Trypsin mutant with improved heat stability
阅读说明：本技术 热稳定性提高的胰蛋白酶突变体 (Trypsin mutant with improved heat stability ) 是由 刘松 彭文坚 陈坚 周景文 于 2021-08-03 设计创作，主要内容包括：本发明公开了热稳定性提高的胰蛋白酶突变体,属于基因工程技术领域。本发明在高酶活灰色链霉菌胰蛋白酶基础上,通过定点突变生物技术改造胰蛋白酶分子结构,分析了SGT的柔性区域,并通过半理性设计最终获得两株热稳定性与比酶活提高的突变菌株S40P,Q124P。这些突变体能够在较高温度下进行工业生产,利于生产工艺的灵活性,具有良好的工业应用前景。(The invention discloses a trypsin mutant with improved thermal stability, belonging to the technical field of genetic engineering. On the basis of the high-enzyme-activity streptomyces griseus trypsin, the invention modifies the molecular structure of the trypsin through site-directed mutagenesis biotechnology, analyzes the flexible region of SGT, and finally obtains two mutant strains S40P, Q124P with improved thermal stability and specific enzyme activity through semi-rational design. The mutants can be industrially produced at higher temperature, are beneficial to the flexibility of the production process and have good industrial application prospect.)
1. The trypsin mutant is characterized in that the trypsin mutant takes trypsin with an amino acid sequence shown as SEQ ID NO.1 as a parent, and the amino acid at the 40 th position or the 124 th position of the trypsin mutant is mutated respectively.
2. The mutant according to claim 1, wherein serine at position 40 and glutamine at position 124 of the parent are mutated to proline, respectively.
3. A gene encoding the mutant of claim 1 or 2.
4. A vector carrying the gene of claim 3.
5. A microbial cell expressing the mutant of claim 1 or 2 or containing the gene of claim 3.
6. The microbial cell of claim 5, wherein the microbial cell comprises, but is not limited to, Pichia pastoris, Escherichia coli, and Bacillus subtilis.
7. A method for improving the heat stability of trypsin is characterized in that the amino acid at the 40 th position of the trypsin shown in SEQ ID NO.1 is mutated into proline or the amino acid at the 124 th position is mutated into proline.
8. A method for improving the enzyme activity of trypsin is characterized in that the amino acid at the 40 th position of the trypsin shown in SEQ ID NO.1 is mutated into proline or the amino acid at the 124 th position is mutated into proline.
9. A method for improving the specific activity of trypsin is characterized in that the amino acid at the 40 th position of the trypsin shown in SEQ ID NO.1 is mutated into proline or the amino acid at the 124 th position is mutated into proline.
10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the vector according to claim 4, or the host cell according to claim 5 in industrial, pharmaceutical, biochemical, food applications.
Technical Field
The invention relates to a trypsin mutant with improved specific enzyme activity and thermal stability, belonging to the technical field of genetic engineering.
Background
Trypsin, a polypeptide hydrolase, is an important component of industrial proteases and can specifically cleave the carboxyl terminal of arginine or lysine in a peptide chain, and the consumption of the trypsin accounts for about 3 percent of the industrial enzyme preparation market. The trypsin has wide application prospect in the fields of leather processing, medicine, food processing and agriculture.
Trypsin is one of important enzyme preparations in the leather processing process and is applied to the steps of soaking, unhairing, local treatment and deliming and softening of leather. The characteristic of specific decomposition of protein by trypsin makes it have the function of promoting the decomposition and dissolution of pus, sputum and blood clot, and the trypsin inhibitor contained in the serum can prevent or inhibit the damage of trypsin to normal tissues. In addition, the trypsin can be used as a tool enzyme for cutting a human insulin precursor produced by genetic engineering to produce mature insulin with activity. In the field of food processing, trypsin can be used for hydrolyzing various animal and vegetable proteins and can also be used for enhancing the properties of the whey protein such as the gelling property, the heat stability, the emulsifying property and the like in the industries of health food and food additives. In addition, trypsin is an important tool enzyme for polypeptide mass spectrum and proteomics analysis.
The heterologous expression microorganism-derived Streptomyces Griseus Trypsin (SGT) can avoid the problems of immunogenicity and the like of animal-derived trypsin and has important application value. Compared with the commonly used commercial porcine trypsin and bovine trypsin, SGT has more remarkable cleavage efficiency. However, the heat stability of various trypsin including SGT at present is poor, and the trypsin is extremely easy to inactivate and cannot be applied to high-temperature production conditions.
