CN110819612A - Screening of novel autocleavage-resistant alkaline protease - Google Patents
Screening of novel autocleavage-resistant alkaline protease Download PDFInfo
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Abstract
The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to screening of a novel alkaline protease mutant capable of resisting autogenous cutting. The invention obtains the mutant with excellent anti-autocleaving performance by modifying and screening the alkaline protease from the Bacillus clausii, the gene of the mutant is mutated into GAT at the 493-and 495-positions, GAT at the 799-and 801-positions, the corresponding amino acid sequences are respectively mutated into Thr at the 161-position and Asp at the 183-position. After incubation for 48h at the constant temperature of 40 ℃, the enzyme activity retention rate of the alkaline protease mutant is improved by more than 40 percent compared with the enzyme activity of wild alkaline protease. The screening of the anti-self-cutting alkaline protease molecule is beneficial to improving the storage and application effects of the alkaline protease.
Description
Technical Field
The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to screening of novel anti-autogenous cutting alkaline protease
Background
Alkaline protease (alkaline protease) is an enzyme that hydrolyzes peptide bonds of proteins at a pH slightly in the alkaline range, has an optimum pH of usually 9 to 11, belongs to the class of serine proteolytic enzymes in endopeptidases, and has a molecular weight of about 27 kDa. The alkaline protease is mainly applied to the enzyme-added detergent industry, is also widely applied to the industries of food, medical treatment, brewing, silk, leather making and the like, and is the enzyme occupying the largest proportion in industrial enzymes.
However, the self-cutting phenomenon of the alkaline protease in the substrate hydrolysis is also intensified due to the wide range of the hydrolysis sites, particularly, after the amino acid existing in the loose secondary structure of β -turn and loop is cut, the active center of the alkaline protease is exposed and is rapidly inactivated after being recognized and cut, the self-cutting easily occurs, a great amount of loss of enzyme activity is caused, and the production cost is increased for maintaining the activity of the alkaline protease for a long time.
The invention aims at searching and analyzing the surface specific amino acid of the alkaline protease to determine the easy self-cutting site, and then performs virtual amino acid mutation by using Discovery Studio software to determine the type of the optimally mutated amino acid. The present invention provides an alkaline protease mutant which is resistant to spontaneous cleavage without decreasing the enzyme activity. The mutant is obtained by starting from a mature peptide of an alkaline protease gene aprE of Bacillus clausii (Bacillus clausii) through technologies such as molecular simulation, site-directed mutagenesis and the like.
Disclosure of Invention
The invention aims to provide an alkaline protease mutant capable of resisting autogenous cutting. The invention is based on alkaline protease aprE from Bacillus clausii, determines an easy self-cutting site by searching and analyzing hydrophobic amino acid on the surface of the alkaline protease, and then performs virtual amino acid mutation by using Discovery Studio software to determine the type of the optimal mutated amino acid. Then, 2 alkaline protease mutants pQY-5 and pQY-6 are obtained by designing primers and introducing mutation sites by reverse PCR technology, recombinant bacteria of bacillus subtilis QY-5 and QY-6 for expressing the mutants are respectively constructed, the enzyme activities are respectively 27U/mL and 31U/mL after fermentation for 48 hours, and the enzyme activities of wild alkaline protease aprE expressed by the recombinant bacteria are respectively improved by 17 percent and 38 percent. The enzyme activity retention rate of the alkaline protease mutant is improved by 41 percent and 70 percent compared with the enzyme activity of the wild alkaline protease.
The technical route for realizing the purpose of the invention is summarized as follows:
the easy self-cutting site is determined by the search and analysis of the hydrophobic amino acids on the surface of the alkaline protease, and then the type of the amino acid with the optimal mutation is determined by virtual amino acid mutation performed by Discovery Studio software. Taking a genome of bacillus clausii as a template, and performing amplification according to GenBank: the method comprises the steps of cloning FJ940727.1 to obtain an alkaline protease gene aprE, constructing the alkaline protease gene aprE on an expression vector pWB600, introducing a mutation site through an inverse PCR technology, carrying out site-specific mutation on the wild alkaline protease gene to obtain a mutant gene (SEQ ID NO: 3, 5), constructing a recombinant vector, and transforming the recombinant vector into Bacillus subtilis WB600 for fermentation enzyme production experiment verification to obtain the alkaline protease mutant capable of effectively resisting autogenous cutting.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of alkaline protease mutants
"amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in the alkaline protease mutant. Like Leul61Thr, indicates that the amino acid at position 161 is replaced by Leu to Thr of the wild-type alkaline protease. The numbering of positions corresponds to SEQ ID NO: 2, the amino acid sequence of the wild-type alkaline protease. Nucleotide changes are also denoted by "original nucleotide position substituted nucleotide" and the position numbering corresponds to that of SEQ ID NO: 1, nucleotide sequence number of wild-type alkaline protease.
