Self-cutting-resistant and high-specific-activity chymotrypsin mutant

Abstract

The invention relates to a self-cutting-resistant and high-specific-activity chymotrypsin mutant, in particular to a self-cutting-resistant and high-specific-activity chymotrypsin mutant obtained through molecular modification and screening and a coding gene and application of the self-cutting-resistant and high-specific-activity chymotrypsin mutant, and belongs to the technical field of protein and gene engineering. The chymotrypsin mutant provided by the invention is obtained by mutation of at least one of the following sites on the basis of an amino acid sequence as shown in SEQ ID NO.1: L97A, L162I, F71I, L83I, F114I and W207H. Compared with original chymotrypsin, the chymotrypsin has better self-cutting resistance and higher specific activity, compared with wild type chymotrypsin, the self-cutting resistance is improved by 82% at most, the specific enzyme activity is improved by 37% at most, and the problem that the activity is easily reduced due to self-cutting in the storage and use process of the chymotrypsin is solved.

Description

Technical field:

[0001] The invention relates to a chymotrypsin self-cleavage-resistant and high-specific activity mutant obtained by molecular modification and screening, and its encoding gene and application, belonging to the technical field of protein and genetic engineering. Background technology:

[0002] Chymotrypsin (EC 3.4.21.1), also known as chymotrypsin, belongs to the serine protease family and is widely used in food processing, biocatalysis, biotechnology and medicine. Chymotrypsin can hydrolyze casein in milk and dairy products to solve the problem of protein allergy; Chymotrypsin can efficiently catalyze substrates to generate products, which is both environmentally friendly and simple; Chymotrypsin can be used as an anti-inflammatory agent to reduce tissue damage caused by inflammation, and can also be used to treat symptoms such as indigestion, nutritional deficiencies and abdominal distension. Chymotrypsin can hydrolyze proteins into peptides and is an important part of protein processing in proteomics analysis.

[0003] Traditionally, chymotrypsin is mainly isolated and extracted from the pancreas of animals, and its source and quality are limited. Various pancreatic proteases have similar properties, and separation and purification are difficult and costly. The use of genetic engineering technology to express and prepare chymotrypsin through microbial recombinant expression can increase its yield and purity, reduce the difficulty of separation and production costs. Animal-derived proteins can be expressed through prokaryotic expression systems or eukaryotic expression systems. The commonly used host bacteria for prokaryotic expression systems are Escherichia coli, which has the advantages of short cycles, low costs, and a variety of expression vectors. However, it also has disadvantages such as difficulty in inclusion body denaturation and protein renaturation, difficulty in purification, and difficulty in expressing the target protein in large quantities and continuously. Animal-derived chymotrypsin is more suitable for preparation and production using eukaryotic expression systems, and Pichia pastoris is currently the most commonly used and most complete host for eukaryotic expression systems.

[0004] Chymotrypsin specifically hydrolyzes peptide bonds with carboxyl ends of tyrosine, tryptophan, phenylalanine and leucine, and has strong amino acid site specificity. Chymotrypsin can not only hydrolyze other protein molecules, but also attack the amino acid sites on its own surface, affecting the stability of the enzyme, resulting in a decrease in enzyme activity, and thus affecting its application efficiency and level. Improving the anti-self-cleavage ability of chymotrypsin through molecular modification helps to improve its stability and hydrolysis efficiency.

[0005] In view of the problems of natural chymotrypsin being easily self-cleaved and inactivated, difficult to purify and having high production costs, obtaining new enzyme molecules with better performance through microbial recombination expression and molecular mutation modification is of great significance for promoting its application in scientific research, medicine and food. Summary of the invention:

[0006] In order to solve the technical problem that chymotrypsin is easily inactivated by self-cleavage, the present invention performs site-directed mutagenesis on the tyrosine, tryptophan, phenylalanine and leucine sites in the bovine chymotrypsin sequence (the amino acid sequence of wild bovine chymotrypsin is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2). Studies have confirmed that the obtained chymotrypsin mutant significantly improves the ability to resist self-cleavage and also has a higher specific activity. These advantages enable it to be better applied in the fields of medicine, scientific research, etc.