At present, no good solution is provided for the problem of poor stability in the process of SGT heterologous secretion expression. If the stability of SGT in heterologous expression can be improved, the production and application values of the enzyme can be obviously improved, and the method is more beneficial to industrial production.
Disclosure of Invention
In order to solve the problems, the invention provides a trypsin mutant with improved thermal stability and a pichia pastoris engineering bacterium capable of expressing the trypsin mutant.
In the previous research work, the inventor has obtained a trypsin recombinant pichia pastoris engineering bacterium with high enzyme activity, wherein the yeast expresses trypsin (called WT1) with an amino acid sequence shown as SEQ ID NO.1, and a nucleotide sequence for coding the trypsin is shown as SEQ ID NO. 2. The invention carries out proper mutation on the parent amino acid on the basis of the parent amino acid, thereby obtaining the mutant with improved stability and enzyme activity and providing wide prospect for the industrial application of the trypsin.
The invention provides a trypsin mutant, which takes trypsin with an amino acid sequence shown as SEQ ID NO.1 as a parent and mutates the amino acid at the 40 th position or 124 th position of the trypsin.
In one embodiment, the nucleotide sequence of the gene encoding said parent is as shown in SEQ ID No. 2.
In one embodiment, the serine at position 40 of the parent is mutated to proline.
In one embodiment, the glutamine at position 124 of the parent is mutated to proline.
The present invention provides a gene encoding the mutant.
The present invention provides a vector carrying the gene of claim 3.
In one embodiment, the vector is any one of pPIC9k, pHIL-S1, pPICZA, and Pyram 75p 6.
The invention provides a microbial cell expressing the mutant, or containing the gene.
In one embodiment, the microbial cells include, but are not limited to, pichia, escherichia coli, bacillus subtilis.
In one embodiment, the microbial cell is pichia pastoris GS115, KM71, KM71H, and/or X33.
The invention provides a method for improving the heat stability of trypsin, which is characterized in that the 40 th amino acid of the trypsin is mutated into proline, or the 124 th amino acid is mutated into proline.
The invention provides a method for improving the enzyme activity of trypsin, which is characterized in that the 40 th amino acid of the trypsin shown in SEQ ID NO.1 in amino acid sequence is mutated into proline, or the 124 th amino acid is mutated into proline.
The invention provides a method for improving the specific activity of trypsin, which is characterized in that the amino acid at the 40 th position of the trypsin shown in SEQ ID NO.1 in amino acid sequence is mutated into proline, or the amino acid at the 124 th position is mutated into proline.
The invention provides the application of the mutant, the gene, the vector or the host cell in the fields of industry, medicine, biochemistry and food.
The invention provides the application of the mutant, the gene, the vector or the host cell in cutting the carboxyl terminal of arginine or lysine in a peptide chain.
The invention has the beneficial effects that:
on the basis of a Streptomyces griseus trypsin with high enzyme activity, the invention modifies the molecular structure of the trypsin by site-directed mutagenesis biotechnology, and finally obtains two mutant strains S40P and Q124P with improved thermal stability after actual mutagenesis verification. The heat stability of the two mutants is obviously improved, and simultaneously the activity of the enzyme is not influenced or even improved. The improvement of residual enzyme activity and specific enzyme activity of the mutant Q124P after being subjected to water bath at 50 ℃ for 30min are most remarkable, and are respectively 43.71 percent and 2453.59 U.mg-1Compared with the control, the improvement is respectively 598% and 60.58%. The residual enzyme activity and the specific enzyme activity of the mutant S40P after being subjected to water bath at 50 ℃ for 30min are 43.74 percent and 1506.17 +/-38.35 U.mg respectively-1. The obtained mutant canCan carry out industrial production at higher temperature, is beneficial to the flexibility of the production process and has good industrial application prospect.
Drawings
FIG. 1 is a diagram of a site-directed mutagenesis-modified trypsin expression vector construction;
FIG. 2 shows the shake flask enzyme activities of trypsin mutants S40A, S40C, S40P, P74A, N77T, E120H, E120L, E120P, Q124F, Q124I, Q124L, Q124M, Q124P, Q124V, Q124Y, D161C, D161E, D161P, D161T, D161S, D161V, T162A, T162Q, N181A and N181S;
FIG. 3 shows the residual enzyme activity of mutants S40A, S40C, S40P, E120P, Q124F, Q124L, Q124M, Q124P, Q124V, Q124Y, D161C, D161E, D161P, D161T, D161S, T162A and T162Q after incubation in 50 ℃ water bath for 30 min.
