Protease K mutant with high catalytic activity and high thermal stability

Abstract

The invention discloses a protease K mutant with high catalytic activity and high thermal stability. The amino acid sequence of the protease K mutant is as shown in SEQ ID NO.1; the protease K mutant is used for hydrolyzing keratin, and oligopeptide or amino acid generated after hydrolysis can be used as a feed additive; after the protease K mutant is treated at 75 DEG C for 30 minutes, the residual enzyme activity is 55%; the specific activity of the protease K mutant is 324U / mg; reasonable design and molecular modification are carried out on protease K from Tritirachium album, site-directed mutagenesis and experimental verification are combined, a protease K mutant with improved thermal stability is obtained through screening, a G226F mutant enzyme is incubated for 30 min at 75 DEG C, the residual enzyme activity is 55%, and the residue of a wild type is 20% under the same treatment condition; in addition, the specific activity of the G226F mutant enzyme is 2.3 times that of a wild type, so that the problem of poor thermal stability of protease K is solved, and the catalytic activity is also improved.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and enzyme engineering, and in particular relates to a proteinase K mutant with high catalytic activity and high thermal stability. Background Art

[0002] Proteinase K (ProK) is an alkaline serine protease originally secreted by Tritirachium album Limber. It was named Proteinase K due to its ability to hydrolyze native keratin. Proteinase K exhibits broad substrate specificity, with particular efficiency at the carboxyl-terminal peptide bonds of aliphatic and aromatic amino acids. Its stability makes it particularly effective in a variety of applications, particularly in molecular biology research (such as DNA / RNA extraction) and industry (such as protein degradation and short peptide production). In recent years, the application of Proteinase K has expanded beyond medical diagnostics and scientific research, and its application in industry has also been growing. This is particularly true in production processes involving high-temperature processes, which place higher demands on its thermal stability and catalytic activity. Despite extensive research in recent years on the molecular engineering and efficient expression of Proteinase K, currently commercialized Proteinase K still suffers from limited thermal stability and catalytic activity, severely limiting its industrial application. Summary of the Invention

[0003] The purpose of the present invention is to provide a proteinase K mutant with high catalytic activity and high thermal stability, so as to solve the problems of low thermal stability and catalytic activity of commercial proteinase K.

[0004] The present invention adopts the following technical solution: a proteinase K mutant with high catalytic activity and high thermal stability, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] Furthermore, its nucleotide sequence is shown in SEQ ID NO.2.

[0006] Furthermore, the proteinase K mutant is obtained by mutating the wild type, the amino acid sequence of the wild type is shown in SEQ ID NO.3, and the mutation site is the glycine at position 226 of the wild type, which is mutated to phenylalanine.

[0007] Furthermore, the wild-type nucleotide sequence is shown in SEQ ID NO.4.

[0008] Furthermore, the proteinase K mutant is used to hydrolyze keratin, and the short peptides or amino acids produced after hydrolysis can be used as feed additives.

[0009] Furthermore, the residual enzyme activity of the proteinase K mutant after treatment at 75° C. for 30 minutes was 55%; and the specific activity of the proteinase K mutant was 324 U / mg.

[0010] The beneficial effects of the present invention are:

[0011] The present invention screens and obtains a proteinase K mutant with improved thermal stability by rationally designing and molecularly modifying proteinase K derived from Candida albicans, combining site-directed mutagenesis and experimental verification. The G226F mutant has a residual enzyme activity of 55% when incubated at 75°C for 30 minutes, while the wild type has a residual activity of 20% under the same treatment conditions. Furthermore, the specific activity of the G226F mutant is 2.3 times that of the wild type, which not only solves the problem of poor thermal stability of proteinase K but also improves its catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure is the SDS-PAGE electrophoresis of wild-type proteinase K recombinantly expressed in Pichia pastoris in the examples (M: Maker; WT: wild-type protein);

[0013] Figure 2 This is the minimum free energy heat map after FoldX predicts the virtual saturation mutation of amino acid residues in the embodiment;

[0014] Figure 3 The minimum free energy heat map after Rosetta predicts virtual saturation mutation of amino acid residues in the embodiment;

[0015] Figure 4 The residual enzyme activity of wild-type proteinase K in the examples after incubation with various mutant enzymes at 75°C for 30 min (WT: wild-type protein; T166I: mutant protein in which threonine at position 166 is mutated to isoleucine; G192Y: mutant protein in which glycine at position 192 is mutated to tyrosine; Q193D: mutant protein in which glutamine at position 193 is mutated to asparagine; Q193E: mutant protein in which glutamine at position 193 is mutated to glutamate; G200A: mutant protein in which glycine at position 200 is mutated to alanine; G226F: mutant protein in which glycine at position 226 is mutated to phenylalanine; T273I: mutant protein in which threonine at position 273 is mutated to isoleucine; T273M: mutant protein in which threonine at position 273 is mutated to methionine);