[0007] The purpose of the present invention is to obtain a chymotrypsin mutant with improved anti-self-cleavage ability and activity. Based on the wild chymotrypsin sequence SEQ ID NO.1, the present invention selects its tyrosine, tryptophan, phenylalanine and leucine sites as target mutation sites, and performs site-directed mutagenesis by reverse PCR technology. Single-point mutation is performed on the target mutation site, and recombinant Pichia pastoris is used for heterologous expression. By measuring the anti-self-cleavage ability and specific activity of the mutant enzyme, 9 single-point mutants that can effectively improve the anti-self-cleavage ability and specific activity are obtained, which are named MT1, MT2, MT3, MT4, MT5, MT6, MT7, MT8, and MT9, respectively. The mutation sites are shown in Table 1.

[0008] Table 1 Mutation site comparison table

[0009]

[0010] The constructed mutant engineering strain ferments and synthesizes chymotrypsinogen. After the zymogen is activated by trypsin, chymotrypsin is prepared by ultrafiltration concentration and purification. The present invention uses N-benzoyl-L-tyrosine-ethyl-ester (BTEE) as a substrate and measures the enzyme activity by measuring the change rate of absorbance at a wavelength of 256nm using an ultraviolet spectrophotometer. The residual enzyme activity rate after incubation at 37°C for 6 hours is used as an evaluation index for the anti-self-cleavage performance of chymotrypsin.

[0011] The initial specific activities of the single point mutants MT1, MT2, MT3, MT4, MT5, MT6, MT7, MT8 and MT9 were 1.23 times, 1.37 times, 0.89 times, 0.31 times, 0.78 times, 0.39 times, 0.18 times, 0.14 times and 0.12 times that of the wild-type chymotrypsin, respectively, indicating that the single point mutants MT1 and MT2 effectively improved the specific activity; the 6h enzyme activity residual rates were 1.62 times, 1.70 times, 0.71 times, 1.05 times, 1.82 times, 1.60 times, 1.68 times, 0 times and 0 times that of the wild-type chymotrypsin, respectively, indicating that the single point mutants MT1, MT2, MT4, MT5, MT6 and MT7 had different degrees of improvement in anti-self-cleavage ability compared with the wild-type enzyme molecules, and the point mutants MT1 and MT2 had both improved specific activity and anti-self-cleavage ability.

[0012] The present invention also provides a recombinant vector comprising the gene encoding the chymotrypsin mutant having improved anti-self-cleavage ability and specific activity;

[0013] Furthermore, the expression vector used by the recombinant vector can be pHBM905A, pPIC9K, pPIC9K-His, pPIC3.5K, pPIC9, pPICZαA, pAO815, pPIC9k-His, pHIL-S1, pPink hc; pGADT7, pGBKT7, pWB980, pT3, etc.;

[0014] Preferably, the expression vector is pPIC9K;

[0015] The present invention also provides a recombinant strain comprising the above-mentioned chymotrypsin mutant encoding gene with improved anti-self-cleavage ability and specific activity;

[0016] Furthermore, the host cell used by the recombinant strain can be Pichia pastoris hosts X33, KM71, KM71H, GS115; Saccharomyces cerevisiae strains YM4271, AH109, ​​Y187, Y190; E. coli BL 21, E. coli X90; Bacillus subtilis SCK6, etc.;

[0017] Preferably, the host cell is Pichia pastoris GS115.

[0018] The present invention also provides a method for preparing the above-mentioned chymotrypsin mutant with improved anti-self-cleavage ability and specific activity:

[0019] (1) Shake flask fermentation of recombinant Pichia pastoris

[0020] The inoculated strain was cultured in YPD liquid medium at 30°C and 200 rpm for 24 h; the seed solution cultured in YPD liquid medium was inoculated with 2% inoculum into BMGY medium and cultured at 30°C and 200 rpm until the bacterial solution OD 600 2-6, then collect the bacteria; transfer to BMMY medium, then add 500 μL of methanol every 24 hours to induce culture for 108 hours;

[0021] (2) Activation of chymotrypsinogen

[0022] The fermentation supernatant was collected by centrifugation at 12000 rpm for 10 min, replaced with 50 mM ammonium bicarbonate containing 1 mM CaCl2 as buffer, and trypsin was added at a ratio of 500:1 between chymotrypsinogen and trypsin, and incubated at 37 °C for 4 h to activate chymotrypsinogen;

[0023] The chymotrypsin may also be a sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function;

[0024] The chymotrypsin may also be a chymotrypsin sequence from animals such as pigs, cattle, sheep and rabbits containing the same mutation sites as MT1-MT9.

