CN117925582A - Nattokinase mutant with high activity and high thermal stability and application thereof - Google Patents
Nattokinase mutant with high activity and high thermal stability and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of enzyme genetic engineering, and discloses a high-activity high-thermal-stability nattokinase mutant and application thereof. The invention aims at wild type nattokinase, the 90 th amino acid, the 95 th amino acid and the 300 th amino acid are replaced by L, S, P th amino acid respectively, the enzyme activity of the obtained nattokinase mutant is improved by 88.24%, the heat treatment is carried out at 60 ℃ for 30 min, and the relative residual enzyme activity is 48.75%, which is obviously superior to that of the wild type nattokinase, and the invention provides a theoretical basis for the large-scale production and application of the nattokinase.
Description
Technical Field
The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a high-activity high-thermal-stability nattokinase mutant and application thereof.
Background
Nattokinase (Nattokinase, NK) is a fibrinolytic enzyme capable of dissolving thrombus and preventing thrombosis in vivo. Thrombolytic drugs such as urokinase and streptokinase commonly used in the market at present have certain thrombolytic efficacy, but have the limitations of insufficient safety, short half-life and low thrombolytic efficiency. The nattokinase is a special protein bioactive substance which can be absorbed by human body after being orally taken, has no obvious toxicity and side effect, can be used as medicines or functional foods, and can prevent thrombosis and reduce blood pressure and blood fat after being orally taken for a long time. With the wide application of nattokinase, efficient production is necessary, and the use of microorganism to produce nattokinase can use inexpensive culture medium to obtain higher yield in shorter time. At present, more expression systems are adopted, namely escherichia coli, pichia pastoris and bacillus, compared with the escherichia coli for expressing inclusion bodies without enzyme activity, the bacillus is an initial expression system, overcomes the defects of the two expression systems, and has the advantage of biosafety (GRAS).
The DNA sequence of nattokinase produced by Bacillus subtilis shows that there is a leader peptide sequence between the signal peptide sequence and the mature peptide sequence. The leader peptide has self-cleavage activity and plays an important role in the correct folding, expression and secretion, stability, substrate selectivity and the like of the mature peptide. Thus, by mutation of the leader peptide site, the folding efficiency of the mature peptide can be aided, changing the enzymatic properties of the protease. In addition, the leader peptide can also act as a competitive inhibitor of the mature peptide, forming a stable and inactive leader peptide-enzyme complex by forced binding to the active site, so that the leader peptide must be cleaved and degraded after the completion of the targeting process, releasing the active enzyme.
Disclosure of Invention
The invention aims to provide a nattokinase mutant with high activity and high thermal stability, wherein the amino acid sequence of the nattokinase is shown as SEQ ID NO. 12.
Another object of the invention is to provide the use of high activity and high thermal stability nattokinase mutant in preparing fibrinolytic enzyme.
In order to achieve the above purpose, the invention adopts the following technical scheme:
Nattokinase aprN (Gene ID: 14100838) derived from Bacillus subtilis, the applicant replaced the original signal peptide with signal peptide SPsacC (shown as SEQ ID NO.2, encoded by the nucleotide shown as SEQ ID NO. 1) which is a highly efficient secretion protein in Bacillus licheniformis to obtain a signal peptide-replaced nattokinase, and further, amino acids at positions 90, 95 and 300 of the nattokinase were replaced with L, S, P, respectively, to obtain a nattokinase triple mutant K90L-A95S-S300P (shown as SEQ ID NO. 12).
The protection scope of the invention also comprises:
a gene encoding a protein represented by SEQ ID NO. 12;
An expression vector containing the above gene;
expressing a recombinant microorganism comprising the protein shown in SEQ ID NO. 12;
the use of the above-mentioned substances for the preparation of fibrinolytic enzymes.
A method for preparing a nattokinase three mutant, comprising: inserting the gene encoding the protein shown in SEQ ID NO.12 into an expression vector, and transforming into bacillus licheniformis BL10 for fermentation expression.