In the present invention, aprE represents the gene sequence of wild type alkaline protease, i.e., the original sequence (shown in SEQ ID NO: 1), and aprEm represents the gene sequence of alkaline protease mutant (shown in SEQ ID NO: 3, 5); APRE represents wild type alkaline protease (the amino acid sequence is shown as SEQ ID NO: 2), APREM represents alkaline protease mutant (the amino acid sequence is shown as SEQ ID NO: 4, 6)
| Alkaline protease | Base | Amino acids |
| APREM-1 | 733 + 735 th position ACC | Thr at position 161 |
| APREM-2 | 799 along position 801 as GAT | Asp at position 183 |
The expression host of the alkaline protease mutant is Bacillus subtilis WB60, and the expression vector is pWB 980.
The experimental steps of the invention are as follows:
1. downloading analyzed crystal structures 4cfz, 4cfy, 3bx1 and 1TK2 from a PDB structure database, and finding out an amino acid residue with the surface containing F, Y, W as a candidate mutation site;
2. the virtual amino acid mutations at positions 161 and 183 of 4cfz were performed by using the discovery studio software, and the best results were calculated as candidate mutant amino acids. The specific parameters are that in a queue status module in the status Energy of the discovery studio, the status: single details, details: candidate sites, All, CHARMm Polar H is selected in a force field, the influence of PH and temperature on mutation performance is ignored, and sites which are easy to self-cut by alkaline protease are obtained.
3. Construction of recombinant Bacillus subtilis strain containing wild type alkaline protease gene
Carrying out enzyme digestion on the wild type alkaline protease coding gene aprE obtained by amplification (figure 3), and connecting the wild type alkaline protease coding gene aprE with an expression vector pWB980 (figure 4) subjected to enzyme digestion to obtain a new recombinant vector; and (3) transforming the recombinant vector into the bacillus subtilis WB600, and obtaining a wild alkaline protease expression strain by screening kana resistance and determining enzyme activity of the obtained recombinant strain.
4. Construction of Bacillus subtilis recombinant Strain containing mutant alkaline protease Gene
Designing a mutation site in a primer, and introducing the mutation site by reverse PCR by using a recombinant plasmid aprE-pWB980 containing a wild-type alkaline protease coding gene as a template. Obtaining mutant alkaline protease aprEM gene, and performing self-connection on the linear vector obtained by amplification to obtain the recombinant vector of the alkaline protease mutant gene. And (3) transforming the recombinant vector into the bacillus subtilis WB600, and sequencing the obtained recombinant strain to obtain the alkaline protease mutant.
5. Fermentation preparation of wild type alkaline protease and mutant alkaline protease
6. Comparing the enzyme activity retention rates of different mutants through a stability investigation experiment to obtain the mutant with higher enzyme activity stability
7. Sequencing the obtained mutant strain plasmid with higher enzyme activity stability to obtain the high-stability alkaline protease mutant gene sequence.
The invention has the beneficial effects that: the wild alkaline protease from the Bacillus clausii has fast enzyme activity loss in the storage or use process due to autogenous cutting, and the mutant gene with excellent autogenous cutting resistance is obtained through gene mutation and screening, so that the application level of the alkaline protease is improved.
Drawings
FIG. 1 alkaline protease surface specific amino acid search
FIG. 2 alkaline protease surface A: 161(Y), B: 183(F)
FIG. 3 is a PCR amplification electrophoretogram of the wild-type alkaline protease gene of the present invention
Wherein: m is DNA Marker, 1 is aprE gene;
FIG. 4 is a diagram illustrating the restriction enzyme digestion of pWB980 of the present invention
Wherein: m is DNA Marker, 1 is pWB 980-aprE and is subjected to double enzyme digestion by BamHI and XbaI;
FIG. 5 enzyme activities of wild-type alkaline protease and alkaline protease mutant
FIG. 6 enzyme activity retention rates of wild-type alkaline protease and alkaline protease mutant
The specific implementation mode is as follows:
the technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.