[0016] Figure 5 This is the SDS-PAGE electrophoresis diagram of the proteinase K mutant G226F protein in the examples (M: Maker; G226F: mutant protein in which glycine at position 226 is mutated to phenylalanine);

[0017] Figure 6This is a graph comparing the thermal stability of the proteinase K mutant G226F and the wild-type enzyme in the examples;

[0018] Figure 7 Graph comparing the pH tolerance of the proteinase K mutant G226F and the wild-type enzyme in the examples. DETAILED DESCRIPTION

[0019] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] The present invention discloses a proteinase K mutant with high catalytic activity and high thermal stability, the amino acid sequence of which is shown in SEQ ID NO.1.

[0021] APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVK VLDDNGSGQYSTIIAGMDFVASDHNRNRCPKGVVASLSLGGFYSSSVNSAAARLQSSGVMVAVAAGNNADARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGSTSSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGVLSNIPFGTVNLLAYNNYQA

[0022] The nucleotide sequence of the proteinase K mutant is shown in SEQ ID NO.2, which is:

[0023]

[0024] The proteinase K mutant is obtained by mutating the wild type. The amino acid sequence of the wild type is shown in SEQ ID NO. 3. The mutation site is the glycine at position 226 of the wild type, which is mutated to phenylalanine.

[0025] SEQ ID NO.3 sequence is:

[0026] APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVK VLDDNGSGQYSTIIAGMDFVASDHNRNRCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNADARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGSTSSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGVLSNIPFGTVNLLAYNNYQA

[0027] The wild-type nucleotide sequence is shown in SEQ ID NO.4, which is:

[0028]

[0029] The invention also discloses that a proteinase K mutant is used for hydrolyzing keratin, and the short peptides or amino acids produced after the hydrolysis can be used as feed additives.

[0030] Example 1

[0031] The required reagents and materials are as follows:

[0032] Escherichia coli Top10 and expression plasmid pGAPZαA were both preserved in our laboratory.

[0033] Low-salt B culture medium formula: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L.

[0034] YPD medium formula: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0035] BSM medium formula: 85% H₃PO₄ 26.7ml / L, CaSO₄·2H₂O 0.93g / L, K₂SO₄ 18.2g / L, MgSO₄·2H₂O 14.9g / L, KOH 4.13g / L, PMT1 4.0ml / L, glycerol 40g / L (or alternative carbon source). Dissolve in water and stir until completely dissolved. Adjust pH to 5.0 with aqueous ammonia. Autoclave BSM and add 4.0ml / L of filter-sterilized PMT1.

[0036] The steps for proteinase K expression and purification are as follows:

[0037] The amino acid sequence of wild-type proteinase K is shown in SEQ ID NO. 3. The proteinase K gene sequence was artificially synthesized based on the codon preference of Pichia pastoris (nucleotide sequence shown in SEQ ID NO. 4). The proteinase K gene was ligated into the expression vector pGAPZαA via the EcoR I and Not I restriction sites to construct the recombinant plasmid pGAPZαA-ProK, which was then transformed into Escherichia coli Top10.

[0038] The top 10 Escherichia coli monoclonal strain containing the recombinant plasmid pGAPZαA-ProK was inoculated into low-salt LB medium containing 25 μg / mL bleomycin and cultured overnight at 37°C for 12-16 hours. The plasmid was extracted and the concentration of the plasmid DNA was determined. Subsequently, the endonuclease Avr II was added and digested at 37°C for 3 hours. The completeness of the plasmid was verified by nucleic acid electrophoresis.

[0039] The plasmid was digested and purified to obtain a high-concentration linearized plasmid. The specific purification method was as follows: the digested plasmid was added to 1 mL of phenol-chloroform and mixed thoroughly. The mixture was centrifuged at 10,000 rpm for 3 minutes. The supernatant was carefully aspirated and transferred to a clean 1.5 mL centrifuge tube. 1 mL of anhydrous ethanol was added and the tube was centrifuged at 10,000 rpm for 3 minutes. The supernatant was discarded. 1 mL of 70% ethanol was added and the tube was centrifuged at 10,000 rpm for 3 minutes. The supernatant was discarded. This step was repeated three times. The residual ethanol was evaporated and the tube was resuspended in 10 μL of ultrapure water. The DNA concentration was determined to obtain a purified and high-concentration linearized plasmid.