Compared with the prior art, the invention has the following advantages:
The wild nattokinase has low enzyme activity and poor thermal stability, and particularly when the temperature exceeds 60 ℃, enzyme molecules can be rapidly deactivated, so that the method is unfavorable for production under a high-temperature environment process, and is difficult to produce on a large scale. According to the invention, molecular transformation is carried out on nattokinase, on one hand, the folding of mature peptide is better assisted through leader peptide optimization, and the improvement effect on the enzyme activity and thermal stability of nattokinase is achieved; on the other hand, through predicting the evolution site of the mature peptide, single site amino acid is replaced by amino acid which promotes the structure of the mature peptide to be more stable, and the enzymatic activity of nattokinase is improved to a certain extent. The mutant strain constructed by the invention effectively promotes the high-efficiency expression of nattokinase and obtains a high-temperature-resistant nattokinase mutant, thereby providing a theoretical basis for the large-scale production and application of nattokinase.
Detailed Description
The technical content of the present invention is further described below with reference to examples, but the present invention is not limited to these examples, and the scope of the present invention is not limited to the following examples, and the technical solutions of the present invention are all conventional in the art unless specifically described otherwise; the reagents or materials, unless otherwise specified, are commercially available. The high-activity high-thermal-stability nattokinase mutant can be obtained by adopting a direct synthesis mode or by constructing engineering strains of the high-activity high-thermal-stability nattokinase mutant and fermenting the engineering strains.
Example 1:
expression of the 90 th amino acid mutant of the nattokinase original sequence:
The bacillus subtilis-derived nattokinase aprN (Gene ID: 14100838) is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretion proteins in bacillus licheniformis, and the 90 th amino acid of the original amino acid sequence is changed from K to I, L or G to obtain mutants K90I (SEQ ID NO. 3), K90L (SEQ ID NO. 4) and K90G (SEQ ID NO. 5) of 3 nattokinase.
In the present invention, the above three mutants were obtained in the following manner:
a nattokinase gene aprN which is mediated by a Prbs promoter and terminated by an amylase terminator TamYL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-aprN, and a mutant skeleton is amplified by using a plasmid pHY-aprN as a template and using a designed mutation primer.
The primers for the mutation vector pHY-aprN-K90I are as follows:
K90I-KF：AATTGAAAATCGATCCGAGCGTTG
K90I-KR：TCGGATCGATTTTCAATTCTTTTACA
the primers for the mutation vector pHY-aprN-K90L are as follows:
K90L-KF：AATTGAAACTGGATCCGAGCGTTG
K90L-KR：TCGGATCCAGTTTCAATTCTTTTAC
the primers for the mutation vector pHY-aprN-K90G are as follows:
K90G-KF：AATTGAAAGGCGATCCGAGCGTTG
K90G-KR：TCGGATCGCCTTTCAATTCTTTTA
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Plasmid was then extracted and tested, and the correctly sequenced plasmids (pHY-aprN-K90I, pHY-aprN-K90L and pHY-aprN-K90G) and template plasmids (pHY-aprN) were respectively transformed into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, and the recombinant expression strains BL10/aprN-K90I, BL10/aprN-K90L, BL/aprN-K90G and the control strain BL10/aprN were obtained by resistance plate and PCR screening.
Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of nattokinase culture medium at 37 ℃ at 230rpm/min, and culturing for 48 hours. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a fibrinogen dissolution method.
LB medium: peptone 10g/L, sodium chloride 10g/L, yeast powder 5g/L
Nattokinase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate and 2g/L, pH of glucose are regulated to 7.2, and 30mL of liquid in each bottle is subjected to a fibrinogen dissolution method to detect the activity of nattokinase, and the experimental results are as follows:
from the table, the 90 th amino acid is changed from K to I, L or G, and the nattokinase activity is improved by at least 21.57%.
Example 2:
expression of the 95 th amino acid mutant of the nattokinase original sequence:
The bacillus subtilis-derived nattokinase aprN (Gene ID: 14100838) is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretion protein in bacillus licheniformis, and the 95 th amino acid of the original amino acid sequence is changed from A to S, P or L to obtain 3 mutants A95S (SEQ ID NO. 6), A95P (SEQ ID NO. 7) and A95L (SEQ ID NO. 8) of the nattokinase. The specific preparation method of the invention is as follows:
a nattokinase gene aprN which is mediated by a Prbs promoter and terminated by an amylase terminator TamYL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-aprN, and a mutant skeleton is amplified by using a plasmid pHY-aprN as a template and using a designed mutation primer.
