A proteinase K mutant and its preparation method

Abstract

The present invention provides a proteinase K mutant and a preparation method thereof. The amino acid sequence of the proteinase K mutant is based on the amino acid sequence of the parent proteinase K, in which methionine (Met) at position 244 is replaced with phenylalanine (Phe). Under suitable conditions, the fermentation broth of the proteinase K mutant exhibits an enzyme activity as high as 4325.54±51.76 U / mL, which is approximately 32.03% higher than that of the parent proteinase K (3276.24 U / mL).

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a proteinase K mutant and a preparation method thereof. Background Art

[0002] Proteinase K (EC 3.4.21.64) is a serine protease from Tritirachium albumlimber, with a typical catalytic triad structure Asp 39 -His 69 -Ser 224 Proteinase K is one of the most active known endopeptidases, capable of non-specifically hydrolyzing native and denatured proteins, preferentially breaking down ester and peptide bonds adjacent to the C-termini of hydrophobic, sulfur-containing, and aromatic amino acids. The mature protease is composed of 279 amino acids, with two disulfide bridges and two bound calcium ions stabilizing the compact structure. Under normal conditions, the pH range for proteinase K is 7-10.0, and its activity can reach over 80% at temperatures between 20°C and 60°C. In scientific research, this enzyme is widely used as a tool in nucleic acid extraction. With the development of nucleic acid detection technology, proteinase K has seen a surge in market demand as a key enzyme for protein degradation and RT-qPCR template extraction. The enzyme also exhibits excellent resistance to SDS, urea, and other agents, demonstrating promising applications in the laundry and feed industries, as well as in sewage treatment, papermaking, and food processing.

[0003] Early commercially available Proteinase K was primarily obtained from Candida albicans. Candida albicans grows slowly, making high-density culture difficult, resulting in low Proteinase K yields. Furthermore, Candida albicans also secretes other proteases, complicating downstream separation and purification. Later, companies such as Roche Diagnostics (US7368274B2 and WO2002072634A2) utilized a Pichia pastoris expression system to achieve secretory expression of Proteinase K, reducing purification costs and increasing Proteinase K yields. However, this enzyme still lags behind commercially available proteases. Therefore, it is crucial to engineer Proteinase K to improve its expression and activity. Yang H et al. (Yang H, Zhai C, Yu X, et al. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains [J]. Protein Expression and Purification, 2016: 38-44.) used Pichia pastoris GS115 as a host to recombinantly express a mutant of T. album limber proteinase K. The mutant carries six mutations and has higher activity than the wild-type protein. Summary of the Invention

[0004] In response to current industry needs and the shortcomings of existing technologies, the present invention aims to provide a proteinase K mutant with improved enzyme activity and a preparation method thereof. The present invention mainly constructs a proteinase K mutant library through error-prone PCR and screens mutants with improved proteinase K enzyme activity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a proteinase K mutant, wherein the amino acid sequence of the proteinase K mutant is that methionine (Met) at position 244 is replaced by phenylalanine (Phe) based on the amino acid sequence of the parent proteinase K, and the amino acid sequence of the parent proteinase K is shown in SEQ ID NO: 1, 2 or 3.

[0007] According to one embodiment of the present invention, the amino acid sequence of the proteinase K mutant is shown in SEQ ID NO: 4.

[0008] The present invention also provides a gene encoding the proteinase K mutant.

[0009] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above-mentioned encoding gene.

[0010] The present invention also provides a host cell comprising the above encoding gene or recombinant vector.

[0011] The present invention also provides the use of the above-mentioned proteinase K mutant for enzymatic hydrolysis of proteins, preferably for decomposing ester bonds and / or peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids.

[0012] The present invention also provides a method for preparing the proteinase K mutant, which comprises performing gene recombination and expression using the coding gene of the proteinase K mutant or an expression vector comprising the coding gene.

[0013] The present invention also provides a method for enzymatically hydrolyzing a protein, which comprises the step of contacting the protein with the proteinase K mutant of the present invention, under conditions where the proteinase K mutant can catalyze the enzymatic hydrolysis of the protein.

[0014] The present invention also provides a method for improving the enzymatic activity of proteinase K, which is by introducing an M244F substitution into a parent proteinase K having an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3.