Example 1: search and analysis of surface specific site of alkaline protease
The sequences of alkaline proteases were searched in the PDB structure database, and four models with the highest consistency were used as the study objects to observe hydrophobic amino acid residues that are present outside the structure together, FIG. 1-A shows all hydrophobic amino acids on the surface of alkaline proteases, but the number of factors is large, and the alkaline proteases themselves are more prone to cut aromatic amino acids, so that three amino acids of FYW were selected as the major focus sites, and P was not selected because of the spatial structure of P, and as shown in FIG. 1-B, the sequences containing FYW in the sequence were set to X and then aligned with the original sequence, and the white part is the difference part, and FIG. 2 also shows the secondary structure of the original sequence (red- α helix, blue- β fold, gray-random coil), and the 3D structure 4cfz resolved by alkaline proteases was used as the observation objects to see whether these difference sites are present on the surface of proteases, and finally 161(Y), 183(F) as the candidate mutation site, as shown in FIG. 2, were determined.
Example 2: alkaline protease virtual amino acid mutations
The DiscoveryStudio software can use the MODELER program to modify amino acid residues to other amino acids of a specified type, while disulfide bonds, cis-proline and additional constraints can be set to add modeling constraints and optimize the modified amino acid residues. The present study performed single-site virtual amino acid saturation mutagenesis of protein complexes. For each mutant, the difference in binding free energy between the wild type and mutant structures was calculated. All electrostatic energy terms are calculated from the protonation state of the input structure, which takes into account the influence of the solvent using a generalized born implicit film approximation. The electrostatic term is approximated as the sum of coulombic interaction and the contribution of polarity to solvation energy. The energy functional also includes van der waals interaction energy, side chain entropy terms, and non-polar surface terms. Mutations can be negative for stabilization of the mutation and vice versa. Table 1 shows the best three mutation results after saturation mutation at each site, and the candidate mutation types provide guidance for subsequent molecular experiments.
TABLE 1 virtual amino acid mutation energies and evaluation results
Example 3: construction of wild type alkaline protease aprE recombinant strain
1. The wild type alkaline protease gene aprE is derived from Bacillus clausii (Bacillus clausii), the genome DNA of the wild type alkaline protease gene aprE is extracted, and the extraction process refers to an operation manual of a kit.
The aprE amplification primers were designed with the following sequences:
primer 1: F5'-CGGGATCCCGGCTGAAGAAGCAAAAGAAAAATATTTAATTG-3'
Primer 2: R5'-GCTCTAGAGCTTAGCGTGTTGCCGCTTC-3'
The reaction conditions of PCR were: PrimeSTAR Max Premix (2X) 25. mu.l, F (10 pmol/. mu.L) 2. mu.L,
R(10pmol/μL)2μL，Template 2μL，ddH2O 50μL。
the setting of the amplification program is as follows: pre-denaturation: 5min at 95 ℃; denaturation: 30s at 95 ℃; annealing: 45s at 57 ℃; extension: 2min at 72 ℃; reacting for 30 cycles; extension: 10min at 72 ℃.
The PCR product was subjected to agarose gel electrophoresis to visualize the band of the wild-type alkaline protease gene, 1062bp in total (see FIG. 3), and then the PCR product was recovered by a small amount of DNA recovery kit to obtain the wild-type alkaline protease gene, i.e., aprE.
2. Construction of expression vector containing wild type alkaline protease gene
The plasmid pWB980 was extracted, and the extraction process was carried out according to the manual of the kit. After BamHI and XbaI double digestion, agarose gel electrophoresis was carried out to see the band of the wild type alkaline protease gene, which was 5080bp (see FIG. 4), and the product was recovered by a small amount of DNA recovery kit to obtain the vector gene sequence.
3. The target fragment (aprE) which is subjected to double enzyme digestion by BamHI and XbaI is connected with the vector fragment to form a recombinant vector, and the connection condition is 16 ℃ for 4 h.
4. And (3) transforming the recombinant vector into bacillus subtilis WB600 host bacteria, and screening positive transformants to obtain a recombinant strain expressing wild-type alkaline protease aprE, which is named as bacillus subtilis QY. The recombinant plasmid in Bacillus subtilis was extracted and designated pQY (containing the wild-type aprE gene).
Example 4 construction of alkaline protease mutants by inverse PCR
1. Directional mutation based on reverse PCR technology
The reaction conditions of the inverse PCR were: PrimeSTAR Max Premix (2X) 25. mu.l, F (10 pmol/. mu.L) 2. mu.L,
R(10pmol/μL)2μL，Template 2μL，ddH2O 50μL。
primer 3: F5'-AATTCAGGTGCAGGCTCAATCAGCACCCCGGC-3'
Primer 4: R5'-CCCAGATGCCGCTACAACAAGAACGCCTCTAGAAG-3'
Primer 5: F5'-AACAACAACCGCGCCAGCGATTCACAGTATGGC-3'
Primer 6: R5'-TTGGTCAGTAGCTCCGACTGCCATTGCGTTCG-3'
Taking primers 3 and 4 and primers 5 and 6 as an upstream primer and a downstream primer respectively, wherein the amplification conditions are as follows: 10min at 94 ℃; 60s at 94 ℃, 1.5min at 58 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃.