The primers for the mutation vector pHY-aprN-A95S were as follows:
A95S-KF：CGAGCGTTAGCTATGTGGAAGAAG
A95S-KR：CCACATAGCTAACGCTCGGATCGC
the primers for the mutation vector pHY-aprN-A95P were as follows:
A95P-KF：CGAGCGTTCCGTATGTGGAAGAAG
A95P-KR：CCACATACGGAACGCTCGGATCGC
the primers for the mutation vector pHY-aprN-A95L were as follows:
A95L-KF：GAGCGTTCTGTATGTGGAAGAAGA
A95L-KR：TCCACATACAGAACGCTCGGATCG
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Plasmid was then extracted and tested, and the plasmids with correct sequence (pHY-aprN-A95S, pHY-aprN-A95P and pHY-aprN-A95L) and template plasmid (pHY-aprN) were respectively transformed into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, and the recombinant expression strains BL10/aprN-A95S, BL10/aprN-A95P, BL10/aprN-A95L and the control strain BL10/aprN were obtained by resistance plate and PCR screening. Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of nattokinase culture medium at 37 ℃ at 230rpm/min, and culturing for 48 hours. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a fibrinogen dissolution method.
LB medium: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast powder;
Nattokinase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate and 2g/L, pH of glucose are regulated to 7.2, and 30mL of liquid in each bottle is subjected to a fibrinogen dissolution method to detect the activity of nattokinase, and the experimental results are as follows:
from the table, the 95 th amino acid is changed from A to S, P or L, and the nattokinase activity is improved by at least 15.69%.
Example 3:
expression of the amino acid mutant at position 300 of the original sequence of nattokinase:
The method comprises the steps of replacing original signal peptide with signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretion protein in bacillus licheniformis by using nattokinase aprN (Gene ID: 14100838) derived from bacillus subtilis as a template, and changing the 300 th amino acid of the original amino acid sequence from S to M, P or V to obtain mutants S300M (SEQ ID NO. 9), S300P (SEQ ID NO. 10) and S300V (SEQ ID NO. 11) of 3 nattokinase. The specific preparation method of the invention is as follows:
a nattokinase gene aprN which is mediated by a Prbs promoter and terminated by an amylase terminator TamYL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-aprN, and a mutant skeleton is amplified by using a plasmid pHY-aprN as a template and using a designed mutation primer.
The primers for the mutation vector pHY-aprN-S300M were as follows:
S300M-KF：GCGTAGGTATGGAGCTTGATGTAA
S300M-KR：CAAGCTCCATACCTACGCTGGAGA
The primers for the mutation vector pHY-aprN-S300P were as follows:
S300P-KF：GCGTAGGTCCGGAGCTTGATGTAA
S300P-KR：CAAGCTCCGGACCTACGCTGGAGA
the primers for the mutation vector pHY-aprN-S300V were as follows:
S300V-KF：GCGTAGGTGTCGAGCTTGATGTAA
S300V-KR：CAAGCTCGACACCTACGCTGGAGA
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Plasmid was then extracted and tested, and the plasmids with correct sequencing (pHY-aprN-S300M, pHY-aprN-S300P and pHY-aprN-S300V) and template plasmid (pHY-aprN) were respectively transformed into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, and the recombinant expression strains BL10/aprN-S300M, BL10/aprN-S300P, BL/aprN-S300V and the control strain BL10/aprN were obtained by resistance plate and PCR screening. Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of nattokinase culture medium at 37 ℃ at 230rpm/min, and culturing for 48 hours. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a fibrinogen dissolution method.
