol / L CaCl2 solution to prepare a 10% skim milk solution as a reaction substrate. Leave at room temperature for 40 minutes. Take 5 mL of skim milk solution in a test tube and place it in a constant temperature water bath at 35°C for 10 minutes. Then take 0.5 mL of the enzyme solution to be tested, which has been diluted to an appropriate multiple, and add it to the test tube containing 5 mL of skim milk solution. Shake it up immediately and start timing. The reaction endpoint is observed when flocculated particles begin to appear on the tube wall, and the coagulation time is recorded. Under the above reaction conditions, the coagulation of 1 mL of 10% skim milk powder in 40 minutes is defined as one Soxhlet unit.
[0059] Enzyme activity calculation formula: Enzyme activity (SU / mL) = test milk volume / rennet volume*D*2400 / T
[0060] Where D: dilution multiple T: coagulation time
[0061] Figure 4 Shown are the recombinant expression strain of the original chymosin gene btchy (left) and the recombinant expression strain of the modified chymosin gene btchy-sup (right) in this example, and the SDS-PAGE test results of the supernatant after fermentation. In the figure: M is a protein marker, and lanes 1-6 are different strains. Figure 5 The following is a bar graph showing the changes in enzyme activity in the fermentation supernatant of the original chymosin and the modified and optimized chymosin recombinant expression strain. It can be seen that the enzyme activity in the fermentation supernatant of the modified and optimized chymosin recombinant expression strain is significantly higher than that of the original chymosin.
[0062] Example 5
[0063] This example is used to illustrate the temperature characteristic analysis of mutant chymosin.
[0064] First, the rennet solution was adjusted to pH = 2 with 1 mol / L H2SO4 and placed at room temperature for 2 hours. Then, the pH was adjusted to pH = 6 with 2 mol / L Tris. After the treated rennet was kept at 70°C for 30 minutes, samples were taken every 0.5 hours to measure the enzyme activity and determine its temperature stability.
[0065] Figure 6 Shown are the enzyme activities of the original chymosin BtCHY and the optimized chymosin BtCHY-sup at different temperatures, as well as the change in enzyme activity over time measured by sampling every 0.5 h after being placed at 70°C for 30 min.
[0066] Depend on Figure 6 As can be seen from the left figure, the activity of the optimized chymosin BtCHY-sup in the embodiment of the present invention is higher than that of the original chymosin BtCHY, and the activity of the optimized calf abomasum chymosin BtCHY-sup is increased by 79% compared with the unoptimized enzyme. Figure 6 As can be seen from the right figure, the temperature sensitivity of the optimized chymosin BtCHY-sup in the embodiment of the present invention is significantly better than that of the original chymosin BtCHY, which is specifically manifested in that: after being placed at 70°C for 30 minutes, the enzyme activity of the original chymosin BtCHY still has high stability under high temperature conditions, while the enzyme activity of the optimized chymosin gene BtCHY-sup is reduced to 0 at each time period after high temperature treatment. It can be seen that under high temperature, the temperature tolerance of the optimized calf abomasum chymosin BtCHY-sup decreases by more than 90%.
[0067] Example 6
[0068] This example is used to illustrate the flocculation reaction of mutant chymosin with milk-containing materials.
[0069] The mutant fermentation supernatant was treated according to the method in Example 5 (the chymosin solution was first adjusted to pH = 2 with 1 mol / L H2SO4, left at room temperature for 2 h, and then adjusted to pH = 6 with 2 mol / L Tris), the treated chymosin solution was contacted with the milk-containing material, and rapidly shaken at 32°C. Figure 7 As shown, obvious floccules appeared on the tube wall of the parallel experimental groups (serial numbers 2, 3, and 4), which indicated the occurrence of flocculation reaction, while no floccules appeared in the control group (serial number 1, which differed from the experimental group only in that it did not contain chymosin), indicating that the mutant chymosin of the present invention had significant milk clotting ability.
[0070] In summary, the present invention obtains a highly active, temperature-sensitive chymosin BtCHY-sup and gene by artificially designing and modifying the original chymosin BtCHY, and produces chymosin for use in dairy production, thereby improving the defects of chymosin in industrial production, avoiding the production of bitter substances in the produced cheese, significantly reducing the cost of industrial cheese production while also ensuring the taste of the cheese.
[0071] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. A highly active calf rennet BtCHY-sup suitable for dairy processing, characterized in that: Its amino acid sequence is shown in SEQ ID NO:
1.
2. A calf abomasal chymosin gene btchy-sup, used to encode the chymosin according to claim 1, characterized in that: The nucleotide sequence of the calf abomasum chymosin gene btchy-sup has more than 95% identity with the nucleotide sequence shown in SEQ ID NO:
2. Preferably, the nucleotide sequence of the calf abomasum chymosin gene btchy-sup is as shown in SEQ ID NO:
2.
3. The method for obtaining rennet according to claim 1, characterized in that: The following steps are involved: The proline at position 84 of the original calf abomasum chymosin was mutated to alanine (P84A), and the glutamine at position 85 was mutated to glutamic acid (Q85E) by site-directed mutagenesis; the amino acid sequence of the original calf abomasum chymosin is shown in SEQ ID NO:
3.
4. A constitutive expression vector, characterized in that: It comprises the calf abomasal chymosin gene btchy-sup as claimed in claim 2.
5. The method for constructing the constitutive expression vector according to claim 4, characterized in that: The following steps are involved: (1) The original chymosin pPIC-btchy plasmid was reversely amplified by PCR using primers pPTDH3-btchy-F and pPTDH3-btchy-R to obtain a vector reverse amplification fragment, and the desired TDH3 promoter fragment was amplified using primers TDH3-F and TDH3-R with the X-33 genome as a template; the vector reverse amplification fragment and the TDH3 promoter fragment were purified respectively, and then connected by T5 enzyme to construct a constitutive expression vector pPTDH3-btchy; the nucleotide sequences of pPTDH3-btchy-F and pPTDH3-btchy-R are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the nucleotide sequences of TDH3-F and TDH3-R are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; (2) Using the constitutive expression vector pPTDH3-btchy containing the original chymosin as a template, PCR was performed using the specific primer CHY P84A-Q85E -F and CHY P84A-Q85E -R introduced the mutation site into the constitutive expression vector pPTDH3-btchy, and then constructed the constitutive expression vector pPTDH3-btchy-sup with the mutation site by seamless cloning technology; PCR specific primer CHY P84A-Q85E -F and CHY P84A-Q85E The nucleotide sequences of -R are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
6. A recombinant expression strain, characterized in that: Comprising the constitutive expression vector as claimed in claim 4; the host cell of the recombinant expression strain is yeast.
7. The method for preparing the recombinant expression strain according to claim 6, characterized in that: The steps include: Linearizing the constitutive expression vector according to claim 4 by enzyme digestion; The linearized recombinant expression vector is electroporated into the host cell to obtain a recombinant expression strain.
8. The method for producing chymosin according to claim 1, characterized in that: The following steps are involved: The recombinant expression strain according to claim 6 is fermented, and rennet is extracted from the supernatant obtained by fermentation.
9. Use of the rennet according to claim 1 in dairy processing, especially in the production of cheese.
10. The use according to claim 9, characterized in that: The application method comprises: contacting the rennet according to claim 1 with a milk-containing material.