[0015] Beneficial effects of the present invention:

[0016] The present invention utilizes error-prone PCR technology to conduct directed evolution of proteinase K derived from Candida albicans in vitro, thereby obtaining a proteinase K mutant M244F with enhanced enzyme activity and a recombinant strain TCCC 31261 thereof. Under suitable conditions, the enzyme activity of the fermentation broth of the proteinase K mutant of the present invention is as high as 4325.54±51.76 U / mL, which is approximately 32.03% higher than that of its parent (3276.24 U / mL). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Enzyme activities of parent TCCC 31259 and mutant TCCC 31261.

[0018] Figure 2a and 2b : Optimum temperatures of parent TCCC 31259 and mutant TCCC 31261.

[0019] Figure 3a and 3b : Optimal pH of parent TCCC 31259 and mutant TCCC 31261.

[0020] Figure 4a and 4b : Temperature stability of parent TCCC 31259 and mutant TCCC 31261.

[0021] Figure 5a and 5b : pH stability of parental TCCC 31259 and mutant TCCC 31261. DETAILED DESCRIPTION

[0022] The present invention will be further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and appropriate ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications made to the material composition and dosage in these embodiments also fall within the scope of protection of the present invention.

[0023] The present invention provides a proteinase K mutant, wherein the amino acid sequence of the proteinase K mutant is that methionine (Met) at position 244 is replaced by phenylalanine (Phe) based on the amino acid sequence of the parent proteinase K, and the amino acid sequence of the parent proteinase K is shown in SEQ ID NO: 1, 2 or 3.

[0024] According to one embodiment of the present invention, the amino acid sequence of the proteinase K mutant is shown in SEQ ID NO: 4.

[0025] The present invention also provides a gene encoding the proteinase K mutant.

[0026] According to one embodiment of the present invention, the nucleotide sequence of the encoding gene is shown as SEQ ID NO: 5.

[0027] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above-mentioned encoding gene, for example, a recombinant vector composed of the vector plasmid pPIC9K and the encoding gene of the present invention.

[0028] The present invention also provides a host cell comprising the above encoding gene or recombinant vector. The host cell can be any host suitable for producing the proteinase K mutant of the present invention from the gene or vector of the present invention, such as Pichia pastoris, Escherichia coli, or Bacillus subtilis.

[0029] The present invention also provides the use of the above-mentioned proteinase K mutant for enzymatic hydrolysis of proteins, preferably for decomposing ester bonds and / or peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids.

[0030] The above-mentioned proteinase K mutants of the present invention have a specific improved enzyme activity compared to their parent proteinase K, for example: an increase of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100%.

[0031] The present invention also provides a method for preparing the above-mentioned Proteinase K mutant, which method is carried out by genetic recombination and expression using the coding gene of the Proteinase K mutant of the present invention or a recombinant vector comprising the coding gene. Gene recombination methods and expression hosts known to those skilled in the art can be used, and culture media and culture conditions suitable for host expression can be selected. The method can also include a step of recovering the Proteinase K mutant, which recovery step may involve a step of separating or purifying the Proteinase K mutant from the host's culture or expression product, and can be carried out using any method known to those skilled in the art.

[0032] The present invention also provides a method for enzymatically hydrolyzing a protein, which comprises the step of contacting the protein with the proteinase K mutant of the present invention, under conditions where the proteinase K mutant can catalyze the enzymatic hydrolysis of the protein.

[0033] The present invention also provides a method for improving the enzymatic activity of proteinase K, which is by introducing an M244F substitution into a parent proteinase K having an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3.

[0034] The following definitions are used in the present invention:

[0035] 1. Nomenclature of amino acid and DNA sequences

[0036] Amino acid residues are referred to using the generally accepted IUPAC nomenclature, using either three-letter abbreviations or single-letter symbols. DNA sequences are referred to using the generally accepted IUPAC nomenclature.

[0037] 2. Identification of Proteinase K Mutants

[0038] The term "amino acid substituted at the original amino acid position" is used to represent the mutated amino acid in a proteinase K mutant. For example, M244F (or Met244Phe) indicates that the amino acid at position 244 is replaced by phenylalanine (F) instead of methionine (M) in the parent proteinase K. The amino acid numbering scheme for the proteinase K of the present invention is based on the sequence set forth in SEQ ID NO: 2.