[0040] The linearized plasmid was electroporated into Pichia pastoris competent cells, and the cells were spread on YPD solid plates containing 100 μg / mL bleomycin and cultured at 30° C. for 48-72 h.

[0041] Use a sterilized toothpick tip or pipette tip to pick up clones and gently tap them onto a casein plate. Place the plate in a 30°C incubator and incubate for approximately 12 to 36 hours. The presence of a hydrolysis zone indicates that proteinase K is secreted and active. The absence of a hydrolysis zone indicates that proteinase K is not secreted and inactive.

[0042] The single clone that formed a transparent circle was inoculated into 5 mL of YPD medium and cultured overnight at 30°C and 200 rpm. This was the primary seed solution. The primary seed solution was inoculated into a shake flask containing 20 mL of fresh BSM inorganic salt medium at a ratio of 2%, and cultured at 30°C and 200 rpm for 72 to 96 hours. The supernatant was collected by centrifugation at 10,000 rpm for 10 minutes.

[0043] Transfer the collected supernatant to ice, add ammonium sulfate while stirring until the saturation in the solution is 80%, let it stand at low temperature for more than 4 hours, centrifuge at 10000 rpm for 5 minutes at 4°C, discard the supernatant, and resuspend the precipitate in 1 mL of 50mM PBS buffer, pH 7.4. The result after desalting is proteinase K. The purity of the purified protein reaches 95% by SDS-PAGE detection. The results are as follows: Figure 1 shown.

[0044] Proteinase K catalyzes the cleavage of peptide bonds at the carboxyl termini of aliphatic and aromatic amino acids. Under certain temperature and pH conditions, casein hydrolysis produces tyrosine, which contains a phenolic group. Under alkaline conditions, this can be reduced with the Folin-phenol reagent to produce molybdenum blue and tungsten blue, the color of which is proportional to the tyrosine content.

[0045] The absorbance at 680 nm was measured to determine the amount of tyrosine produced by enzymatic hydrolysis, and the activity of proteinase K was calculated. One unit of enzyme activity (U) was defined as the amount of L-tyrosine produced by casein hydrolysis per minute at pH 8.0 and 60°C.

[0046] 100 μg / mL L-Tyrosine Standard Solution: Weigh 0.01 g of L-tyrosine with a purity of ≥95% and dissolve it in 2 mL of 1 mol / L HCl. Then dilute to 10 mL with water to create a 1 mg / mL L-Tyrosine standard stock solution. Store at 4°C. When ready to use, take 1 mL of the stock solution and dilute to 10 mL with 0.1 mol / L HCl to obtain a 100 μg / mL L-Tyrosine standard solution.

[0047] Standard curve drawing table

[0048]

[0049]

[0050] Each concentration was repeated three times. 500 μL of tyrosine was added to 2.5 mL of Na2CO3 and then 500 μL of disulfide reagent. The mixture was incubated at 40°C for 20 min to develop color. The color was then measured using a microplate reader after cooling to room temperature. 680nm absorbance.

[0051] Control group: 500 μL of the enzyme solution to be tested was placed at 60°C for 10 min and then 500 μL of preheated 1.0% (W / V) casein sodium salt was added;

[0052] Sample group: 500 μL of the enzyme solution to be tested and 500 μL of preheated 1.0% (W / V) casein sodium salt, react at 60°C for 10 min;

[0053] For both the control and sample groups, 1 mL of a mixture of 0.1 mol / L trichloroacetic acid, 0.2 mol / L sodium acetate, and 0.3 mol / L acetic acid was added. After standing for 2-3 minutes, the mixture was centrifuged at 12,000 rpm for 3 minutes. After centrifugation, 500 μL of the reaction solution was added to 2.5 mL of Na₂CO₃, followed by 500 μL of dichlorophenol reagent, and incubated at 40°C for 20 minutes to develop color.