Detailed Description
1. Purification of Trypsin
1) Centrifuging the sample, collecting supernatant, adjusting pH to 7.4 with 1M NaOH, centrifuging at 9000 Xg for 15min, and removing thallus precipitate; centrifuging at 12000 Xg for 20min, removing impurities, and collecting supernatant; the sample was filtered through a 0.22 μm filter and placed on ice until use.
2) A loading buffer (solution A) 50mM Tris-HCl (pH7.4 containing 0.5M NaCl) and an elution buffer (solution B) 50mM glycine-HCl buffer (pH 3.0) were prepared.
3) And (3) purification process: cleaning a pipeline and a purification column by using ultrapure water, and then cleaning an elution system by using the ultrapure water, wherein the volume of the column is 2; respectively washing A, B pumps with a loading buffer solution and an elution buffer solution, washing the loading pumps with the loading buffer solution, and then washing the system with a loading buffer solution column by 2 column volumes; switching to a sample loading pump for automatic sample loading, and performing column washing by using a sample loading buffer solution after the sample loading is finished, and performing column washing by one column volume until the UV absorption value is reduced to an initial value, so that trypsin is fully combined with the Benzamidine in the filler; column washing with 20% elution buffer for 1 column volume and column washing with 40% elution buffer for 1.5 column volumes; column washing 1.5 column volumes with 60% elution buffer; column washing 1.5 column volumes with 80% elution buffer; column wash 2 column volumes with 100% elution buffer.
4) A trypsin sample was collected, the pH of the collected sample was adjusted to 3.0 with 1MTris buffer and dialyzed.
2. Trypsin amidase enzyme activity determination method
The change in absorbance at 410nm for 10min in a reaction cell having an optical path of 0.5cm was measured at 37 ℃ for 100. mu.L of the crude enzyme solution and 900. mu.L of BAPNA (Na-benzoyl-DL-arginine-p-nitroamide hydrochloride) solution to give A410 nm/min. The enzyme activity is defined as: the amount of enzyme required to raise Δ A410nm/min by 0.1 at 37 ℃ was 1 amidase hydrolysis unit.
Example 1: construction of recombinant vector for Trypsin mutant
1) Construction of mutant recombinant plasmids
Taking trypsin with an amino acid sequence shown as SEQ ID NO.1 as a parent, respectively mutating S at the 40 th position to A, C, P, P at the 74 th position to A, N at the 77 th position to T, E at the 120 th position to H, L, P, Q at the 124 th position to F, I, L, M, P, V, Y, D at the 161 th position to C, E, P, T, S, V, A, Q, and N at the 181 th position to A, S.
Taking a plasmid with the amino acid sequence of SEQ ID NO.1 for mutating the 40 th position of trypsin into proline (S40P) as an example, taking a Ppic-9K vector (purchased from Saimer Feishell science and technology Co., Ltd.) connected with the sequence shown in SEQ ID NO.2 as a template (shown in figure 1), taking S40P-F, S40-R as a primer, and carrying out PCR to obtain a nucleotide sequence of a mutant (S40P) for coding the amino acid sequence with the 40 th position of serine being mutated into proline;
secondly, performing enzyme digestion on the PCR product containing the recombinant gene obtained in the last step by using Dpn I to remove a template, purifying the enzyme digestion product, and chemically converting the purified product into JM109 competent cells to obtain a conversion solution;
thirdly, the transformation liquid is coated on LB culture medium containing 100 mug/L kanamycin, and is cultured at 37 ℃ until a single colony grows out, the single colony is picked up and is cultured in LB liquid culture medium containing 100 mug/L kanamycin at 37 ℃ for 8-10h, plasmids in the bacterial liquid are extracted, sequencing verification is carried out, the correct recombinant plasmid is established, and the recombinant plasmid is named as pPIC9K-Exmt S40P.
