Heat-resistant alkaline protease K mutant and application thereof

Abstract

The invention discloses a heat-resistant alkaline protease K mutant and application thereof. Based on the amino acid sequence of wild type alkaline protease K, the amino acid sequence of the mutant comprises substitution of P175C and D200C, and the amino acid sequence of the wild type alkaline protease K comprises a sequence as shown in SEQ ID No.1. Compared with wild type alkaline protease K, the alkaline protease K mutant disclosed by the invention has the advantages that the activity maintaining capability under a high-temperature condition is obviously enhanced, so that the alkaline protease K mutant is particularly important for industrial processes needing to be operated under a high-temperature environment, such as the fields of food processing, medicine production, feed production, leather processing and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a heat-resistant alkaline proteinase K mutant and its applications. Background Technology

[0002] Alkaline proteinase K (Pro K) is a serine protease with broad substrate specificity and strong proteolytic ability. It typically exhibits high enzymatic activity under alkaline conditions and is widely used in various industrial sectors, including detergents, leather processing, food industry, feed additives, and organic waste treatment. Due to its efficient hydrolytic ability, proteinase K is particularly suitable for removing protein-based stains and breaking down complex protein structures, making it valuable in commercial detergents and industrial enzyme preparations.

[0003] Under normal conditions, proteinase K is used in the pH range of 7.5-9.0, and its activity can reach over 80% between 20-60℃. Although alkaline proteinase K exhibits good catalytic activity in alkaline environments, its native form is prone to conformational instability and loss of enzyme activity at high temperatures, making it difficult to meet the thermostability requirements of certain industrial processes. For example, in high-temperature processes such as detergent, feed pellet preparation, and food enzymatic hydrolysis, temperatures are typically above 50℃, which places higher demands on the thermostability of proteinase K. However, after years of verification by scholars both domestically and internationally, only a few mutation sites have been found to improve the specific activity of proteinase K. For instance, CN117025575A discloses a thermostability-enhanced proteinase K mutant, Prok-T206M, based on the sequence of the wild-type Prok alkaline serine protease from *Nanococcus limber albus*, with a threonine mutation at position 206 replaced by methionine. The half-life of the proteinase K mutant Prok-T206M at 65℃ is extended by 30 minutes compared to the wild-type Prok. However, this patent requires the addition of methanol for induction during proteinase K expression, and methanol has certain toxicity, posing certain safety issues.

[0004] Therefore, improving the thermal stability of alkaline proteinase K is of great significance for expanding its application range and extending its service life under high temperature environments. Summary of the Invention

[0005] To address the shortcomings of existing alkaline proteinase K in terms of poor heat resistance, this invention provides a heat-resistant alkaline proteinase K mutant and its applications.

[0006] A first aspect of the present invention protects an alkaline proteinase K mutant based on the amino acid sequence of wild-type alkaline proteinase K, wherein the amino acid sequence of the mutant includes substitutions of P175C and D200C, and the amino acid sequence of the wild-type alkaline proteinase K includes the sequence shown in SEQ ID No. 1.

[0007] SEQ ID No. 1:

[0008] AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDHNNRNCPKGVVASLSLGGGYSSSVNS AAARLQSSGVMVAAGNNNADARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGDLSNIPFGTVNLLAYNNYQAHHHHHH*

[0009] This application combines wild-type alkaline proteinase K with Ca. 2+ The Pro175 and Asp200 at the binding site are simultaneously mutated to Cys, introducing disulfide bonds, thereby enhancing its thermal stability. In some embodiments, the amino acid sequence of the mutant includes the sequence shown in SEQ ID No. 2.

[0010] SEQ ID No. 2:

[0011]

[0012] In some embodiments, the wild-type alkaline proteinase K is derived from filamentous fungi (Parengyodontium album).

[0013] The alkaline proteinase K mutant of this application has good heat resistance and does not require methanol induction during subsequent expression in host cells, thus avoiding the safety issues caused by the high toxicity and flammability of methanol. It is safer and more suitable as a food or feed additive.

[0014] Another aspect of the present invention protects an isolated polynucleotide encoding the mutant described above.

[0015] In some embodiments, the nucleotide sequence of the polynucleotide comprises the sequence shown in SEQ ID No. 3.

[0016] SEQ ID No. 3:

[0017]

[0018] Another aspect of the present invention protects a nucleic acid construct comprising the polynucleotides described above.

[0019] Another aspect of the present invention protects a host cell comprising a nucleic acid construct as described above or a genome in which polynucleotides as described above are integrated.