[0039] 3. Definition of parent proteinase K

[0040] The term "parent" or "parent sequence" refers to a sequence that existed prior to the mutation of the present invention, upon which amino acid substitutions at specific positions are made to generate new mutants. The amino acid sequence of the parent proteinase K of the present invention is shown in SEQ ID NO: 1, 2, or 3. SEQ ID NO: 1 is derived from Tritirachium album limber GenBank: P06873.2 and carries six mutations (Y151A, K208H, S273T, G293A, K332R, S337N). Positions 1-15 represent a signal peptide, positions 16-105 represent a leader peptide, and positions 106-384 represent a mature peptide. SEQ ID NO: 2 is the amino acid sequence of proteinase K without the signal peptide, i.e., positions 16-384 of SEQ ID NO: 1. SEQ ID NO: 3 is the mature peptide sequence of proteinase K, i.e., positions 106-384 of SEQ ID NO: 1.

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] The present invention will be described in more detail below through specific examples. Unless otherwise specified, in the following examples:

[0043] The culture medium and enzyme activity determination method used in the present invention are as follows:

[0044] LB medium: yeast powder 10 g / L, peptone 10 g / L, sodium chloride 5 g / L.

[0045] MD solid medium: YNB 26.8, glucose 40.0, 8×10 -5 % biotin.

[0046] YPD medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L glucose, and 18 g / L agar for solid culture medium.

[0047] YPG medium: peptone 20 g / L, yeast powder 10 g / L, glycerol 20 g / L.

[0048] Casein medium: 10g / L yeast powder, 20g / L peptone, 15g / L agar powder, 10g / L casein. Preparation: Taking 100mL of casein medium as an example, dissolve 1g yeast powder and 2g peptone in 50mL of water, then add 1.5g agar powder to form Solution A. Add 1g casein to 50mL of pH 9.0 buffer and heat to a milky dispersion to form Solution B. Mix Solutions A and B, sterilize at 110°C for 15 minutes, and allow to solidify by pouring onto a plate.

[0049] BMGY medium: 10 g yeast powder and 20 g peptone dissolved in 700 mL water. When used, add 100 mL pH 6.0 1 mol / L potassium phosphate buffer, 100 mL 10× YNB, 2 mL 500× biotin, and 10 mL glycerol.

[0050] BMMY medium: 10 g yeast powder and 20 g peptone were dissolved in 700 mL of water. When used, 100 mL of pH 6.0 1 mol / L potassium phosphate buffer, 100 mL of 10× YNB, 2 mL of 500× biotin, and 2.5 mL of methanol were added.

[0051] Proteinase K activity assay method:

[0052] (1) Definition of enzyme activity

[0053] Under certain temperature and pH conditions, the amount of enzyme that hydrolyzes casein to produce 1 μg of tyrosine in 1 minute is defined as one unit of enzyme activity, expressed in U. The determination method mainly refers to the national standard GB / T 23527-2009.

[0054] (2) Reagent configuration

[0055] Reagent I: 0.4 M sodium carbonate solution: 42.4 g Na2CO3 solid is dissolved in 1000 mL water.

[0056] Reagent II: boric acid buffer: dissolve 9.54 g of solid sodium tetraborate and 1.6 g of solid sodium hydroxide in 800 mL of deionized water, adjust the pH to 10.59 ± 0.05 with NaOH solution, and dilute to 1000 mL with deionized water.

[0057] Reagent III: 0.4 M trichloroacetic acid solution: Dissolve 65.4 g trichloroacetic acid in 1000 mL water and store in the dark.

[0058] Reagent IV: Folin phenol reagent: Folin reagent: deionized water = 1:2, store at 4℃ away from light

[0059] Reagent V: 1% casein solution: Add 1 g of casein to 80 mL of boric acid buffer and place in a boiling water bath until the casein dissolves. Adjust the pH to 9 with NaOH solution and dilute to 100 mL with boric acid buffer.

[0060] (3) Measurement method

[0061] Add 1 mL of diluted enzyme solution to a test tube and preheat at 65°C for 2 minutes. Add 1 mL of the preheated casein solution, shake well, and incubate at 65°C in a water bath for 10 minutes. Add 2 mL of trichloroacetic acid solution and shake well (for a blank control, add trichloroacetic acid first, then casein solution). Remove and cool to room temperature. Centrifuge at 12,000 rpm for 2 minutes. Take 0.5 mL of the supernatant, add 2.5 mL of sodium carbonate solution and 0.5 mL of forlin reagent, develop at 40°C for 20 minutes, and measure absorbance at 680 nm using a microplate reader.