Recombinant plasmids of mutants S40, P74, N77, E120, Q124, D161, T162, N181 and N181 were constructed by using the primers in Table 1 and the same method as in step (1), and named pPIC 9-Exmt S40, pPIC 9-ExmtS 40, pPIC 9-ExmtP 74, pPIC 9-ExmtN 77, pPIC 9-ExmtE 120, pPIC 9-ExmtQ 124, pPIC 9-ExmtPIC 9-ExmtP 181, ExmtPIC 9-ExmtP 161, pPIC 9-ExmtN 161, pPIC 9-ExmtP 181, pPIC 9-ExmtP 124, pPIC 9-ExmtP 161, pPIC 9-7, pPIC-ExmtP-9-7, pPIC-9-ExmtP 33, pPIC-7, pPIC-9-7, pPIC-9-7, pPIC-ExmtP-7, pPIC-9-7, pPIC-9-7, pPIC-ExmtP-9-7, pPIC-9-ExmtP-9-7, pPIC 9-7, pPIC-9-X-9-7, pPIC-9-X-9-7, and N-X-7, and N-9-X-9-7.
TABLE 1 primers used in example 1
Example 2: construction of yeast engineering bacteria producing mature trypsin mutant
The recombinant plasmid pPIC9K-Exmt S40P obtained in example 1 was linearized with Sal I, and the linearized fragment was recovered and used to electrically shock transform Pichia pastoris GS115 competent cells, as follows:
1) inoculating YPD plate-activated Pichia pastoris GS115 into a 25mL/250mL Erlenmeyer flask containing YPD medium, and culturing overnight at 30 ℃; the overnight-cultured bacterial solution was inoculated into a 50mL/500mL Erlenmeyer flask containing YPD medium at an inoculum size of 1mL/100mL, and the culture cell concentration OD was determined6001.3 to 1.5;
2) centrifuging at 4 ℃ for 10min at 5000r/min, collecting thalli, and suspending the cells with 50mL and 25mL of sterile water respectively;
3)5mL of 1M sorbitol is used for resuspending the cells, and the cells are centrifuged at 5000r/min and 4 ℃ for 10min to collect thalli;
4)500 μ L of 1M sorbitol resuspended the cells and aliquoted into 80 μ L/1.5mL EP tubes for electroporation of competent cells;
5) mixing 20 μ L linearized plasmid with 80 μ L competent cells, and standing on ice for 15 min;
6) adding the mixture into a pre-cooled sterile electric conversion cup (0.2cm), electrically shocking at 1500V and 25 muF once at 200 omega, and adding 1mL of 1M sorbitol into the mixture;
7) coating 150 mu L of the mixture obtained in the step 6 on an MD plate, and culturing for 3 days at the temperature of 30 ℃;
8) picking white colonies in the plate, and verifying correct recombinant bacteria: respectively dibbling the cells in 1, 2, 3 and 4mg/mL (geneticin) YPD plates, selecting a single colony in the 4mg/mL geneticin plate for shake flask fermentation, measuring the activity of trypsin, selecting a recombinant bacterium with the highest activity, and naming the recombinant bacterium GS 115-S40P.
Other recombinant plasmids constructed in the example 1 are transformed into competent cells of Pichia pastoris GS115 by the same method to construct a recombinant Pichia pastoris strain GS115S 40A, GS115S40C, GS115S40P, GS115P74A, GS115tN77T, GS115tE120H, GS115E120L, GS115E120P, GS115Q124F, GS115Q I, GS115Q124L, GS115Q124M, GS115Q124P, GS115Q124V, GS115Q Y, GS115D161C, GS115D E, GS115D 695161 161 2, GS115D161T, GS115D161S, GS115D V, 115T A, GS115T 161T 181Q, GS115N181 3687458 and GS115N S containing trypsin mutant.
Example 3: enzyme activity and heat stability of trypsin mutant
The recombinant pichia pastoris containing the trypsin mutant, which is constructed in the example 2, is respectively inoculated into 50mL of YPD culture medium, activated for 24 hours at the temperature of 30 ℃, the activated bacterial liquid is centrifuged for 5min at 3000g to collect thalli, the supernatant is discarded, and 35mL of fermentation culture medium is added for resuspension.