[0025] The present invention also provides the application of the self-cleavage resistant high specific activity chymotrypsin mutant in the fields of food processing, biocatalysis, biotechnology and medicine.

[0026] Beneficial effects:

[0027] The mutant screened by the present invention has better anti-self-cleavage performance and higher specific activity than the original enzyme. Compared with the wild-type chymotrypsin, the anti-self-cleavage performance is improved by up to 82%, and the specific enzyme activity is improved by up to 37%, thereby improving the problem of easy self-cleavage and reduced activity of chymotrypsin during storage and use. Description of the drawings:

[0028] Figure 1 :Specific activities of wild-type chymotrypsin and its point mutants MT1-MT9

[0029] Figure 2 :Residual enzyme activity of wild chymotrypsin and its point mutants MT1-MT9 after incubation for 6 h Specific implementation method:

[0030] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0031] In the following examples, the processes and methods not described in detail are conventional methods commonly used in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

[0032] The following definitions are used in the present invention:

[0033] 1. Nomenclature of amino acid and DNA sequences

[0034] The generally accepted IUPAC nomenclature for amino acid residues is used, using the single-letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0035] 2. Identification of chymotrypsin mutants

[0036] The mutated amino acid in the chymotrypsin mutant is represented by "amino acid replaced by the original amino acid position", such as L97A, which means that the amino acid at position 97 is replaced by A from the wild-type L. The position number corresponds to the amino acid sequence number of the wild-type chymotrypsin in SEQ ID NO: 1.

[0037] Experimental materials and reagents:

[0038] 1) Strains and vectors

[0039] Escherichia coli JM109 and Pichia pastoris GS115 were preserved in our laboratory and can be purchased through commercial channels; pMD19 (T-simple) vector was purchased from Invitrogen; pPIC9K vector was purchased from TaKaRa.

[0040] 2) Enzymes and kits

[0041] Plasmid extraction kit and genome extraction kit were purchased from Beijing Solebow Technology Co., Ltd.; restriction endonuclease was purchased from TaKaRa.

[0042] 3) Culture medium

[0043] The Escherichia coli culture medium was LB, the yeast seed liquid culture medium was YPD, the yeast enrichment culture medium was BMGY, and the yeast induction culture medium was BMMY.

[0044] The method for determining the activity of chymotrypsin used in the present invention is as follows:

[0045] The present invention uses N-benzoyl L-tyrosine ethyl ester (BTEE) as a substrate, and measures the enzyme activity by measuring the rate of change of absorbance at a wavelength of 256nm using an ultraviolet spectrophotometer. The specific method is: using 0.5mM BTEE as a substrate, at pH 7.8 and temperature 25°C, the reaction system is 3.0mL (optical path 1cm), and the change value of absorbance at 256nm is measured. Δ256nm, the amount of enzyme required to convert 1μM BTEE into BT per minute is defined as one enzyme activity unit. The chymotrypsin enzyme activity and specific activity are calculated by the following formulas:

[0046]

[0047] The present invention obtains the amino acid sequence of wild bovine chymotrypsinogen (Entry number: P00766, SEQ ID NO.1) through Uniprot database, sends the amino acid sequence to Jinweizhi Biotechnology Co., Ltd., selects Pichia pastoris as the expression host for codon optimization, and obtains the chymotrypsinogen Pichia pastoris optimized gene (SEQ ID NO.2). The chymotrypsinogen gene is amplified by PCR technology. The amplified product is purified and recovered, connected to the pPIC9K cloning vector, and transferred into Escherichia coli JM109 competent cells. The 97th, 162nd, 94th, 71st, 83rd, 114th, 207th and 215th amino acids are mutated by inverse PCR technology to obtain the mutant of chymotrypsin with improved anti-self-cleavage ability and specific activity.