[0020] In some embodiments, the host cell can be any cell capable of being used for proteinase K recombinant production, such as prokaryotic or eukaryotic cells. The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. Gram-negative bacteria include, but are not limited to, *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell can also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. Preferably, the host cell is *Pichia pastoris*, specifically *Pichia pastoris* PpHR3.

[0021] Another aspect of the present invention protects a method for producing an alkaline proteinase K mutant, comprising: (a) culturing the host cells described above under conditions suitable for expressing the alkaline proteinase K mutant; and (b) recovering the alkaline proteinase K mutant.

[0022] Another aspect of the present invention protects a composition comprising the mutant described above. The composition may comprise the alkaline proteinase K mutant of the present invention as the major enzyme component, for example, a single-component composition. Alternatively, the composition may comprise a variety of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: proteases, glucosylamylases, β-amylases, and amylopectinases.

[0023] Another aspect of the present invention protects the use of the mutants, polynucleotides, nucleic acid constructs, host cells, or compositions described above in at least one of the following: 1) hydrolyzed proteins; 2) as feed additives or raw materials for detergents; 3) food processing; 4) the use of recognizing the carboxyl terminus of phenylalanine and hydrolyzing the amide bond between phenylalanine and nitroaniline.

[0024] In some embodiments, in 4), the substrate is selected from N-succinyl-L-phenylalanine-P-nitro-aniline during hydrolysis.

[0025] Another aspect of the present invention protects a method for improving the thermal stability of alkaline proteinase K, based on the sequence shown in SEQ ID No. 1, by mutating proline at position 175 to cysteine ​​and aspartic acid at position 200 to cysteine.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1) This invention obtains an alkaline proteinase K mutant (labeled as mutant alkaline proteinase (PD)) by mutating the amino acid sequence of alkaline proteinase K as shown in SEQ ID No. 1 at P175C and D200C. The enzyme activity of the mutant alkaline proteinase (PD) is increased by 20.4% compared with that of the wild-type alkaline proteinase (WT); and the enzyme activity of the mutant alkaline proteinase decreases slowly at 65°C, and even after treatment at 65°C for 60 min, the heat resistance of the mutant alkaline proteinase (PD) is significantly higher than that of the wild-type enzyme (WT). Therefore, the alkaline proteinase K mutant of this application is beneficial for expanding its application range in industrial scenarios.

[0028] 2) The alkaline proteinase K mutant of the present invention has a significantly enhanced ability to maintain activity under high temperature conditions and does not require methanol induction, thus it has broad application prospects in food processing (cheese making, bread fermentation), pharmaceutical production, feed production and leather processing. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the protein structure of wild-type alkaline proteinase K in Example 1 of the present invention.

[0030] Figure 2 This is a diagram illustrating the construction of the expression cassette for the alkaline proteinase K protein expression system in Example 1 of the present invention.

[0031] Figure 3 The image shows the results of shake-flask fermentation and enzyme activity assay of the alkaline proteinase K mutant in Example 3 of the present invention.

[0032] Figure 4 This is a graph showing the thermostability test results of the alkaline proteinase K mutant treated at 65°C in Example 4 of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. The following examples are merely specific embodiments of the invention, but the scope of protection of the invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. In the quantitative experiments in the following embodiments, three replicate experiments were set up, and the results were averaged.

[0034] Example 1: Obtaining the alkaline proteinase K mutant

[0035] First, a search was conducted in the NCBI database for the accession number (QHB21810.1) of alkaline proteinase K from *P. album*. Protein structure analysis revealed that proteinase K possesses the typical catalytic triplet Asp39-His69-Ser224 characteristic of serine proteases. The proteinase K contains two Ca²⁺ ions. 2+ The binding site is mainly involved in the thermal stability of proteinase K, but not in proteolytic activity.

[0036] like Figure 1 As shown, the Ca1 site is located inside alkaline proteinase K, coordinated by the carbonyl groups of Pro175 and Val177, two carboxylic acid oxygen Asp200s, and a carbonyl octahedron of four water molecules, and has a more critical impact on overall stability. The Ca2 site is located on the protein surface, coordinated by Thr16, Asp260, and a carbonyl group of one water molecule; the binding is weaker, and its impact on thermal stability is smaller. Therefore, to improve the thermal stability of proteinase K, the Pro175 and Asp200 sites of the Ca1 site were simultaneously mutated to Cys through rational design, thereby forming a P175C–D200C disulfide bond to replace the original Ca2 site. 2+ The mediated coordination stabilizes the overall protein structure and is expected to improve the thermal stability of proteinase K.

