Preparation method and application of milk enzymatic protein polypeptide

Abstract

This invention discloses a method for preparing and applying enzymatically hydrolyzed protein peptides from bovine milk. The preparation method involves using skim milk as raw material and preparing the peptide through a mixture of alkaline protease mutant and trypsin. The alkaline protease mutant with high specific activity and high catalytic efficiency is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 by R121K, S250A, G192S / E221R, or A182E / P238D. The method of preparing the peptide by using the above-mentioned alkaline protease mutant and trypsin through mixed hydrolysis can simultaneously improve the degree of hydrolysis of bovine milk and reduce the allergenicity of bovine milk.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering, and in particular to a preparation method and application of milk enzymatic protein polypeptide. Background Art

[0002] In the food industry, the processing and modification of cow's milk is one of the important means to improve the functionality and nutritional value of dairy products. Alkaline protease, as an important proteolytic enzyme, is widely used in the enzymatic hydrolysis of dairy products. Alkaline protease can hydrolyze proteins in cow's milk to produce a variety of bioactive peptides, thereby improving the functional properties of dairy products, such as antioxidant, antihypertensive and immunomodulatory effects. However, the efficiency and specificity of traditional alkaline protease in the enzymatic hydrolysis of cow's milk still need to be improved, which limits its application in the deep processing of dairy products.

[0003] At present, studies have shown that by modifying the molecular structure of alkaline protease, its enzymatic activity and substrate specificity can be significantly improved. Especially in the hydrolysis of milk protein, the activity and selectivity of alkaline protease are crucial for the generation of specific functional peptides. However, the application effect of existing alkaline protease mutants in milk hydrolysis has not been fully optimized, and there are still problems such as low enzymatic efficiency and complex peptide mixtures generated, which affect the quality and functionality of dairy products.

[0004] In order to solve the above problems, it is urgent to develop new alkaline protease mutants to improve their performance in the enzymatic hydrolysis of milk. These mutants should have higher catalytic efficiency and better substrate specificity, and be able to accurately and fully hydrolyze milk proteins to generate more functional peptides, reduce immunogenicity, and improve the nutritional value and market competitiveness of dairy products. Summary of the invention

[0005] The invention uses improved alkaline protease and trypsin to enzymatically hydrolyze cow's milk to prepare polypeptides, aiming to improve the hydrolysis degree of cow's milk and reduce the allergenicity of cow's milk.

[0006] The first object of the present invention is to provide a method for preparing a milk enzymatic protein polypeptide, wherein skimmed milk is used as a raw material and the polypeptide is prepared by mixed enzymatic hydrolysis with an alkaline protease mutant and trypsin, wherein the alkaline protease mutant is prepared by subjecting the amino acid sequence as shown in SEQ ID NO.1 to a single mutation of R121K, a single mutation of S250A, a double mutation of G192S / E221R or a double mutation of A182E / P238D.

[0007] The second object of the present invention is to provide an alkaline protease mutant, wherein the mutant is subjected to R121K single mutation, S250A single mutation, G192S / E221R double mutation or A182E / P238D double mutation in the amino acid sequence as shown in SEQ ID NO.1.

[0008] The third object of the present invention is to use the alkaline protease mutant in the enzymatic hydrolysis of milk to prepare polypeptides.

[0009] The fourth object of the present invention is to use the alkaline protease mutant in improving the hydrolysis degree of milk and reducing the allergenicity of milk.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects:

[0011] 1. The invention is based on Shouchella clausii The gene sequence of alkaline protease GenBank accession number WP_325956049.1 (SEQ ID NO: 1) was subjected to R121K single mutation, S250A single mutation, G192S / E221R double mutation or A182E / P238D double mutation, and mutants that can significantly improve the specific activity and catalytic efficiency of the enzyme were successfully screened;

[0012] 2. The invention is based on Shouchella clausii The single mutant and double mutant of alkaline protease are combined with trypsin to enzymatically hydrolyze milk to prepare polypeptides. The preparation method can significantly improve the hydrolysis degree of milk and reduce the allergenicity of milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Analysis of the degree of hydrolysis of defatted milk products using different alkaline protease mutants and trypsin;

