Glycosylation-free human enterokinase mutant as well as recombinant expression and application thereof

Abstract

The invention discloses a glycosylation-free human enterokinase mutant as well as recombinant expression and application thereof, belongs to the field of genetic engineering and protein technical engineering, and solves the problems of disordered glycosylation modification, low enzymatic activity and low protein expression quantity during recombinant expression of enterokinase in an enterokinase microbiological cell factory. The method comprises the following steps: firstly, carrying out single-site mutation, double-site mutation, three-site mutation and four-site mutation on the basis of a human enterokinase wild type, and constructing a pichia pastoris cell factory to obtain a series of human enterokinase mutants without glycosylation modification. Through calculation design and rational transformation, partial or non-glycosylation is subjected to deep fixed-point transformation, and pichia pastoris cell factory expression is constructed, so that the expression quantity and the total enzyme activity of the mutant are greatly improved. The highest enzyme activity of the glycosylation-free enterokinase mutant generated by fed-batch fermentation in a pichia pastoris cell factory reaches 7.8 * 10 < 4 > U / L, and the enterokinase mutant has the capacity of large-scale industrial fermentation generation and application.

Description

Technical Field

[0001] The present invention relates to a glycosylation-free human enterokinase mutant and its recombinant expression and application, and belongs to the fields of genetic engineering and protein technology engineering and biotechnology. Background Art

[0002] Enterokinase (EK) is a serine protease found in the mammalian duodenum. It consists of two subunits, a light chain and a heavy chain, linked by a pair of intermolecular disulfide bonds. The light chain of EK expressed alone possesses biocatalytic activity. Enterokinase specifically recognizes the DDDDK sequence and acts on the carboxyl-terminal peptide bond of lysine, producing a peptide chain with a first amino acid identical to the native protein, with no residual extra amino acids. Compared to other specific protein-cleaving enzymes such as TEV protease, thrombin, and HRV3C protease, enterokinase cleaves proteins with a first amino acid identical to the native protein, leaving no extra amino acids. This specific, residue-free cleavage protease has a wide range of applications in the processing and production of proteins and peptides related to pharmaceuticals, food, and health supplements.

[0003] Because enterokinase can specifically and residue-freely cleave proteins, it has a wide range of applications in the biotechnology, pharmaceutical, and food industries. In biotechnology, enterokinase is widely used in the processing of recombinant proteins, including the removal of purification tags such as the His tag, as well as N-terminal fusion proteins such as thioredoxin (TrxA), heavy-chain binding protein (Bip), two-base endopeptidase (Kex2), and ubiquitin-like protein modifier (SUMO). Tag removal facilitates the production of high-quality protein products. In the pharmaceutical field, enterokinase is used in the production of monoclonal antibodies, vaccine proteins, and recombinant enzymes to remove non-native protein fragments to improve drug purity and activity. Enterokinase is also used in the production of insulin to cleave insulin precursors to obtain active insulin. In the food industry, enterokinase is used in cheese preparation and meat processing to improve the taste of food.

[0004] The production of enterokinase can be extracted from animal tissues, such as the duodenum of pigs and cattle, but the disadvantages are that it is easily contaminated by other proteolytic enzymes and that animal-derived products are very limited in the medical field. Using microbial fermentation, compared with animal sources, it is safer, has higher purity, and has the potential for large-scale industrial production and cost reduction. Currently, human enterokinase has been expressed in various microbial expression systems, such as Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and CHO cells, but the problems faced in different cell expression systems and the expression amount and enzyme activity of human enterokinase are different. The light chain of human enterokinase contains 4 pairs of disulfide bonds, which makes the heterologous expression of enterokinase in E. coli exist in the form of inactive inclusion bodies, and in vitro renaturation is required to obtain active enterokinase, but the in vitro renaturation process is complex and cannot be used for large-scale industrial production. On the contrary, using P. pastoris to express human enterokinase can obtain active enterokinase, but the expression amount is low, the enzyme activity is low, and it is difficult to meet the requirements of industrial production. In addition, due to the endogenous glycosylation modification of P. pastoris, the enterokinase carries high-mannose type and non-uniform glycosylation modification from P. pastoris, which affects the protein purity and application safety in drug protein processing and other application scenarios. SUMMARY

[0005] To solve the above problems, the present application designs human enterokinase by rapid site-directed mutagenesis, sequence alignment, and analysis of mutant amino acid types and distribution characteristics, and uses a yeast cell factory for secretory expression to obtain enterokinase mutants with significantly reduced or completely eliminated glycosylation modification, while improving the expression amount and enzyme activity of the enzyme. Such mutants can be used for hydrolytic cleavage and processing of recombinant proteins containing the DDDDK sequence.