[0062] The plasmids involved in the examples are shown in Table 1:

[0063] Table 1

[0064]

[0065] Note: pPIC9K plasmid is a commercial plasmid.

[0066] The primer information involved in the embodiment is shown in Table 2:

[0067] Table 2

[0068]

[0069] Example 1: Obtaining Proteinase K Mutants

[0070] (1) Acquisition of mutant genes

[0071] The gene sequence of the parent proteinase K (SEQ ID NO: 2) reported in the literature was synthesized, and its gene was used as a template to construct the plasmid of the parent proteinase K on pPIC9K using tprk-R and tprk-F primers. At the same time, EP-tprk-F and EP-tprk-R were combined with 0.05mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, and 0.5mM of Mn 2+ , amplified by error-prone PCR, the amplification reaction system is as follows:

[0072] <![CDATA[Mn 2+ ]]> 21 μL template 1 μL Taq enzyme 25 μL Primer tprk-F 2μL Primer tprk-R 2μL

[0073] The PCR product was subjected to agarose gel electrophoresis, and the size of the proteinase K electrophoresis band was between 1000-1500 bp. The PCR product was then recovered using a small amount of DNA recovery kit to obtain the mixed mutant fragment PRKMn.

[0074] (2) Construction of recombinant vector

[0075] 1) The pPIC9K plasmid was extracted and double-digested with EcoR I and Not I, followed by agarose gel electrophoresis. The product was then recovered using a DNA gel recovery kit to obtain a linearized vector sequence.

[0076] The double enzyme digestion system is as follows:

[0077] <![CDATA[ddH2O]]> 25 μL Plasmid template 15 μL Q.cut Buffer 5μL Restriction endonuclease EcoRI 2.5 μL Restriction enzyme Not I 2.5 μL

[0078] 2) The linear vector fragment obtained by enzyme digestion and the mixed target gene fragments were connected by seamless cloning to form recombinant plasmids pPIC9K-PRK (parent) and pPIC9K-PRKMn (mutant).

[0079] The seamless cloning enzyme reaction system is as follows:

[0080] Seamless cloning enzyme 5μL Linear vector xμL Insert yμL

[0081] After mixing evenly, react in a 50°C water bath for 15 min.

[0082] PS: First, use NanoDrop 2000C ultra-micro-volume spectrophotometer to measure the DNA concentration of linear vector and target gene according to the formula:

[0083] pmols = mass in ng / (fragment length in bp × 0.65 kDa),

[0084] x:y=1:2;x+y=5,

[0085] In addition, the amount of x and y added should be between 0.01 and 0.25 pmols, and the amount of linear vector and insert added can be calculated based on this.

[0086] 3) Extract the recombinant pPIC9K plasmid according to the kit's instruction manual. After single enzyme digestion with Sal I, perform agarose gel electrophoresis and recover the product using a DNA gel recovery kit to obtain a linearized recombinant plasmid.

[0087] The single enzyme digestion system is as follows:

[0088] <![CDATA[ddH2O]]> 27 μL Plasmid template 15 μL Q.cut Buffer 5μL Restriction endonuclease Sal I 3μL

[0089] After mixing, enzyme digestion was performed in a 37°C water bath for 2 h. After the reaction was completed, the enzyme digestion products were subjected to agarose gel electrophoresis and then recovered using a small amount of DNA recovery kit: linear recombinant plasmid pPIC9K-PRK and mixed pPIC9K-PRKMn.

[0090] Example 2: Transformation and screening of recombinant proteinase K mutant plasmids in Pichia pastoris

[0091] Take 15 μL of linear fragment and mix it with 80 μL of GS115 yeast competent cells, transfer it to an ice-cold electroporation cup (2 mm gap between the two electrodes), and place it in an electroporator for electroporation. The electroporation parameters are: 1500 V, 25 μF, 200 Ω; then quickly add 200 μL of 1 M sorbitol solution 1 mL to the electroporation cup, and then add 800 μL of MD medium. After mixing, transfer it to a 1.5 mL EP tube and activate it on a shaker at 30°C for 2 h; centrifuge the bacterial suspension to remove the supernatant, add 80 μL of MD medium to resuspend it, spread it on an MD plate, place it upside down in a 30°C incubator, and culture it until a single colony appears.