[0037] That is, by rationally designing and mutating Ca1-related sites and introducing disulfide bonds, a calcium ion-independent proteinase K is obtained, thereby enhancing its thermal stability.

[0038] The α-factor signal peptide and the original alkaline proteinase K (ProK, hereinafter also referred to as WT, amino acid sequence as shown in SEQ ID NO.1) were synthesized by Qingke Biotechnology Co., Ltd., and the position encoding the mCherry sequence in the Pichia pastoris synthetic expression system vector pCE3Blunt Vector-An201cp-mCherry (this plasmid was obtained by constructing the nucleotide sequence of An201cp-mCherry (SEQ ID No.17) into pCE3 Blunt Vector, which was purchased from Novizan) was replaced to obtain the recombinant plasmid pCE3 Blunt Vector-An201cp-ProK. Figure 2 ).

[0039] The structure of proteinase K was observed using PyMOL 3D molecular visualization and analysis software, such as... Figure 1 As shown in Table 1, suitable primers were designed for the Pro175 and Asp200 sites. After two rounds of whole plasmid PCR, the P175C-D200C mutation was successfully used to obtain the PD mutant.

[0040] Table 1 Primers used to construct proteinase K mutants

[0041]

[0042] The primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0043] The PCR amplification system is shown in Table 2.

[0044] Table 2

[0045] Components Dosage PrimeSTAR Max DNA Polymerase 25μL F(10μM) 1μL R(10μM) 1μL Template (5ng / μL) 1μL <![CDATA[ddH2O]]> 22μL

[0046] The PCR amplification conditions are as follows:

[0047] 1) Pre-denaturation: 98℃ for 3 min;

[0048] 2) Denaturation: 98℃ for 10s; Annealing: 60℃ for 15s; Extension: 72℃ for 1min; 30 cycles in total;

[0049] 3) Post-extension: 72℃ for 5 minutes;

[0050] 4) Store at 4℃.

[0051] Transformation and validation:

[0052] The PCR product was directly transformed into E. coli DH5α competent cells using the heat shock method. After sequencing verification, the expression cassette plasmid pCE3 Blunt Vector-An201cp-PD of the PD mutant was obtained.

[0053] Example 2: Secretory expression of alkaline proteinase K mutant in Pichia pastoris

[0054] Alkaline proteinase K and its mutant were secreted and expressed using the Pichia pastoris protein expression system. The protein expression system of Pichia pastoris reference 1 (Anssi Rantasalo et al. A universal gene expression system for fungi. Nucleic Acids Res. 2018 Oct 12; 46(18):e111. doi:10.1093 / nar / gky558.) was commissioned to Qingke Biotechnology Co., Ltd. to synthesize the nucleotide sequences of CP32-STFpp (SEQ ID No. 16) and An201cp-mCherry (SEQ ID No. 17) and construct them into pCE3 Blunt Vector (ClonExpress UltraOne Step Cloning Kit V2, Novizan), and pCE3 Blunt Vector-CP32-STFpp was obtained.

[0055] CP32-STFpp sequence

[0056]

[0057] An201-mCherry sequence

[0058]

[0059] Donar-DNA, used for constructing a SES protein expression system in Pichia pastoris, was amplified from the pCE3 Blunt Vector-CP32-STFpp plasmid (related to the Pichia pastoris synthetic expression system) and the pCE3 Blunt Vector-An201cp-PD plasmid (constructed in Example 1) using KeyPo high-fidelity enzyme (2×KeyPo Master Mix, Vazyme). After purification using an Omega gel extraction kit, the DNA was used for Pichia pastoris transformation. The amplification primers are shown in Table 3. The primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0060] Reference 2 (Hao Fang et al. Engineering Pichia pastoris for Efficient DeNovo Synthesis of 2′-Fucosyllactose. Journal of Agricultural and Food Chemistry, 2025 Apr 9; 73(14):8555-8566. doi:10.1021 / acs.jafc.5c00598.) constructed the HGP-sgRNA-Int1-Int6 plasmid for Pichia pastoris gene editing and, with its assistance, integrated the corresponding Donar-DNA expression cassette into the Int1 and Int6 sites of the Pichia pastoris PpHR3 genome via electroporation.

[0061] Table 3 Primers used for amplifying Donor-DNA for transforming Pichia pastoris.