[0014] Figure 2 Analysis of IgG inhibition rate of defatted bovine milk products hydrolyzed by different alkaline protease mutants and trypsin. DETAILED DESCRIPTION

[0015] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1 Preparation of alkaline protease SCAP and its mutants

[0017] Alkaline protease wild-type pET-28a(+) -SCAP-wt expression vector construction:

[0018] According to NCBI Shouchella clausiiThe gene sequence of alkaline protease of GenBank accession number WP_325956049.1 was optimized by codon optimization of the nucleotide sequence encoding the mature peptide of alkaline protease using the online software Codon Adaptation Tool (http: / / www.jcat.de / ), and then commissioned Shanghai Sangon Biotechnology Co., Ltd. to chemically synthesize the obtained pET-28a(+)-SCAP expression vector with HIS tag to construct.

[0019] Amino acid sequence of SCAP: MKKPLGKIVASTALLISVAFSSSIASAAEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNN SIGVLGVAPNAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVGASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO:1);

[0020] Construction of expression vectors of alkaline protease mutants pET-28a(+)-SCAP-R121K, pET-28a(+)-SCAP-S250A, pET-28a(+)-SCAP-G192S / E221R, and pET-28a(+)-SCAP-A182E / P238D:

[0021] The alkaline protease SCAP mutant was constructed by site-directed mutagenesis technology. The primer pairs were designed according to the Fast Mutagenesis System of Beijing Quanshijin Biotechnology Co., Ltd. (Table 1). The constructed recombinant plasmid pET-28a(+)-SCAP was used as a template to introduce single mutation (single primer method), double mutation (single primer method for two-step site-directed mutagenesis) and triple mutation (single primer method for three-step site-directed mutagenesis) for PCR amplification. The amplification reaction system was: 2×PCR Super Mix 25μL, primer F (10μM) 1μL, primer R (10μM) 1μL, template (5ng) 1μL, and water was added to 30μL. Amplification reaction conditions: initial denaturation: 98°C, 30 seconds; denaturation: 98°C, 10 seconds; annealing: 58°C, 30 seconds; extension: 72°C, 1 kb / min; number of cycles: 25-30 times; final extension: 72°C, 5 minutes; after completion, store at 4°C; take 10μL PCR product, detect by 1% agarose gel electrophoresis, directly sequence the target bands for identification, and mark them as pET-28a(+)-SCAP-R121K, pET-28a(+)-SCAP-S250A, pET-28a(+)-SCAP-G192S / E221R, and pET-28a(+)-SCAP-A182E / P238D, respectively.

[0022] Table 1 Mutation primer sequences

[0023]

[0024] Transfection experiment of expression vectors of alkaline protease and its mutants: Place 50 µL of competent cells BL21(DE3) on ice, add 1-5 µL (about 50-100 ng) of pET-28a(+)-SCAP-wt, pET-28a(+)-SCAP-R121K, pET-28a(+)-SCAP-S250A, pET-28a(+)-SCAP-G192S / E221R, pET-28a(+)-SCAP-A182E / P238D plasmids to the competent cells respectively; let stand on ice for 30 minutes; perform heat shock: heat at 42°C for 45 seconds, then immediately place on ice for 2 minutes; add 450 µL of LB medium without antibiotics to the tube; shake and culture at 37°C, 180 rpm for 1 hour; spread on a plate containing 50 1 μg / mL kanamycin on LB plates and culture at 37°C for 12-16 hours;