[0048] The site-directed mutagenesis specifically includes the following steps: using the constructed pPIC9K-bctA (obtained by connecting the wild-type bovine chymotrypsin to pPIC9K) as a template, performing reverse PCR amplification with the corresponding mutant primers, decomposing the original template with the restriction endonuclease DpnI, verifying the amplified product by agarose electrophoresis and purifying and recovering the product, and simultaneously performing double enzyme digestion treatment on the constructed recombinant plasmid pPIC9K-M-bctA (M represents the corresponding mutant) and the expression vector pPIC9K with EcoI and NotI, respectively, recovering the target gene fragment and the vector fragment and connecting them with a seamless cloning enzyme to obtain the expression vector pPIC9K-M-bctA; the constructed expression vector is transformed into Escherichia coli by the heat shock method, the recombinant transformant is verified by the bacterial liquid, and the plasmid of the verified correct transformant is extracted and sequenced to determine the corresponding mutant. The mutant plasmid with correct sequencing was extracted, pPIC9K-M-bctA was linearized with Sac I, and electroporated into the competent state of Pichia pastoris GS115. The transformants were selected and spread on MD plates for high copy screening, and the transformants with correct sequencing were kept at -80°C.

[0049] The present invention will be further explained below through specific examples.

[0050] Example 1: Cloning of bovine chymotrypsinogen gene.

[0051] According to the amino acid sequence of wild bovine chymotrypsinogen, Pichia pastoris was selected as the expression host for codon optimization, and the chymotrypsinogen Pichia pastoris optimized gene (SEQ ID NO.2) was obtained, the wild-type chymotrypsinogen gene (SEQID NO.2) was synthesized, primers were designed and the gene was amplified by PCR technology, and the amplified gene fragment was connected to the vector pPIC9K by homologous recombination to obtain the recombinant cloning vector pPIC9K-M-bctA. The vector was transformed into Escherichia coli JM109 competent cells.

[0052] Example 2: Site-directed mutagenesis

[0053] The process of site-directed mutagenesis was as follows: using the constructed pPIC9K-M-bctA as a template, corresponding primers were designed for reverse PCR, and single-point or combined mutations were introduced at the corresponding amino acid sites 97, 162, 94, 71, 83, 114, 207, and 215. The primer sequences are shown in Table 2. The original template was removed by using Dpn I restriction endonuclease, the amplified product was verified by agarose electrophoresis and the product was purified and recovered, the recovered plasmid was transformed into Escherichia coli, the plasmid of the constructed strain was extracted, single and double enzyme digestion were performed for verification and sequencing, and the recombinant plasmid pPIC9K-M-bctA (M represents the corresponding mutant) was extracted; the constructed recombinant plasmid pPIC9K-M-bctA (M represents the corresponding mutant) and the expression vector pPIC9K were treated with EcoI and Not I for double enzyme digestion at the same time, the target gene fragment and the vector fragment were recovered, and they were connected by seamless cloning enzyme to obtain the expression vector pPIC9K-M-bctA; pPIC9K-M-bctA was linearized with Sac I, and electroporated to the competent state of Pichia pastoris GS115, the transformants were selected and coated on MD plates for high copy screening, and the transformants with correct sequencing were saved.

[0054] Table 2 Primer comparison table

[0055]

[0056] Example 3: Shake flask fermentation of recombinant Pichia pastoris

[0057] Inoculate the strain onto YPD solid medium for three-zone striping and culture at 30°C for 48h; pick a single colony from the YPD solid plate onto YPD liquid medium and culture at 30°C, 200rpm for 24h; inoculate the seed solution cultured in YPD liquid medium with 2% inoculum into BMGY medium at 30°C, 200rpm until the bacterial solution OD 6002-6, then transfer it to a sterile centrifuge tube and centrifuge at 5000rpm for 8min to retain the bacteria; add appropriate amount of BMMY and repeat centrifugation to remove residual glycerol; transfer to BMMY medium, and then add 500μL of methanol every 24h to induce culture for 108h.