Fermentation medium (g/L): k2HPO4·3H2O 1.51；KH2PO45.91; 0.2 parts of biotin; YNB (yeast without amino acid nitrogen source) 13.4; tryptone 10; 5, yeast powder; biotin 4X 10-4(ii) a Methanol 1mL/100mL。
Adding methanol every 24h at 30 deg.C and 220rpm to make methanol concentration in fermentation system 1mL/100mL, culturing for 120h, collecting fermentation broth and measuring crude enzyme activity (see figure 2), selecting enzyme activity greater than 20 U.mL-1Purifying the mutant to obtain the purified protein. Respectively measuring the enzyme activity of the protein, taking another certain amount of pure enzyme liquid for water bath heat treatment at 50 ℃ in a Tris-HCL buffer solution with the pH value of 8.0, respectively measuring the enzyme activity of the residual enzyme before and after 30min of treatment, and taking the enzyme activity of the pure enzyme liquid which is not subjected to high-temperature treatment as a reference to obtain the percentage of the residual enzyme activity, wherein the specific enzyme activity of the mutant and the residual enzyme activity after 30min of heat treatment are shown in figure 3.
The mutants S40P and Q124P have improved specific enzyme activity on the premise of remarkably improving thermal stability, and the catalytic activity and the thermal stability are shown in Table 2.
TABLE 2 trypsin mutants remaining enzyme activity after heat treatment (U/mL)
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> Trypsin mutant with improved thermostability
<130> BAA210980A
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<170> PatentIn version 3.3
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gtcgtcggcg gaacccgcgc cgcccagggc gagttcccct tcatggtccg gctctccatg 60
ggctgcggcg gcgccctcta cgcccaggac atcgtcctca ccgccgccca ctgcgtgagc 120
ggatcgggca acaacacctc gatcaccgcc accggcggcg tcgttgatct ccagtcgtcc 180
agcgccgtca aggtccgctc caccaaggtc ctccaggccc ccggctacaa cggcaccggc 240
gctgactggg cgctcatcaa gctcgcccag cccatcaacc agcccacgct gaagatcgcc 300
accaccaccg cctacaacca gggcacgttc accgtcgccg gctggggcgc caacattgag 360
ggcggcagcc agcagcgcta cctgctcaag gccaacgtcc cattcgtctc cgacgccgcc 420
tgccgctccg cctacggcaa cgagcttgtg gccaacgagg agatttgcgc cggatacccc 480
gacactggtg gcgttgatac ctgccagggt gactccggcg gcccgatgtt cgttaaggac 540
aacgccgacg agtggattca ggtcggcatc gtcagctggg gctacggctg cgcccggccc 600
ggctacccgg gtgtctacac cgaggtctcg accttcgctt ccgccatcgc ctcggccgcc 660
cgcacgctct ga 672
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Val Val Gly Gly Thr Arg Ala Ala Gln Gly Glu Phe Pro Phe Met Val
1 5 10 15
Arg Leu Ser Met Gly Cys Gly Gly Ala Leu Tyr Ala Gln Asp Ile Val
20 25 30
Leu Thr Ala Ala His Cys Val Ser Gly Ser Gly Asn Asn Thr Ser Ile
35 40 45
Thr Ala Thr Gly Gly Val Val Asp Leu Gln Ser Ser Ser Ala Val Lys
50 55 60
Val Arg Ser Thr Lys Val Leu Gln Ala Pro Gly Tyr Asn Gly Thr Gly
65 70 75 80
Ala Asp Trp Ala Leu Ile Lys Leu Ala Gln Pro Ile Asn Gln Pro Thr
85 90 95
Leu Lys Ile Ala Thr Thr Thr Ala Tyr Asn Gln Gly Thr Phe Thr Val
100 105 110
Ala Gly Trp Gly Ala Asn Ile Glu Gly Gly Ser Gln Gln Arg Tyr Leu
115 120 125
Leu Lys Ala Asn Val Pro Phe Val Ser Asp Ala Ala Cys Arg Ser Ala
130 135 140
Tyr Gly Asn Glu Leu Val Ala Asn Glu Glu Ile Cys Ala Gly Tyr Pro
145 150 155 160
Asp Thr Gly Gly Val Asp Thr Cys Gln Gly Asp Ser Gly Gly Pro Met
165 170 175
Phe Val Lys Asp Asn Ala Asp Glu Trp Ile Gln Val Gly Ile Val Ser
180 185 190
Trp Gly Tyr Gly Cys Ala Arg Pro Gly Tyr Pro Gly Val Tyr Thr Glu
195 200 205
Val Ser Thr Phe Ala Ser Ala Ile Ala Ser Ala Ala Arg Thr Leu
210 215 220