[0006] The first technical solution provided by the present application is a human enterokinase mutant, which is a human enterokinase parent with an amino acid sequence as shown in SEQ ID NO. 1, and at least one mutation as follows:

[0007] (1) mutating the 64th asparagine to glutamine;

[0008] (2) mutating the 103rd asparagine to glutamine;

[0009] (3) mutating the 125th asparagine to glutamine;

[0010] (4) mutating the 165th asparagine to glutamine.

[0011] Specifically, the four N-glycosylation site asparagines Asn of the wild-type human enterokinase are mutated to structurally similar glutamines Gln, including the following mutation combinations:

[0012] (1) single-site mutations N64Q, N103Q, N125Q, N165Q;

[0013] (2) double-site mutations N64Q / N103Q, N64Q / N125Q, N64Q / N165Q, N103Q / N125Q, N103Q / N165Q, N125Q / N165Q;

[0014] (3) triple-site mutations N64Q / N103Q / N125Q, N64Q / N103Q / N125Q, N64Q / N125Q / N165Q, N103Q / N125Q / N165Q;

[0015] (4) quadruple-site mutations N64Q / N103Q / N125Q / N165Q.

[0016] In an embodiment: the amino acid sequence of the wild-type human enterokinase is shown as SEQ ID NO. 1, the amino acid sequence of the single-site mutation is shown as SEQ ID NO. 2-5, the amino acid sequence of the double-site mutation is shown as SEQ ID NO. 6-11, the amino acid sequence of the triple-site mutation is shown as SEQ ID NO. 12-15, and the amino acid sequence of the quadruple-site mutation is shown as SEQ ID NO. 16.

[0017] The second technical solution provided by the present application is a human enterokinase mutant protein, which is a S5Q, G17A, L24H, E49D, I56Y, M61T, K62Q, T70V, L74P, E77Q, V79I, F100S, K101P, Y104F, E116P, V134T, V135S, T139P, N142D, R156Q single-point mutation or combined mutation of the human enterokinase mutant of the first technical solution.

[0018] In some embodiments, the sequence alignment is determined by analyzing the amino acid sequence of human enterokinase to determine which protein family it belongs to, and then performing sequence alignment on the amino acid sequences of the proteins in the family, and then analyzing the amino acid properties and position distribution. According to the amino acid position-specific scoring matrix and the evolutionary consensus sequence, multiple-site synergistic mutation is carried out to improve the expression level of the completely sugar-free plan modified N64Q / N103Q / N125Q / N165Q, and obtain N64Q / N103Q / N125Q / N165Q-EKP1, N64Q / N103Q / N125Q / N165Q-EKP2, N64Q / N103Q / N125Q / N165Q-EKP3, N64Q / N103Q / N125Q / N165Q-EKP4, N64Q / N103Q / N125Q / N165Q-EKP5 mutants.

[0019] In some embodiments, the amino acid sequence of the human enterokinase mutant protein is as shown in any one of SEQ ID NO. 17-21.

[0020] The third technical solution provided by the present application is a gene encoding the human enterokinase mutant of the first technical solution or the human enterokinase mutant protein of the second technical solution.

[0021] The fourth technical solution provided by the present application is a recombinant plasmid carrying the gene of the third technical solution.

[0022] In some embodiments, the expression vector of the recombinant plasmid includes but is not limited to pPIC9K, pPICZα, pHIL-S1, pGAPZα, pPIC3.5K, pPICZ, pHIL-D2, pGAPZ.

[0023] The fifth technical solution provided by the present application is a recombinant cell expressing the human enterokinase mutant of the first technical solution or the human enterokinase mutant protein of the second technical solution, or containing the gene of the third technical solution, or transformed with the recombinant plasmid of the fourth technical solution.

[0024] In some embodiments, the recombinant cell takes Pichia pastoris as the host.

[0025] Further, the Pichia pastoris is Pichia pastoris GS115 (K. phaffii GS115).

[0026] The sixth technical solution provided by the present application is a recombinant yeast bacterium, which takes Pichia pastoris GS115 as the host and takes plasmid pPIC9K as the expression vector to express the human enterokinase mutant of the first technical solution or the human enterokinase mutant protein of the second technical solution.