[0054] Blank group: 500 μL of water was added with 2.5 mL of Na2CO3, and then 500 μL of dichlorophenol reagent was added, and incubated at 40°C for 20 min to develop color;

[0055] Cool to room temperature and measure A 680nm The absorbance is calculated as follows:

[0056]

[0057] X: Enzyme activity of proteinase K sample (U / mL or U / g)

[0058] C: Tyrosine concentration of the sample tube, in μg / mL

[0059] C0: Tyrosine concentration in the control tube, in μg / mL

[0060] 4: Total volume of the reaction system, in mL

[0061] N: dilution factor of the sample solution

[0062] M: molar mass of L-tyrosine (181.20 g / mol)

[0063] 10: Reaction time, unit is 10min

[0064] C S : Concentration of proteinase K sample solution, in g / mL

[0065] First, the 3D structure of the enzyme was constructed using Swiss-Model, using PDB ID: 3prk. Following homology modeling, molecular dynamics (MD) simulations were performed to assess the stability of the proteinase K model in a dynamic environment. MD simulations were performed using Gromacs 2019.6, employing the amber99sb force field and the TIP3P water model, under constant temperature and pressure (NVT and NPT) conditions, with periodic boundary conditions considered.

[0066] First, the optimized crystal structure was placed in the center of a cubic box, ensuring that the distance between the protein surface and the edge of the box was at least 1 nm, and the system was structurally optimized. The final result was used as the initial structure for subsequent MD simulations. Next, position-restrained equilibrium was performed using 100 ps of NVT equilibrium and NTP equilibrium, and molecular dynamics simulations were performed at 300 K for 100 ns each. During the simulation, all bond lengths were constrained using the LINCS algorithm with a time step of 2 fs. Electrostatic interactions were calculated using the particle-mesh Ewald (PME) method.

[0067] The entire sequence of proteinase K was subjected to virtual saturation mutation using FoldX and Rosetta software, and the free energy change ΔΔG was calculated. The overall structural stability of the protein (ΔG) can be evaluated by free energy, and the lower the value, the better the thermal stability. The change in thermal stability caused by the mutation can be calculated by the ΔG change between the wild type and the mutant (i.e., ΔΔG). If ΔΔG is less than -1 kcal / mol, it means that the mutation has improved the protein stability.

[0068] Select appropriate mutation sites based on the magnitude of the free energy change. The minimum free energy heat map after virtual saturation mutation of candidate amino acid sites is shown in the figure below. Figure 2 and Figure 3The results of virtual screening of mutation sites are as follows: T166I, G192F / Y, S195P, Q193D / E, G200A, D202M, S210M / V, G226F, S228D, S230E, T273I / M, and P318A.

[0069] Primers were designed to amplify the 16 mutants using the plasmid pGAPZαA-Prok expressing the wild-type proteinase K gene as a template. The PCR protocol was as follows: initial denaturation at 98°C for 2 minutes; 30 cycles of denaturation at 98°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds; and finally, extension at 72°C for 5 minutes. The PCR products were digested with Dpn I at 37°C for 4 hours. The digested products were transformed into competent E. coli Top10 cells and cultured on low-salt LB plates at 37°C for 12-16 hours until single colonies emerged. Single colonies were then selected for sequencing to obtain the different proteinase K mutants. The recombinant plasmids for the 16 mutants were then isolated and used for future reference. The primer sequences for PCR amplification of the proteinase K mutants are shown in Table 2.

[0070] Table 2 Primers used for site-directed mutagenesis

[0071]

[0072]

[0073] Expression and screening of proteinase K mutants:

[0074] The proteinase K mutant plasmid obtained in Example 2 was electroporated into Pichia pastoris X33 competent cells for expression. The specific steps are as follows:

[0075] (1) The proteinase K mutant plasmid was linearized by AvrⅡ digestion and then electroporated into Pichia pastoris X33. The plasmid was cultured on YPD plates containing 100 μg / mL bleomycin at 30°C for 48-72 h until a single colony was grown.

[0076] (2) Use a sterilized toothpick tip or a pipette tip to pick up clones and gently tap them onto a casein plate. Place the plate in a 30°C incubator and incubate for about 12 to 36 hours. The appearance of a hydrolysis zone indicates that proteinase K is secreted and active. The absence of a hydrolysis zone indicates that proteinase K is not secreted or active. Experimental verification shows that the eight mutation sites T166I, G192Y, Q193D / E, G200A, G226F, and T273I / M form transparent zones after plate screening. The single clones that form transparent zones are inoculated into 5 mL of YPD medium and cultured overnight at 30°C and 200 rpm. This is the primary seed solution. The primary seed solution is inoculated into a shake flask containing 20 mL of fresh BSM inorganic salt medium at a ratio of 2%, cultured at 30°C and 200 rpm for 72 to 96 hours, and centrifuged at 10,000 rpm for 10 minutes to collect the supernatant.