[0062]

[0063]

[0064] The specific steps for Pichia pastoris electroporation are as follows:

[0065] 1) Obtaining competent cells of Pichia pastoris

[0066] 1-1) Inoculate a single fresh Pichia pastoris colony into 5 mL of YPD medium (YPD medium consists of 1% yeast extract, 2% peptone, and 2% glucose) and incubate overnight at 30°C to obtain the bacterial solution.

[0067] 1-2) Transfer the bacterial culture obtained in step 1-1) to a 50 mL YPD shake flask, initial OD 600 Incubate at 30℃ for 4-5 hours, maintaining a temperature of 0.15-0.2.

[0068] 1-3) When OD 600 When the bacterial concentration reaches 0.8-1.0, prepare competent Pichia pastoris cells from the bacterial culture. Transfer the bacterial culture to a 50 mL sterile centrifuge tube and centrifuge at 4000 rpm for 5 min.

[0069] 1-4) Discard the supernatant, add 8.55 mL Beds (10 mM N-diglycine, 3% (v / v) ethylene glycol, 1 M sorbitol, pH adjusted to 8.3 with sodium hydroxide), 1 mL of 1 M dithiothreitol (DTT), and 450 μL of dimethyl sulfoxide (DMSO). Gently resuspend the precipitate by pipetting to obtain a resuspended liquid.

[0070] 1-5) Incubate the resuspended liquid obtained in step 1-4) at room temperature for 5 min at 100 rpm, and then centrifuge at 4000 rpm for 5 min.

[0071] 1-6) Discard the supernatant, add 950 μL of Beds and 50 μL of DMSO, and gently resuspend the precipitate by pipetting. Centrifuge at 4000 rpm for 30 seconds.

[0072] 1-7) Discard the supernatant, add 190 μL of Beds and 10 μL of DMSO, and gently resuspend the precipitate by pipetting. The cells obtained after resuspension are Pichia pastoris competent cells, which should be stored in an ultra-low temperature freezer.

[0073] 2) Electroporation of Pichia pastoris competent cells

[0074] Take 40 μL of Pichia pastoris competent cells obtained in step 1), add 500 ng HGP-sgRNA-Int1-Int6 plasmid and 1 μg Donor-DNA. After mixing, transfer to a pre-chilled electroporation cuvette (2.0 mm spacing) and place on ice for 2 min. The electroporation instrument parameters are set as follows: voltage 1500 V, resistance 400 Ω, capacitance 25 μF.

[0075] 3) After electroporation, quickly add 1 mL of pre-cooled incubation solution to obtain a mixture. If the transformant is selected using defective screening, the incubation solution is 1 M sorbitol; if the transformant is selected using antibiotic screening, the incubation solution is a 1:1 mixture of YPD and 1 M sorbitol.

[0076] 4) Transfer the mixture to a 1.5 mL pre-cooled sterile EP tube and incubate at 30°C in a shaking incubator for 2-4 hours. Spread an appropriate amount of bacterial culture (refer to the transformation efficiency) onto a plate containing the screening antibiotic or the defective strain. Incubate the plate at 30°C in a biochemical incubator for 2-3 days.

[0077] 5) Use the primers Int1-F1 / R1 or Int6-F1 / R1 in Table 3 to screen positive clones by colony PCR. After sequencing the PCR products, the strain is constructed.

[0078] Example 3: Enzyme activity assay of alkaline proteinase K mutant

[0079] In this embodiment, the strain constructed in Example 2 was cultured in shake flasks to obtain a crude enzyme solution, and the enzyme activity of the crude enzyme solution was measured. This included the following:

[0080] 3.1 Shake flask culture

[0081] 1) The recombinant Pichia pastoris strain carrying the alkaline proteinase K mutant gene obtained in Example 2 was inoculated into a glass test tube containing 5 mL of YPD medium and cultured at 30 °C and 220 rpm / min for about 12 hours.

[0082] 2) Then, the OD of the recombinant Pichia pastoris strain cultured in step 1) 600 Adjust the value to 2 to obtain a cell suspension. Transfer 1 mL of the cell suspension to a shake flask containing 50 mL of YPD medium and continue culturing at 30 °C and 220 rpm / min for 72 hours to obtain crude enzyme solution (PD). Add 5 mL of 20% glucose every 24 hours.

[0083] Meanwhile, a wild-type alkaline proteinase K group (WT) was established, and its expression, shake-flask culture, etc. were the same as those of the mutant.

[0084] 3.2 Enzyme activity assay

[0085] To measure the activity of alkaline proteinase K, N-succinyl-L-phenylalanine-p-nitroaniline (Suc-phe-pNA) was selected as the chromogenic substrate for alkaline proteinase K. After alkaline proteinase K cleaves Suc-phe-pNA, it releases p-nitroaniline (pNA). The activity of alkaline proteinase K can be quantitatively determined by colorimetric detection at 405 nm.