[0027] The seventh technical solution provided by the present application is a method for producing recombinant enterokinase, which is to perform fermentation culture and induction expression by using the recombinant cell of the fifth technical solution or the recombinant yeast bacterium of the sixth technical solution to obtain a fermentation liquor containing recombinant enterokinase.

[0028] In some embodiments, the induction expression condition is to perform induction expression at 30℃ and 220rpm for 96h, and to add 1% methanol every 24h.

[0029] In some embodiments, the enterokinase in the fermentation broth is purified under the following conditions: A solution: 25 mM Tris-HCl, 300 mM NaCl, 25 mM imidazole; B solution: 25 mM Tris-HCl, 150 mM NaCl, 300 mM imidazole; dialysis solution: 25 mM Tris-HCl, 150 mM NaCl.

[0030] The eighth technical solution provided by the present application is the use of the human enterokinase mutant of the first technical solution, or the human enterokinase mutant protein of the second technical solution, or the gene of the third technical solution, or the recombinant plasmid of the fourth technical solution, or the recombinant cell of the fifth technical solution, or the recombinant yeast of the sixth technical solution in the preparation of enterokinase or an enterokinase-containing product.

[0031] The present application also provides the use of the human enterokinase mutant of the first technical solution, or the human enterokinase mutant protein of the second technical solution, or the gene of the third technical solution, or the recombinant plasmid of the fourth technical solution, or the recombinant cell of the fifth technical solution, or the recombinant yeast of the sixth technical solution, or the method of the seventh technical solution in the field of biological industry and medicine:

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1. The present application first constructs human enterokinase without glycosylation, enhances its application in the field of biological industry and medicine, and avoids the problem of immunogenicity.

[0034] 2. The present application obtains a mutant with two glycosylation sites by rapid site-directed mutagenesis deglycosylation, and the total enzyme activity is 2.5×10 4 U / L, which is 4 times that of the wild-type human enterokinase; a mutant with one glycosylation site is obtained, and the specific enzyme activity is 3.6×10 3 U / mg, which is 10 times that of the wild-type human enterokinase.

[0035] 3. The present application uses point mutation through sequence alignment and attribute and position distribution of mutant amino acids, so that the expression amount of the human enterokinase mutant protein without glycosylation reaches 163.8 mg / L, which is 9.8 times that of the wild-type human enterokinase; the total enzyme activity reaches 1.1×10 4 U / L, which is 1.8 times that of the wild-type human enterokinase. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Figure 1 is a diagram of predicting glycosylation sites for the wild-type amino acid sequence of human enterokinase.

[0037] Figure 2 Figure for alignment of wild type amino acid sequence of human enterokinase.

[0038] Figure 3 Figure for enterokinase wild type and mutant protein body protein gel.

[0039] Figure 4 Figure for enterokinase wild type and mutant protein body total enzyme activity.

[0040] Figure 5 Figure for batch-fed fermentation results of mutant N64Q / N103Q / N125Q / N165Q-EKP5 of human enterokinase. DETAILED DESCRIPTION

[0041] The following describes the preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0042] Test method:

[0043] Enzyme activity determination of enterokinase: enzyme activity determination was carried out in 0.18 mL buffer at 37°C, the buffer contained 0.6 mM GD4K-β naphthylamine (GDDDDK-β naphthylamine), 25 mM Tris-HCl (pH 8.4), 10% DMSO and 10 mM CaCl2, and the reaction was started by adding a trace amount of enterokinase. The formation rate of β-naphthylamine was determined by continuously monitoring the fluorescence increment (λex=337 nm and λem=420 nm) for 100 seconds using a fluorescence photometer. The specific steps were as follows: 0.18 mL buffer was taken in a black 96-well plate, and it was placed in a 37°C enzyme marker for 1 min, then 0.02 mL of enzyme solution of different dilution multiples (according to the activity of the sample, to prevent the fluorescence signal from exceeding the range) was added, and the fluorescence value was immediately measured by the enzyme marker as a function of time.

[0044] Raw materials used in the examples:

[0045] Strain: Pichia pastoris GS115 (K. phaffii GS115), plasmid pPIC9K, a commercialized plasmid.

[0046] YPD medium: 10 g / L of yeast powder, 20 g / L of peptone, 20 g / L of glucose.