[0025] Strain activation, induced expression and purification experiments of alkaline protease and its mutants: positive clones were picked and inoculated into 5 mL LB medium containing 50 µg / mL kanamycin, and cultured overnight at 37°C and 180 rpm; the overnight cultured bacteria were inoculated into 500 mL LB medium (containing 50 µg / mL kanamycin), and cultured at 37°C and 180 rpm with shaking until OD600 reached 0.6-0.8 (about 2-3 hours); isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM; induction conditions: 16°C, 180 rpm shaking culture for 16-20 hours; after the induction culture ended, the cells were centrifuged at 4°C and 6000 rpm for 15 minutes to collect the cell pellet; the cells were resuspended in 10 mL lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH8.0); ultrasonic lysate was used in an ice bath for ultrasonic disruption, with ultrasonic parameters as follows: ultrasonic 5 seconds, 5 seconds interval, and a total duration of 5 minutes; after ultrasonication, centrifuged at 4°C, 12000rpm for 30 minutes, and the supernatant was collected to remove cell debris; the supernatant was passed through a Ni-NTA affinity chromatography column, and the flow-through was collected; the column was washed with an equilibrium buffer (50mM Tris-HCl, 300mM NaCl, 10mM Imidazole, pH8.0) to remove non-specific binding proteins, and the volume of the washing solution was about 20-30mL; the column was washed with an elution buffer (50mMTris-HCl, 300mM NaCl, 250mM Imidazole, pH 8.0) was used to elute the His-tagged SCAP protein, and the fractionated eluates containing the target protein were collected. Finally, the prepared alkaline protease and its mutants were labeled as: SCAP-wt, SCAP-R121K, SCAP-S250A, SCAP-G192S / E221R, and SCAP-A182E / P238D, respectively.

[0026] Example 2 Study on the activity of alkaline protease SCAP and its mutants

[0027] Determination of enzyme activity and specific activity of alkaline protease SCAP and its mutants: Casein is a widely used natural protein substrate for determining the activity of proteases; alkaline protease can hydrolyze casein to generate small molecular peptides. Unhydrolyzed casein will precipitate under acidic conditions, while the hydrolyzed peptides remain in the solution; the small molecular peptides produced by hydrolysis can be quantitatively determined by the Folin-phenol reagent method or ultraviolet spectrophotometry, thereby calculating the enzyme activity and specific activity.

[0028] Weigh an appropriate amount of casein powder to prepare a 1% (w / v) casein solution, and dissolve it in 0.05M phosphate buffer (pH 8.0); heat it in a water bath to 60°C and stir gently to ensure that the casein is completely dissolved; add the following components to the reaction tube: 1mL 1% casein solution; 0.5mL 0.6mg / mL alkaline protease SCAP-wt, SCAP-R121K, SCAP-S250A, SCAP-G192S / E221R, SCAP-A182E / P238D; 0.5mL 0.05M phosphate buffer (pH 8.0); the total reaction system is 2mL, and incubate in a 55°C water bath for 10 minutes to allow the enzyme reaction to proceed.

[0029] After the reaction is completed, immediately add 2 mL of 10% (w / v) trichloroacetic acid (TCA) to terminate the reaction; TCA can precipitate unhydrolyzed casein; after terminating the reaction, centrifuge the mixture at 4°C for 10 minutes (10,000 rpm), or filter through filter paper to remove undissolved precipitates and collect the supernatant; take 1 mL of the supernatant, measure its absorbance at 280 nm, and record the amount of small molecule peptides produced by alkaline protease hydrolysis.

[0030] The enzyme activity unit is defined as the amount of enzyme required to hydrolyze casein to release 1 micromole of tyrosine equivalents in 1 minute and is calculated using the following formula:

[0031]

[0032] Specific activity is defined as the enzyme activity per milligram of protein, indicating the purity of the enzyme, and is calculated using the following formula:

[0033]

[0034] The protein concentration can be determined by Bradford method or BCA method, and the results are shown in Table 2.

[0035] Table 2 Specific activity and kinetic parameters of alkaline protease SCAP and its mutants

[0036]

[0037] The results in Table 2 show that the specific activity of SCAP-R121K is 1.56 times that of SCAP-wt, the specific activity of SCAP-S250A is 2.58 times that of SCAP-wt, the specific activity of SCAP-G192S / E221R is 1.09 times that of SCAP-wt, and the specific activity of SCAP-A182E / P238D is 2.18 times that of SCAP-wt.

[0038] The catalytic efficiency (Kcat / Km) of SCAP-R121K increased by 1.58 times, the catalytic efficiency (Kcat / Km) of SCAP-S250A increased by 2.89 times, the catalytic efficiency (Kcat / Km) of SCAP-G192S / E221R increased by 1.32 times, and the catalytic efficiency (Kcat / Km) of SCAP-A182E / P238D increased by 4.67 times.