2. Construction of expression vectors containing mutants
And (3) carrying out self-ligation on the PCR product to obtain a recombinant vector of the alkaline protease mutant gene, and transforming the recombinant vector into the bacillus subtilis WB600 to obtain the alkaline protease mutant.
EXAMPLE 5 screening of alkaline protease mutants
1. Preliminary screening
The recombinant strain expressing the alkaline protease mutant obtained in example 2 was inoculated on a kanamycin-resistant plate, cultured at 37 ℃ for 12 hours, and a single colony was picked and inoculated in 50mL of LB medium (kanamycin-resistant) at 37 ℃ for 48 hours with shaking at 220r/min, and at the same time, Bacillus subtilis QY was inoculated as a control.
And (2) taking AAPF as a substrate, determining the enzyme activity of alkaline protease in the fermentation supernatant, finally screening 2 strains with fermentation liquor enzyme activity obviously higher than that of the bacillus subtilis QY, and respectively naming the strains as bacillus subtilis QY-5 and QY-6.
2. Fermentation and enzyme liquid preparation
The 2 strains (QY-5, QY-6) selected above and the control bacterium Bacillus subtilis QY were inoculated on kanamycin-resistant plates, cultured at 37 ℃ for 12 hours, a single colony was selected and inoculated in 5mL of LB medium (kanamycin-resistant), cultured with shaking at 37 ℃ for 12 hours, inoculated in 2% of the inoculum size in 100mL of LB medium (kanamycin-resistant), and cultured with shaking at 37 ℃ for 48 hours. And (3) performing ultrafiltration purification on the enzyme solution obtained by fermentation, incubating at the constant temperature of 40 ℃ for 48h, sampling at a fixed point, and determining the enzyme activity.
3. Evaluation experiment of anti-autogenous cutting performance
And (2) respectively detecting the enzyme activities of the supernatants of the 2 strains (QY-5 and QY-6) and the contrast bacteria bacillus subtilis QY after fermentation for 48 hours by taking AAPF as a substrate, wherein the enzyme activities of the alkaline protease mutants are respectively 27U/mL and 31U/mL, and the enzyme activities of the wild alkaline protease aprE expressed by the specific gravity group are respectively improved by 17 percent and 38 percent. The enzyme activity retention rate of the alkaline protease mutant is improved by 41 percent and 70 percent compared with the enzyme activity of the wild alkaline protease.
EXAMPLE 6 determination of sequences of alkaline protease mutants
Obtaining the alkaline protease mutant which accords with the virtual amino acid mutation result by sequencing, wherein the sequencing result shows that: the nucleotide sequence of the alkaline protease gene carried by the plasmid in the bacillus subtilis QY-5 is SEQ ID NO: 3, the encoded amino acid sequence is SEQ ID NO: 4, the applicants have named the alkaline protease aprE-M5; the nucleotide sequence of the alkaline protease gene carried by the plasmid in the bacillus subtilis QY-6 is SEQ ID NO: 5, the coded amino acid sequence is SEQID NO: 6, the applicants have named the alkaline protease aprE-M6;
further, the applicants have compared the amino acid sequences of the alkaline proteases aprE-M5 and aprE-M6 obtained as described above with the amino acid sequence of the wild-type alkaline protease aprE of SEQ ID NO: 1 comparative analysis. The results show that: compared with the wild alkaline protease aprE, the 161 th amino acid of the alkaline protease aprE-M5 is mutated from Tyr to Thr; the 183 th amino acid of the alkaline protease aprE-M2 was mutated from Phe to Asp.
Claims (3)
1. Screening of novel autocleavable alkaline protease, characterized in that: novel autocleavage resistant alkaline protease mutants are obtained by virtual amino acid mutation.
2. The novel autocleavage resistant alkaline protease mutant of claim 1 having one or both of the following mutations: the 733-rd and 735-th mutation of the gene sequence is ACC, the 799-th and 801-th mutation is GAT, the corresponding amino acid sequence mutation is Thr at the 161-th mutation and Asp at the 183-th mutation.
3. The novel autocleavage resistant mutant of alkaline protease as claimed in claim 1, wherein: the alkaline protease mutant has enzyme activity retention rates which are respectively improved by more than 40 percent compared with wild type after being incubated for 48 hours under the condition of optimal enzyme activity.
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Cited By (2)
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| WO2022134236A1 (en) * | 2020-12-21 | 2022-06-30 | 天津科技大学 | Alkaline protease mutant, gene thereof, engineering bacteria thereof, preparation method therefor and use thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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