[0047] BMGY medium: 10 g / L of yeast powder, 20 g / L of peptone, 3.6 g / L of K2HPO4.3H2O, 11.8 g / L of KH2PO4, 4×10 -4 g / L of biotin, 10 g / L of glycerol.

[0048] BMMY medium: yeast powder 10 g / L, peptone 20 g / L, K2HPO4.3H2O 3.6 g / L, KH2PO4 11.8 g / L, biotin 4 x 10 -4 g / L, methanol 10 g / L.

[0049] The above reagents are as follows: yeast powder and peptone are from Oxoid (UK) Company imported by Thermo Fisher Scientific (China) Co., Ltd., biotin is from Shenguo Bioengineering (Shanghai) Co., Ltd., and the rest of the reagents are from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0050] The primer information is shown in Table 1.

[0051] Table 1. Primer and sequence

[0052]

[0053]

[0054] Example 1: Construction of recombinant plasmid pPIC9K-HLEK and recombinant Pichia pastoris GS115-pPI-HLEK (K. phaffii GS115-pPI-HLEK)

[0055] The human enterokinase gene HLEK light chain amino acid sequence (IVGGSNAKEGAWPWVVGLYYGGRLLCGASLVSSDWLVSAAHCVYGRNLEPSKWTAILGLHMKSNLTSPQTVPRLIDEIVINPHYNRRRKDNDIAMMHLEFKVNYTDYIQPICLPEENQVFP PGRNCSIAGWGTVVYQGTTANILQEADVPLLSNERCQQQMPEYNITENMICAGYEEGGID SCQGDSGGPLMCQENNRWFLAGVTSFGYKCALPNRPGVYARVSRFTEWIQSFLH) was searched in the UniProt database, and the sequence was then expressed in Pichia pastoris GS115 (K.phaffii GS115) as host, the human wild-type enterokinase gene sequence is obtained by gene synthesis and codon optimization, and is represented by ATTGTTGGTGGTTCTAACGCTAAGGAAGGTGCTTGGCCTTGGGTTGTTGGTTTGTATTATGGTGGTAGATTGTTGTGTGGTGCTTCTTTGGTTTCTTCTGATTGGTTGGTTTCTGCTGCTCATTGTGTTTACGGTAGAAACTTGGAACCATCTAAATGGACTGCTATTTTGGGTTTGCATATGAAGTCTAACCTGACTTCTCCACAAACTGTTCCAAGATTGATTGATGAAATCGTCATCAACCCTCATTACAATAGAAGAAGAAAGGACAACGACATCGCTATGATGCATTTGGAATTTAAGGTCAACTACACCGATTACATCCAACCAATTTGTTTGCCAGAAGAAAACCAAGTTTTCCCACCAGGTAGAAATTGTTCTATTGCTGGTTGGGGTACTGTTGTTTATCAAGGTACTACTGCTAACATCTTGCAAGAAGCTGATGTTCCTTTGTTGTCTAACGAAAGATGTCAACAACAGATGCCAGAATACAATATCACTGAAAACATGATCTGCGCTGGTTATGAAGAAGGTGGTATTGATTCTTGTCAGGGTGATTCTGGTGGTCCATTGATGTGTCAAGAAAACAACAGATGGTTCTTGGCTGGTGTTACTTCTTTTGGTTACAAGTGTGCTTTGCCAAACAGACCAGGTGTTTACGCTAGAGTTTCTAGATTTACTGAGTGGATTCAGTCTTTCTTGCAT (SEQ ID NO. 22).

[0056] The pPIC9K expression plasmid of Pichia pastoris GS115 is double-enzymatically cut with BamH I and EcoR I, and the HLEK gene fragment is one-step connected to the cut pPIC9K by using the Gibson Assembly kit to obtain the recombinant plasmid pPIC9K-HLEK.

[0057] The plasmid pPIC9K-HLEK was transformed into Pichia pastoris GS115 by electroporation. The conditions were as follows: voltage 1.5 kV, 5 ms (the width of the shock cup was 1 mm), and repeated once. The recombinant bacteria were screened on MD plates containing biotin 4 x 10 -4 g / L, YNB 13.4 g / L, glucose 20 g / L, and agar powder 15 g / L. The recombinant bacteria were named K. phaffii GS115-pPI-HLEK.