[0039] Example 3 Method for preparing polypeptides by hydrolyzing defatted milk using different alkaline protease mutants and trypsin

[0040] SCAP-wt group: 500 mL skim milk was placed in a 1000 mL container, the milk was preheated to 50°C, kept for 10 minutes, sterilized and homogenized, 100 mg each of alkaline protease SCAP-wt and trypsin (Sigma-Aldrich, T1426) were weighed and dissolved in 50 mL of 0.05 M phosphate buffer, respectively, and the pH was adjusted to 8.0. After the preparation was completed, the two enzyme solutions were mixed in a 1:1 ratio, and the final enzyme concentration was 1000 U / mL; 500 mL of pretreated milk was heated to 55°C, and 2 mL of the mixed enzyme solution was slowly added to the milk. The hydrolysis reaction was carried out at 55°C, the pH was adjusted to 8.0, and the pH was kept stable by adding 1 M NaOH solution. The hydrolysis time was 2 hours, and samples were taken for subsequent experimental analysis.

[0041] SCAP-R121K group: The operation method was the same as that of the SCAP-wt group, except that the mixed enzyme was alkaline protease SCAP-R121K and trypsin (Sigma-Aldrich, T1426).

[0042] SCAP-S250A group: The operation method was the same as that of the SCAP-wt group, except that the mixed enzyme was alkaline protease SCAP-S250A and trypsin (Sigma-Aldrich, T1426).

[0043] SCAP-G192S / E221R group: The operation method was the same as that of the SCAP-wt group, except that the mixed enzyme was alkaline protease SCAP-G192S / E221R and trypsin (Sigma-Aldrich, T1426).

[0044] SCAP-A182E / P238D group: The operation method was the same as that of the SCAP-wt group, except that the mixed enzyme was alkaline protease SCAP-A182E / P238D and trypsin (Sigma-Aldrich, T1426).

[0045] TNBS (2,4,6-trinitrobenzenesulfonic acid) method to detect the degree of hydrolysis of skim milk enzymatic hydrolysate:

[0046] 1) Sample treatment of enzymatic hydrolysate: Take 0.5 mL of the enzymatic hydrolysate solution (protein concentration is about 1% w / v) of the above SCAP-wt group, SCAP-R121K group, SCAP-S250A group, SCAP-G192S / E221R group and SCAP-A182E / P238D group, place it in a 1.5 mL centrifuge tube, add 0.5 mL of 0.1 M NaHCO3 solution (pH 8.0), add 0.5 mL of 0.1% TNBS solution, mix well, incubate in a 37°C water bath for 60 min, after the reaction is completed, add 1 mL of 1 M HCl to terminate the reaction, centrifuge to remove the precipitate, take the supernatant for testing, and measure the absorbance at 340 nm.

[0047] 2) Preparation of completely hydrolyzed samples: Take 0.5 mL of unhydrolyzed skim milk sample, add 6 M HCl and hydrolyze at 105°C for 24 hours to obtain a completely hydrolyzed product. After the hydrolysis is completed, the sample is concentrated to dryness and redissolved with distilled water to the same volume as the enzymatic sample 0.5 mL. Follow the same steps as 1) for the enzymatic hydrolyzed product: add 0.5 mL of 0.1 M NaHCO3 solution (pH 8.0), add 0.5 mL of 0.1% TNBS solution, mix well, and incubate in a water bath at 37°C for 60 minutes. After the reaction is completed, add 1 mL of 1 M HCl to terminate the reaction, centrifuge to remove the precipitate, take the supernatant for testing, and measure the absorbance at 340 nm as the number of free amino groups (total number of peptide bonds) of the completely hydrolyzed sample.

[0048] 3) Preparation of standard curve: Use L-glutamic acid standard solution of known concentration to prepare standard curve. Dissolve L-glutamic acid into different concentration gradients (0.1, 0.2, 0.3, 0.4, 0.5 mM) in 0.1 M NaHCO3 solution. React each concentration with 0.1% TNBS solution and measure according to the steps in 1). Measure the absorbance of each standard sample at 340 nm and draw a standard curve of absorbance and free amino concentration.