LB medium: peptone 10g/L, sodium chloride 10g/L, yeast powder 5g/L
Nattokinase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate and 2g/L, pH of glucose are regulated to 7.2, and 30mL of liquid in each bottle is subjected to a fibrinogen dissolution method to detect the activity of nattokinase, and the experimental results are as follows:
As can be seen from the table, the nattokinase activity is improved by at least 31.37% by changing the 300 th amino acid from S to M, P or V.
Example 4:
Expression of nattokinase three-site mutant:
the 95 th amino acid of the original amino acid sequence is changed from A to S by a K90L (SEQ ID NO. 4) template, so as to construct a nattokinase double-site mutant vector K90L-A95S. And then taking K90L-A95S as a template, and changing the 300 th amino acid of the original amino acid from S to P to obtain a nattokinase three-locus mutant K90L-A95S-S300P (shown in SEQ ID NO. 12). The specific preparation method of the invention is as follows:
a nattokinase gene aprN which is mediated by a Prbs promoter and terminated by an amylase terminator TamYL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-aprN, and a mutant skeleton is amplified by using a plasmid pHY-aprN as a template and using a designed mutation primer.
The primers for the mutation vector pHY-aprN-K90L are as follows:
K90L-KF：AATTGAAACTGGATCCGAGCGTTG
K90L-KR：TCGGATCCAGTTTCAATTCTTTTAC
the primers for the mutation vector pHY-aprN-K90L-A95S are as follows:
K90L-A95S-KF：CTGGATCCGAGCGTTAGCTATGTGGAAGAA
K90L-A95S-KR：GCTAACGCTCGGATCCAGTTTCAATTCTTT
The primers for the mutation vector pHY-aprN-K90L-A95S-S300P were as follows:
K90L-A95S-S300P-KF：GCGTAGGTCCGGAGCTTGATGTAA
K90L-A95S-S300P-KR：CAAGCTCCGGACCTACGCTGGAGA
and (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Then extracting the plasmids for measurement, respectively transforming the plasmids (pHY-aprN-K90L-A95S-S300P) and the template plasmids (pHY-aprN) which are sequenced correctly into Bacillus licheniformis BL10 (CN 104630123A, CCTCCNO: M2013400) by an electroporation method, and obtaining the nattokinase three-site mutant recombinant strain BL10/aprN-K90L-A95S-S300P and the control strain BL10/aprN by a resistance plate and PCR screening. Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of nattokinase culture medium at 37 ℃ at 230rpm/min, and culturing for 48 hours. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a fibrinogen dissolution method.
In order to detect the overall thermal stability of the mutant, a proper amount of supernatant is taken out and placed on ice immediately after being treated in a water bath at 60 ℃ for 30min, the untreated and heat-treated fermentation broth is diluted to corresponding multiples, and a fibrinogen dissolution method is adopted to detect the nattokinase activity.
LB medium: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast powder;
nattokinase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate and 2g/L, pH of glucose are regulated to 7.2, and the liquid amount per bottle is 30mL. The activity of nattokinase is detected by adopting a fibrinogen dissolution method, and the experimental result is as follows:
From the table, the 90 th amino acid, the 95 th amino acid and the 300 th amino acid are respectively replaced by L, S, P th amino acid, and the enzyme activity of the obtained nattokinase three-site mutant is improved by 88.24%; the heat treatment is carried out for 30min at 60 ℃, the relative residual enzyme activity of the wild type nattokinase is 11.76%, the relative residual enzyme activity of the three-site mutant is 48.75%, and the enzyme activity and the heat stability of the nattokinase three-site mutant are obviously improved.
Claims (6)
1. An artificially synthesized nattokinase three mutant, wherein the mutant is shown in SEQ ID NO. 12.
2. A gene encoding the protein shown in SEQ ID NO. 12.
3. An expression vector comprising the gene of claim 2.
4. Recombinant microorganisms expressing a protein comprising the protein shown in SEQ ID No. 12.
5. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the recombinant microorganism according to claim 4 for the preparation of a fibrinolytic enzyme.
6. The method for preparing the nattokinase three mutant of claim 1, comprising: inserting the gene encoding the protein shown in SEQ ID NO.12 into an expression vector, and transforming into bacillus licheniformis BL10 for fermentation expression.
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