[0058] Example 4, Activation of Chymotrypsinogen

[0059] The fermentation supernatant was collected by centrifugation at 12000 rpm for 10 min, replaced with 50 mM ammonium bicarbonate containing 1 mM CaCl2 as buffer, and trypsin was added at a ratio of 500:1 between zymogen and trypsin, and incubated at 37°C for 4 h to activate chymotrypsinogen.

[0060] Example 5: Analysis of initial specific activity and anti-self-cleavage performance of single mutants

[0061] The concentrated and purified wild-type chymotrypsin and mutants prepared by the method of Example 3-4 were used as experimental objects, and the initial specific activity was determined by the aforementioned method. The results are as follows: Figure 1 As shown: the initial specific activities of the single point mutants MT1, MT2, MT3, MT4, MT5, MT6, MT7, MT8 and MT9 reached 35.83U / mg, 39.86U / mg, 25.96U / mg, 9.00U / mg, 22.87U / mg, 11.34U / mg, 5.16U / mg, 4.12U / mg and 3.15U / mg, respectively, which were 1.23 times, 1.37 times, 0.89 times, 0.31 times, 0.78 times, 0.39 times, 0.18 times, 0.14 times and 0.12 times the specific activity of the wild-type chymotrypsin (29.17U / mg), indicating that the single point mutants MT1 and MT2 effectively improved the specific activity.

[0062] The chymotrypsin solution was diluted 10 times with 50 mM ammonium bicarbonate solution containing 1 mM calcium ions. After incubation at 37 °C for 6 h, the residual enzyme activity was measured, which was used as an evaluation index for the anti-self-cleavage performance. The experimental results are shown in Figure 2 As shown, the 6h residual enzyme activity rates of MT1, MT2, MT3, MT4, MT5, MT6, MT7, MT8 and MT9 were 44.23%, 46.33%, 19.37%, 28.59%, 49.55%, 43.64%, 45.66%, 0% and 0%, respectively, which were 1.62 times, 1.70 times, 0.71 times, 1.05 times, 1.82 times, 1.60 times, 1.68 times, 0 times and 0 times of the residual enzyme activity of wild-type chymotrypsin (27.24%), indicating that the single point mutants MT1, MT2, MT4, MT5, MT6 and MT7 have different degrees of improvement in the anti-self-cleavage ability compared with the wild-type enzyme molecules.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A self-cleaving chymotrypsin-resistant mutant, characterized in that Based on the amino acid sequence shown in SEQ ID NO.1, at least one of the following mutations occurs: L97A, L162I, F71I, L83I, F114I, W207H.

2. A gene encoding the protease mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that As shown in the sequence listing SEQ ID NO. 3, 4, 6, 7, 8, 9.

4. A recombinant vector or recombinant strain comprising the gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression vector is pHBM905A, pPIC9K, pPIC9K-His, pPIC3.5K, pPIC9, pPICZαA, pAO815, pPIC9k-His, pHIL-S1, pPinkhc, pGADT7, pGBKT7, pWB980 or pT3.

6. The recombinant strain according to claim 4, characterized in that The host cells are Pichia pastoris hosts X33, KM71, KM71H, GS115; Saccharomyces cerevisiae strains YM4271, AH109, ​​Y187, Y190; Escherichia coli E. coli BL 21, E. coliX90; or Bacillus subtilis SCK6.

7. A method for producing chymotrypsin using the recombinant strain according to claim 6, characterized in that: The details are as follows: (1) Recombinant Pichia pastoris shake flask fermentation The inoculated strain was cultured in YPD liquid medium at 30°C and 200 rpm for 24 h; the seed solution cultured in YPD liquid medium was inoculated with 2% inoculum into BMGY medium and cultured at 30°C and 200 rpm until the bacterial solution OD 600 2-6, then collect the bacteria; transfer to BMMY medium, then add 500 μL of methanol every 24 hours to induce culture for 108 hours; (2) Activation of chymotrypsinogen The fermentation supernatant was ultrafiltered using an ultrafiltration tube and replaced with 50 mM ammonium bicarbonate containing 1 mM CaCl2 as a buffer until the enzyme solution was colorless and transparent, and trypsin was added and incubated to activate chymotrypsinogen; (3) Purification of chymotrypsin.

8. The chymotrypsin as claimed in claim 1 is widely used in food processing, biocatalysis, biotechnology and medicine.

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