[0086] The total volume of the reaction solution was 200 μL, which contained 2 mM of Suc-phe-pNA substrate, 50 mM of Tris-HCl (pH 8.0) and 10 μL of crude enzyme solution.

[0087] The reaction was carried out at 37°C for 1.5 hours, and the activity of alkaline proteinase K was determined by measuring the absorbance at 405 nm. One enzyme activity unit (U / mL) is defined as the amount of enzyme that increases by 1 μmol pNA per minute at 37°C. Enzyme activity results are shown below. Figure 3 .

[0088] from Figure 3 It was found that both wild-type alkaline protease (WT) and mutant alkaline protease (PD) could be secreted and expressed in Pichia pastoris. The enzyme activities of the crude enzyme solution after shake-flask fermentation were 0.000529 U / mL and 0.000637 U / mL, respectively. The enzyme activity of mutant alkaline protease (PD) was 20.4% higher than that of wild-type alkaline protease (WT).

[0089] Example 4: Determination of the thermal stability of alkaline proteinase K mutant

[0090] Wild-type alkaline protease (WT) and mutant alkaline protease (PD) were incubated in a PCR instrument at 65℃. Samples were taken at 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, and the enzyme activity of alkaline protease was measured at 37℃. The relative enzyme activity was calculated. The relative enzyme activity is the ratio between the enzyme activity of the sample treated at 65℃ for different times and the enzyme activity of the untreated sample. The trend of relative enzyme activity with incubation time is shown in the figure. Figure 4 The reaction system is the same as that used in the enzyme activity assay in Example 3, Section 3.2.

[0091] from Figure 4 It was found that during the entire incubation process, the relative activity of the mutant alkaline protease (PD) decreased significantly less than that of the wild-type alkaline protease over time, and the rate of decrease was relatively slow. Specifically, at 10 min of incubation, the relative activity of the wild-type alkaline protease (WT) had decreased significantly by 80.3%, while the relative activity of the mutant alkaline protease (PD) had only decreased by 25.7%. At 60 min of incubation, the relative activity of the wild-type alkaline protease (WT) had decreased significantly to 4.2%, while the relative activity of the mutant alkaline protease (PD) had decreased to 11.4%. However, at this time point, the relative activity of the mutant alkaline protease (PD) was still 2.7 times that of the wild-type alkaline protease (WT). In summary, this indicates that the thermostability of the mutant alkaline protease (PD) is significantly superior to that of the wild-type alkaline protease (WT).

[0092] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. A heat-resistant alkaline proteinase K mutant, characterized in that, Based on the amino acid sequence of wild-type alkaline proteinase K, the amino acid sequence of the mutant contains substitutions of P175C and D200C, and the amino acid sequence of wild-type alkaline proteinase K contains the sequence shown in SEQ ID No.

1.

2. The mutant as described in claim 1, characterized in that, The amino acid sequence of the mutant contains the sequence shown in SEQ ID No.

2.

3. An isolated polynucleotide, characterized in that, Encode the mutant as described in claim 1 or 2.

4. The polynucleotide as described in claim 3, characterized in that, It contains the nucleotide sequence shown in SEQ ID No.

3.

5. A nucleic acid construct, characterized in that, It contains the polynucleotide as described in claim 3 or 4.

6. A host cell, characterized in that, It comprises a nucleic acid construct as described in claim 5 or a genome in which the polynucleotide as described in claim 3 or 4 is integrated.

7. A composition, characterized in that, It includes the mutant as described in claim 1 or 2.

8. The use of the mutant of claim 1, the polynucleotide of claim 3, the nucleic acid construct of claim 5, the host cell of claim 6, or the composition of claim 7 in at least one of the following: 1) hydrolyzed protein; 2) as a feed additive or raw material for detergents; 3) food processing; 4) in recognizing the carboxyl terminus of phenylalanine and hydrolyzing the amide bond between phenylalanine and nitroaniline.

9. The application as described in claim 8, characterized in that, During hydrolysis, the substrate is selected from N-succinyl-L-phenylalanine-P-nitro-aniline.

10. A method for improving the thermal stability of alkaline proteinase K, characterized in that, Based on the amino acid sequence shown in SEQ ID No. 1, proline at position 175 was mutated to cysteine ​​and aspartic acid at position 200 was mutated to cysteine.

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