[0092] A single yeast colony obtained by transformation was spotted onto a 1% casein plate. Induction was performed by adding 200 μL of methanol to the lid of the culture dish every 12 hours. After 48-60 hours of induction, the hydrolysis zone on the casein plate was observed. The single colony corresponding to the hydrolysis zone was the proteinase K recombinant colony. The screening result was a single mutant colony. The plasmid was extracted and sent to a biotechnology company for sequencing. The mutant was named TCCC 31261, and the parent strain was named TCCC 31259. Sequencing revealed that the amino acid methionine Met at position 244 of the proteinase K mutant was mutated to phenylalanine Phe. The amino acid sequence of the mutant is shown in SEQ ID NO: 4:

[0093] APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDH

[0094] VYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLA

[0095] RISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSR

[0096] DGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFV

[0097] ASDHNNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVFVAVAAGNNNA

[0098] DARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGST

[0099] RSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGDLSNIPFGTVNLLAYNNYQA;

[0100] The mutant gene sequence is shown in SEQ ID NO: 5:

[0101] gctccagccgttgaacaaagatctgaagctgctccattgattgaagctagaggtgaaatggttgcaaacaagtacattgtta

[0102] agtttaaggaaggttctgctctatccgctctggacgctgctatggaaaagatttctggaaagccagatcatgtttacaaaaa

[0103] cgtcttttctggttttgctgccactcttgatgaaaacatggttagagttttgagagctcatcctgacgttgaatacattgagcaa

[0104] gatgccgttgtgactataaacgctgcacaaactaacgctccatggggattggctagaatttcttctacttctccaggtacttc

[0105] aacatactactatgatgaatctgcaggtcagggtagttgtgtttacgttattgatactggtattgaggcttctcatccagaattt

[0106] gaaggtagggctcaaatggtgaagacttattacgcttcatcaagagatggtaacggtcatggtactcattgtgctggtacc

[0107] gttggttctaggacttacggtgttgctaagaagactcaactgtttggtgttaaggttttggatgataatggcagtggtcaatatt

[0108] ctactattattgcaggtatggattttgttgcatctgatcataacaacagaaactgtccaaagggtgttgttgcttctttgtctttgg

[0109] gcggtggttactcttcttctgtgaactctgccgcagcccgtttgcagtctagtggtgtatttgtcgctgttgcagcaggtaaca

[0110] acaacgcagatgctagaaattactctcccgcttctgagccatctgtatgcacggttggagccactgacagatacgatagac

[0111] gttctagtttttctaactacggctctgttcttgacatttttgctccaggaacttctattttgtctacttggattggaggctctacaag

[0112] gtctatatcaggtacatctatggctactccacacgttgccggtttggctgcctacttaatgactttgggtagaactactgctgc

[0113] taacgcttgcagatatattgccgatacagctaataagggtgatttgagtaacattccatttggtactgtcaatttgttggcttac

[0114] aataactaccaagcttaa

[0115] Example 3: Expression of Proteinase K and Its Mutants in Pichia pastoris and Determination of Enzymatic Properties

[0116] Shake flask fermentation: Genetically engineered strains TCCC 31259 and TCCC 31261 were streaked onto YPD plates and inverted at 30°C. Activated single colonies were picked and placed in 5 mL YPG medium, shaken at 30°C and 220 rpm for 24 h, and then inoculated into 50 mL BMGY liquid medium at a 2% inoculum until the OD 600 After reaching 2-6, the mutant was transferred to 50 mL of BMMY liquid medium and cultured at 30°C, 220 rpm, with shaking, supplemented with 0.5% methanol every 12 hours for 6 days of induction. After 144 hours of fermentation, the fermentation broth was collected and centrifuged at 4°C, 12,000 rpm, for 2 minutes. The supernatant was diluted appropriately and assayed for proteinase K activity according to the national standard method. The results showed that after 144 hours of shake flask fermentation, the enzyme activity of the mutant fermentation broth reached 4325.55±51.76 U / mL, an increase of approximately 32.03% compared to the 3276.24 U / mL of the starting strain.