[0058] Example 2: Construction of mutant plasmids with single-site mutation, double-site mutation, triple-site mutation, and four-site mutation by site-directed mutation strategy

[0059] By analyzing the wild-type HLEK amino acid sequence, it was found that the amino acid sequence of HLEK was as follows: Figure 1As shown, the potential glycosylation sites were found to be 64, 103, 125 and 165. In order to remove the glycosylation, the above glycosylation sites were mutated from Asn to Gin by designing primers N64Q-F / N64Q-R, N103Q-F / N103Q-R, N125Q-F / N125Q-R and N165Q-F / N165Q-R, and the base sequences of the primers were shown in Table 1. The four glycosylation sites were combined to obtain N64Q, N103Q, N125Q, N165Q single-site mutants, and the amino acid sequences were shown in SEQ ID NO. 2-5; N64Q / N103Q, N64Q / N125Q, N64Q / N165Q, N103Q / N125Q, N103Q / N165Q, N125Q / N165Q double-site mutants, and the amino acid sequences were shown in SEQ ID NO. 6-11; N64Q / N103Q / N125Q, N64Q / N103Q / N125Q, N64Q / N125Q / N165Q, N103Q / N125Q / N165Q triple-site mutants, and the amino acid sequences were shown in SEQ ID NO. 12-14; N64Q / N103Q / N125Q / N165Q quadruple-site mutant, and the amino acid sequence was shown in SEQ ID NO. 15. The above obtained gene fragments were ligated with the vector pPIC9K according to the method of Example 1, and then transformed into E. coli JM109 competent cells.The transformants were selected for plasmid sequencing reaction. After successful sequencing alignment, they were transformed into Pichia pastoris GS115 by electroporation according to the method of Example 1, and K. phaffii GS115-pPI-N64Q, K. phaffii GS115-pPI-N103Q, K. phaffii GS115-pPI-N125Q, K. phaffii GS115-pPI-N165Q, K. phaffii GS115-pPI-N64Q / N103Q, K. phaffii GS115-pPI-N64Q / N125Q, K. phaffii GS115-pPI-N64Q / N165Q, K. phaffii GS115-pPI-N103Q / N125Q, K. phaffii GS115-pPI-N103Q / N165Q, K. phaffii GS115-pPI-N125Q / N165Q, K. phaffii GS115-pPI-N64Q / N103Q / N125Q, K. phaffii GS115-pPI-N64Q / N103Q / N125Q, K. phaffii GS115-pPI-N64Q / N125Q / N165Q, K. phaffii GS115-pPI-N103Q / N125Q / N165Q, K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q were obtained.

[0060] Example 3: Construction of mutant plasmids obtained by sequence alignment

[0061] Firstly, the wild-type human enterokinase HLEK was analyzed on the interpro website to belong to the trypsin family, and then the proteins belonging to this family were downloaded from the NCBI website. Sequence alignment was performed using PROMALS3D, and a phylogenetic tree was constructed using iTOL. Further screening of proteins with closer genetic relationship to wild-type human enterokinase was performed through the phylogenetic tree, and sequence alignment was performed again using PROMALS3D to analyze the site conservation. Amino acid position-specific scoring matrix and evolutionary consensus sequence were obtained, and position-specific scoring matrix (PSSM) was obtained. WebLogo was used for mapping analysis of mutation site conservative amino acid residues. The conservative amino acid residues of the mutation site were analyzed in terms of attributes and structural position, and the amino acid residues near the active center of the enzyme were excluded, and the rationality of their mutation was analyzed. Figure 2According to the amino acid position-specific scoring matrix and evolutionary consensus sequence, multi-site synergistic mutation is carried out to improve the expression level of completely aglycosylation modified N64Q / N103Q / N125Q / N165Q. The mutation sites are introduced into primers by PCR, and multi-round combination mutation PCR is carried out to obtain N64Q / N103Q / N125Q / N165Q-EKP1, N64Q / N103Q / N125Q / N165Q-EKP2, N64Q / N103Q / N125Q / N165Q-EKP3, N64Q / N103Q / N125Q / N165Q-EKP4, N64Q / N103Q / N125Q / N165Q-EKP5. The N64Q / N103Q / N125Q / N165Q-EKP1 mutant contains S5Q, E49D, I56Y, K62Q, L74P, K101P, T139P mutations, the N64Q / N103Q / N125Q / N165Q-EKP2 mutant contains S5Q, E49D, I56Y, K62Q, L74P, K101P, T139P, G17A, L24H, E77Q, V79I, V135S mutations, the mutant N64Q / N103Q / N125Q / N165Q-EKP3 mutant contains S5Q, E49D, I56Y, K62Q, L74P, K101P, T139P, G17A, L24H, E77Q, V79I, V135S, N142D, R156Q mutations, the mutant N64Q / N103Q / N125Q / N165Q-EKP4 mutant contains S5Q, G17A, L24H, E49D, I56Y, M61T, K62Q, T70V, L74P, E77Q, V79I, F100S, K101P, Y104F, E116P, V134T, V135S, T139P, N142D, R156Q mutations, and the mutant N64Q / N103Q / N125Q / N165Q-EKP5 contains S5Q, G17A, L24H, E49D, I56Y, M61T, K62Q, T70V, L74P, E77Q, V79I, F100S, K101P, Y104F, E116P, V134T, V135S, T139P mutations.