[0049] 4) The degree of hydrolysis (DH) is calculated by the following formula. The results are shown in Figure 1 :

[0050]

[0051] Figure 1The results showed that compared with the SCAP-wt group, the SCAP-R121K group, SCAP-S250A group, SCAP-G192S / E221R group and SCAP-A182E / P238D group could significantly improve the hydrolysis degree of skim milk, among which the hydrolysis degree of the SCAP-G192S / E221R group was better than that of the SCAP-R121K group, SCAP-S250A group and SCAP-A182E / P238D group.

[0052] The IgG inhibition rate of the enzymatic hydrolysate was determined by indirect competitive enzyme linked immunosorbent assay (ELISA) in vitro: 1) Coating: The enzymatic hydrolysate solutions of the above SCAP-wt group, SCAP-R121K group, SCAP-S250A group, SCAP-G192S / E221R group and SCAP-A182E / P238D group were diluted to 10 μg / mL in carbonate buffer (0.05 mol / L, pH 9.6) and 100 μg / mL was added to each well of a 96-well ELISA plate. 1) Blocking: Pour off the coating solution, wash three times with phosphate buffered saline (PBST) containing 0.05% Tween-20 by volume, 5 min each time, block each well with 250 μL of 3% gelatin dissolved in PBS (0.01 mol / L, pH 7.4) at 37°C for 1 h, wash with PBST, and dry three times; 2) Add anti-skim milk allergen serum: Wash the ELISA plate three times, add 100 μL of PBS to each well. Rabbit antiserum and negative serum diluted 1:5000, incubated at 37℃ for 1h, washed with PBST, and tapped dry 3 times; 4) Add enzyme-labeled secondary antibody: wash the ELISA plate 3 times, incubate goat anti-rabbit IgG antibody labeled with horseradish peroxidase diluted with PBS 1:5000 at 37℃ for 1h, washed with PBST, and tapped dry 3 times; 5) Color development: wash 3 times with PBST, add 100μLOPD solution to each well, incubate at 37℃ in the dark for 15min. 6) Stop the reaction: add 50μL 2mol / LH2SO4 to each well to stop the reaction; 7) Use an ELISA reader to measure the absorbance of the sample at a wavelength of 490nm; the IgG inhibition rate is calculated using the following formula, and the results are shown in Figure 2 :

[0053]

[0054] Wherein: B is the absorbance value of each group of enzymatic hydrolyzate sample wells; B0 is the absorbance value of negative serum.

[0055] Figure 2The results showed that compared with the SCAP-wt group, the SCAP-R121K group, SCAP-S250A group, SCAP-G192S / E221R group and SCAP-A182E / P238D group significantly increased the IgG inhibition rate of the enzymatic hydrolysate of skim milk, among which the IgG inhibition rate of the SCAP-A182E / P238D group was the highest. Analysis showed that the SCAP-R121K, SCAP-S250A, SCAP-G192S / E221R, and SCAP-A182E / P238D mutants could cause greater damage to the IgG binding sites in the skim milk allergenic protein, thereby making skim milk less allergenic.

[0056] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing milk enzymatic protein polypeptide, characterized in that: The method uses skimmed milk as a raw material and prepares the alkaline protease mutant by mixed enzymatic hydrolysis with trypsin. The alkaline protease mutant is prepared by subjecting the amino acid sequence shown in SEQ ID NO.1 to double mutation G192S / E221R.

2. The preparation method according to claim 1, characterized in that The ratio of alkaline protease mutant to trypsin was 1:

1.

3. An alkaline protease mutant, characterized in that: The mutant is a double mutation of G192S / E221R in the amino acid sequence as shown in SEQ ID NO.

1.

4. Use of the alkaline protease mutant according to claim 3 in preparing polypeptides by enzymatic hydrolysis of milk.

5. Use of the alkaline protease mutant according to claim 3 in improving the hydrolysis degree of milk and reducing the allergenicity of milk.

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