[0077] (3) The enzyme activity of the wild-type proteinase K and the mutant fermentation supernatant that formed a clear zone was measured respectively, and the activity of each mutant proteinase K that was not heat-treated was set to 100%. At the same time, the wild-type and mutant fermentation supernatants that formed a clear zone were treated at 75°C for 30 minutes, and the residual activity after heat treatment was measured. The ratio of the residual activity after heat treatment to the enzyme activity before heat treatment was calculated, which was the residual enzyme activity rate of the enzyme after heat treatment. The results are shown in the figure. Figure 4 As shown. The residual enzyme activity of wild-type proteinase K after treatment at 75°C for 30 minutes was 20%, while the thermal stability of proteinase K mutant G226F was significantly improved, and the residual enzyme activity after treatment at 75°C for 30 minutes was 55%. Then, mutant G226F with a greater improvement in thermal stability was selected for expression and purification. The purity of the purified protein reached 95% by SDS-PAGE detection. Its amino acid sequence is shown in SEQ ID NO.1 sequence, and its nucleotide sequence is shown in SEQ ID NO.2 sequence; the results are shown in Figure 5 shown.

[0078] (4) The specific activity of the purified proteinase K mutant G226F was determined. The enzyme activity assay conditions are shown in Example 1. The results showed that the specific activity of the proteinase K mutant G226F was 324 U / mg, which is 2.3 times that of the wild-type proteinase K (143 U / mg). Therefore, the enzymatic properties of the proteinase K mutant G226F were analyzed in detail.

[0079] Thermal stability analysis of proteinase K mutant G226F:

[0080] The concentrations of purified wild-type proteinase K and mutant G226F proteins were determined. The cells were then incubated at the same temperature for 10-60 minutes, and the enzyme activity was measured. The residual enzyme activity after treatment at different times was calculated. The activity of untreated wild-type proteinase K and mutant G226F was taken as 100%, and the residual enzyme activity after treatment at different temperatures was calculated. The results are shown in Figure 2. Figure 6 As shown. Compared with the wild-type enzyme, the thermal stability of mutant G226F was significantly improved. When mutant G226F was incubated at 75°C, the melting point temperature (T m value) is about 30min, which is the wild type (T m The results showed that the thermal stability of G226F mutant enzyme was significantly improved compared with wild-type proteinase K.

[0081] pH stability analysis of proteinase K mutant G226F:

[0082] Different pH buffers (50mM) were prepared, including the following: citric acid-sodium citrate buffer (pH 4.0-6.0); phosphate buffer (pH 6.0-8.0); borax-hydrochloric acid buffer (pH 8.0-9.0); and borax-sodium hydroxide buffer (pH 10.0). The enzyme was diluted with different pH buffers and allowed to stand at 4°C for 24 hours. The enzyme activity of wild-type proteinase K and mutant G226F was then measured. The residual enzyme activity of the control group was taken as 100%, and the residual enzyme activity of the treated enzymes was analyzed. The results are shown in Figure 2. Figure 7 The results showed that the wild-type and mutant G226F proteins were very stable at pH 4.0-12.0.

[0083] In addition, the Michaelis constants of the wild type and G226F mutant were measured at pH 8.0. The Michaelis constant of the wild type proteinase K was: m The value is 0.99 mg / mL, V max was 216.2±10.90μmol / min / mg, k cat 110.59s -1 , k cat / K m The K of mutant G226F is 111.70 mL / s / mg. m 0.96 mg / mL, V max is 473.7 μmol / min / mg, k cat 242.30s -1 , k cat / K m The catalytic efficiency of the mutant enzyme (k cat / K m The results showed that all kinetic parameters of the mutant G226F were significantly better than those of the wild type.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A proteinase K mutant with high catalytic activity and high thermal stability, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A proteinase K mutant with high catalytic activity and high thermal stability according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. The proteinase K mutant with high catalytic activity and high thermal stability according to claim 1, characterized in that: The proteinase K mutant is obtained by mutating the wild type. The amino acid sequence of the wild type is shown in SEQ ID NO.

3. The mutation site is the glycine at position 226 of the wild type, which is mutated to phenylalanine.

4. The proteinase K mutant with high catalytic activity and high thermal stability according to claim 1, characterized in that The wild-type nucleotide sequence is shown in SEQ ID NO.

4.

5. The proteinase K mutant with high catalytic activity and high thermal stability according to claim 1, characterized in that: The proteinase K mutant is used for hydrolyzing keratin, and the short peptides or amino acids produced after the hydrolysis can be used as feed additives.

6. The proteinase K mutant with high catalytic activity and high thermal stability according to claim 1, characterized in that The residual enzyme activity of the proteinase K mutant after being treated at 75° C. for 30 minutes is 55%; the specific activity of the proteinase K mutant is 324 U / mg.

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