[0062] The gene fragment obtained above was ligated with vector pPIC9K according to the method of Example 1, and then transformed into competent cells of E. coli JM109. The transformants were selected for plasmid sequencing reaction. After successful sequencing by sequence alignment, they were electroporated into P. pastoris GS115 according to the method of Example 1, to obtain K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q-EKP1, K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q-EKP2, K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q-EKP3, K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q-EKP4, and K. phaffii GS115-pPI-N64Q / N103Q / N125Q / N165Q-EKP5.

[0063] Example 4: Fermentation of recombinant P. pastoris GS115 to synthesize human wild-type enterokinase and mutants thereof

[0064] The strains constructed in Examples 1-3 were activated on YPD solid plates, and incubated in an inverted manner in a 30°C constant-temperature incubator for 48-72 hours. The single colonies activated on the YPD plates were inoculated into 15-20 mL of fresh liquid YPD medium, and incubated at 30°C and 220 rpm for 16-18 hours. The cultured bacterial liquid was inoculated into 45 mL of fresh sterile BMGY liquid medium at an inoculation amount of 10% (V·V-1), and incubated at 30°C and 220 rpm for 24 hours.

[0065] The cultured bacterial liquid was centrifuged at 4°C and 4000 rpm for 10 minutes, and the supernatant was discarded. The bacterial cells were washed once with 30 mL of sterile water. The bacterial cells were resuspended with fresh sterile BMMY medium, and the volume of the bacterial liquid was kept unchanged before and after the medium was replaced. The expression was induced at 30°C and 220 rpm for 96 hours, and 1% pure methanol was added every 24 hours.

[0066] The results are shown in Table 1. Figure 3 As shown in Table 1, it is indicated that the aglycosylated enterokinase mutants were successfully obtained by mutation, and protein bands with single bands were obtained (because all glycosylation modification processes are not uniform, and protein bands will appear diffuse tailing phenomenon as long as there is glycosylation).

[0067] Example 5: Purification and enzyme activity determination of human enterokinase and wild-type thereof

[0068] The fermentation broth obtained in Example 4 was centrifuged at 4500 rpm and 4°C for 20 min to obtain a supernatant. Enterokinase was subsequently purified by nickel column affinity chromatography. First, the 20% ethanol in the nickel column was removed, and then the nickel column was washed with 10 mL of ultrapure water. The nickel column was then balanced with 10 mL of liquid A. The supernatant obtained by the above centrifugation was passed through the nickel column, and then the impurities were washed away with 10 mL of liquid A. Finally, enterokinase was eluted with 2.5 mL of liquid B. The enterokinase solution obtained after elution was placed in a dialysis bag, dialyzed in the dialysate to remove imidazole, and used for subsequent enzyme activity determination.

[0069] Enterokinase activity was assayed in a 200 μL black 96-well plate. The reaction system consisted of 180 μL of buffer containing 0.6 mM GD4K-β-naphthylamine, 25 mM Tris-HCl (pH 8.4), 10% DMSO, and 10 mM CaCl₂, and 20 μL of a specific concentration of enterokinase. The activity was determined by continuously monitoring the fluorescence increment (λex = 337 nm and λem = 420 nm) for 100 seconds using a fluorometer.

[0070] The results are as follows Figure 4 As shown, the total enzyme activity of the N64Q / N103Q / N125Q / N165Q-EKP5 mutant obtained by introducing mutations through multiple rounds of PCR using primers carrying mutation sites was increased by 1.8 times compared with the wild type.