[0117] Optimum temperature test: The fermentation supernatants of mutant recombinant bacteria TCCC 31259 and parent recombinant bacteria TCCC 31261 were respectively measured at different temperatures (45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃) and pH 9.0 for proteinase K activity. The highest enzyme activity at different reaction temperatures was considered 100%. The mutant TCCC 31261 ( Figure 2b ) and the parent TCCC 31259 ( Figure 2a ) had an optimum temperature of 65°C for proteinase K expression, but the activity of proteinase K expressed by mutant TCCC31261 remained above 60% in the range of 45-70°C, while the activity of proteinase K expressed by parent TCCC 31259 remained above 60% in the range of 50-70°C.

[0118] Optimal pH test: The enzyme activities of the fermentation supernatants of the mutant recombinant TCCC 31259 and the parent recombinant bacteria TCCC 31261 were measured at different pH values ​​(7.0, 8.0, 9.0, 10.0, 11.0) at 65°C. The highest enzyme activity at the different reaction pH values ​​was considered 100%. The enzyme activity of the mutant TCCC 31261 ( Figure 3b ) and the parent TCCC 31259 ( Figure 3a ) have an optimum pH of 9.0 for proteinase K, but mutant TCCC 31261 can maintain a relative enzyme activity of about 95% in the pH range of 8.0-10.0, and has higher catalytic activity than its parent TCCC 31259 (relative enzyme activity is above 90% in the pH range of 8.0-10.0).

[0119] Temperature stability test: The fermentation broth of mutant recombinant TCCC 31259 and parent recombinant bacteria TCCC 31261 were kept at different temperatures (45℃, 55℃, 65℃, 70℃) for 100 minutes, and the residual enzyme activity was calculated with the activity of the untreated strain as 100%. Figure 4b ) was kept at 65℃ for 100min, and the residual enzyme activity was maintained at 41.27%, which was higher than that of the parent TCCC 31259 ( Figure 4a )The residual enzyme activity was 7.74% higher.

[0120] pH stability test: The fermentation broth of mutant recombinant TCCC 31259 and parent recombinant bacteria TCCC 31261 were placed in different pH values ​​(7.0, 9.0, 11.0) and stored at room temperature for 8 days. The activity of the untreated fermentation broth was taken as 100% to calculate the residual enzyme activity. Figure 5b) was stored at room temperature at pH 9.0 for 8 days, and its residual enzyme activity was 65.52%, which was significantly higher than that of the parent TCCC 31259 ( Figure 5a )The residual enzyme activity was 10.07% higher.

[0121] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A proteinase K mutant, characterized in that The amino acid sequence of the proteinase K mutant is that the methionine at position 244 of the parent proteinase K is replaced by phenylalanine based on the amino acid sequence shown in SEQ ID NO: 2, or the methionine at position 259 is replaced by phenylalanine based on the amino acid sequence shown in SEQ ID NO: 1, or the methionine at position 154 is replaced by phenylalanine based on the amino acid sequence shown in SEQ ID NO:

3.

2. The proteinase K mutant according to claim 1, characterized in that The amino acid sequence of the proteinase K mutant is shown in SEQ ID NO:

4.

3. A gene encoding the proteinase K mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that Comprising the coding gene according to claim 3.

5. A host cell, characterized in that Comprising the coding gene according to claim 3 or the recombinant vector according to claim 4.

6. The host cell according to claim 5, wherein The host cell is a Pichia pastoris cell.

7. Use of the proteinase K mutant according to claim 1 or 2 for enzymatic hydrolysis of proteins, for decomposing ester bonds and / or peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids.

8. A method for preparing the proteinase K mutant according to claim 1 or 2, characterized in that: The method comprises: culturing the host cell according to claim 5 under suitable conditions and making it express the coding gene according to claim 3, and then isolating the proteinase K mutant from the culture or expression product.

9. A method for enzymatic hydrolysis of protein, characterized in that: The method comprises the step of contacting a protein with the proteinase K mutant according to claim 1 or 2 under conditions that can catalyze the enzymatic degradation of the protein by the proteinase K mutant.

10. A method for improving proteinase K enzyme activity, characterized in that: The method is to introduce an M244F substitution into a parent proteinase K having an amino acid sequence as shown in SEQ ID NO: 2, or to introduce an M259F substitution into a parent proteinase K having an amino acid sequence as shown in SEQ ID NO: 1, or to introduce an M154F substitution into a parent proteinase K having an amino acid sequence as shown in SEQ ID NO: 3.

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