[0071] Example 6: Synthesis of the non-glycosylated enterokinase mutant N64Q / N103Q / N125Q / N165Q-EKP5 by Pichia pastoris batch-fed-batch fermentation

[0072] The K. phaffii GS115-pPIC9k-N64Q / N103Q / N125Q / N165Q-EKP5 engineered bacteria in Example 3 were subjected to a 5 L fed-batch fermentation, specifically comprising the following steps: first, the frozen engineered strain was thawed and activated and cultured on a YPD solid plate at 30°C for 48 h. A single clone was picked and inoculated into 20 mL of liquid YPD medium and cultured at 30°C and 220 rpm for 20 h to serve as the primary seed; then, 5% (v / v) was inoculated into 95 mL of YPD medium and cultured at 30°C and 220 rpm for 24 h to obtain the secondary seed.

[0073] Fermentation was carried out by inoculating 10% (v / v) secondary seeds into 2.25 L of BSM medium at 30°C, pH 5.5 (adjusted with ammonia), and maintaining dissolved oxygen below 30%. The first stage was a glycerol batch fermentation. After glycerol depletion, the second stage began with a 12-h feed of 556 mL of 50% glycerol containing 2.4% PTM1. The fermentation speed was adjusted to 700 rpm, with an aeration rate of 3 vvm. Following the feeding phase, a 2-h starvation phase was followed. The third stage was methanol induction, using methanol containing 2.4% PTM1 at a speed of 700–800 rpm and an aeration rate of 3–4 vvm. Dissolved oxygen was maintained below 30%. The total induction duration was 180 h. Samples were collected every 12 h for analysis of OD600, cell mass, and protein concentration.

[0074] The results are as follows Figure 5 As shown in the figure, the mutant N64Q / N103Q / N125Q / N165Q-EKP5 was obtained by introducing mutations through multiple rounds of PCR using primers carrying mutation sites. After fed-batch fermentation, the total enzyme activity reached 7.8×10 4 U / L, has the ability to produce and apply in large-scale industrial fermentation.

[0075] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A human enterokinase mutant, characterized in that: The mutant is a human enterokinase parent with an amino acid sequence as shown in SEQ ID NO.1, with at least one of the following mutations: (1) Mutate asparagine at position 64 to glutamine; (2) mutating asparagine at position 103 to glutamine; (3) mutating asparagine at position 125 to glutamine; (4) Mutate asparagine at position 165 to glutamine.

2. A human enterokinase mutant protein, characterized in that: The human enterokinase mutant protein is a human enterokinase mutant according to claim 1 subjected to single point mutations or combined mutations of S5Q, G17A, L24H, E49D, I56Y, M61T, K62Q, T70V, L74P, E77Q, V79I, F100S, K101P, Y104F, E116P, V134T, V135S, T139P, N142D, and R156Q.

3. The human enterokinase mutant protein according to claim 2, characterized in that The amino acid sequence of the human enterokinase mutant protein is shown in any one of SEQ ID NOs. 17 to 21.

4. A gene encoding the human enterokinase mutant according to claim 1 or the human enterokinase mutant protein according to claim 2 or 3.

5. A recombinant plasmid carrying the gene according to claim 4.

6. A recombinant cell expressing the human enterokinase mutant according to claim 1 or the human enterokinase mutant protein according to claim 2 or 3, or containing the gene according to claim 4, or transformed with the recombinant plasmid according to claim 5.

7. The recombinant cell according to claim 6, characterized in that The recombinant cell uses Pichia pastoris as a host.

8. A recombinant yeast, characterized in that The recombinant yeast uses Pichia pastoris (Komagataellaphaffii) GS115 as a host and plasmid pPIC9K as an expression vector to express the human enterokinase mutant according to claim 1 or the human enterokinase mutant protein according to claim 2 or 3.

9. A method for producing recombinant enterokinase, characterized in that: The method comprises the steps of using the recombinant cell according to claim 6 or 7 or the recombinant yeast according to claim 8 to perform fermentation culture and induce expression to obtain a fermentation liquid containing recombinant enterokinase.

10. Use of the human enterokinase mutant according to claim 1, or the human enterokinase mutant protein according to claim 2 or 3, or the gene according to claim 4, or the recombinant plasmid according to claim 5, or the recombinant cell according to claim 6 or 7, or the recombinant yeast according to claim 8 in the preparation of enterokinase or a product containing enterokinase.

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