CN115717137B - Lysyl specific endonuclease mutant and preparation method and application thereof - Google Patents
Lysyl specific endonuclease mutant and preparation method and application thereof Download PDFInfo
- Publication number
- CN115717137B CN115717137B CN202211681726.7A CN202211681726A CN115717137B CN 115717137 B CN115717137 B CN 115717137B CN 202211681726 A CN202211681726 A CN 202211681726A CN 115717137 B CN115717137 B CN 115717137B
- Authority
- CN
- China
- Prior art keywords
- lysyl
- specific endonuclease
- mutant
- recombinant
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010042407 Endonucleases Proteins 0.000 title claims abstract description 50
- 102000004533 Endonucleases Human genes 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 125000001288 lysyl group Chemical group 0.000 title abstract 5
- 230000035772 mutation Effects 0.000 claims abstract description 36
- 102220523563 C-C motif chemokine 2_Y51D_mutation Human genes 0.000 claims abstract description 16
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 44
- 241000894006 Bacteria Species 0.000 claims description 27
- 239000013604 expression vector Substances 0.000 claims description 23
- 108091033319 polynucleotide Proteins 0.000 claims description 16
- 239000002157 polynucleotide Substances 0.000 claims description 16
- 102000040430 polynucleotide Human genes 0.000 claims description 16
- 238000003259 recombinant expression Methods 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 11
- 238000010276 construction Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 abstract description 44
- 108090000790 Enzymes Proteins 0.000 abstract description 44
- 230000000694 effects Effects 0.000 abstract description 44
- 150000001413 amino acids Chemical class 0.000 abstract description 10
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 42
- 102100031780 Endonuclease Human genes 0.000 description 22
- 108090000623 proteins and genes Proteins 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108010053229 Lysyl endopeptidase Proteins 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- -1 trypton 10 g/L Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 1
- 241000590020 Achromobacter Species 0.000 description 1
- 241000590035 Achromobacter lyticus Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010019957 Escherichia coli periplasmic proteinase Proteins 0.000 description 1
- GHSJKUNUIHUPDF-BYPYZUCNSA-N L-thialysine Chemical group NCCSC[C@H](N)C(O)=O GHSJKUNUIHUPDF-BYPYZUCNSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 241001478887 unidentified soil bacteria Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of genetic engineering, in particular to a lysyl specific endonuclease mutant, a preparation method and application thereof. The lysyl specific endonuclease mutant is obtained by mutating amino acid at 51 st and/or 137 th in wild lysyl specific endonuclease as follows: Y51D mutation, R137K mutation. Compared with the wild lysyl specific endonuclease, the mutant provided by the invention has obviously improved enzyme activity and enzyme activity stability.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a lysyl specific endonuclease mutant, a preparation method and application thereof.
Background
Lysyl-specific endonucleases (Lysyl endopeptidase, EC 3.4.21.50), also known as lysyl endopeptidases, achromobacter proteinase I, lysine endopeptidases, lysyl endonucleases, lysine C-terminal endonucleases, lys-C endonucleases, are serine proteases originally discovered and isolated from soil bacteria by Masaki et al. Lysyl endopeptidases are highly specific and specifically cleave peptide bonds at the carboxy terminus of lysine residues and S-aminoethylcysteine residues in peptide chains.
Lysyl-specific endonucleases are polypeptides consisting of 268 amino acid residues, in which the peptide chain contains three disulfide bonds (Cys 6-Cys216, cys12-Cys80, cys36-Cys 58) inside, the triplet consisting of His57, asp113 and Ser194 determines the catalytic activity of the enzyme, while Asp225 determines its specific selectivity towards lysine.
Lysyl-specific endonucleases have only 20% homology with bovine trypsin, but the amino acid sequence and spatial structure that determine the catalytic activity of the enzyme and the lysine specificity are completely identical, and thus lysyl-specific endonucleases are classified as trypsin family. Compared with bovine trypsin, lysyl-specific endonucleases have higher selectivity for lysine carbon-terminal, and have 10 times of activity than trypsin, a wider optimal pH range (pH 8.5-10.5), and still normal stability in 4M urea or 0.1% SDS. These properties make lysyl-specific endonucleases a very useful tool enzyme in biomedical production.
The lysyl endonuclease produced by using the natural achromobacter Achromobacter lyticus M497-1 has low expression quantity, long period and obviously higher cost, so that the price of the lysyl endonuclease is high. The escherichia coli is the most widely used protein expression system at present, and the escherichia coli expression system has clear genetic background and physiological characteristic research and is developed and can be used by various commercial engineering bacteria; and the escherichia coli is easy to culture and control, simple in transformation operation, and has the characteristics of high expression level, low cost, short period and the like.
However, due to the self amino acid sequence and structural characteristics of lysyl specific endonuclease, the inclusion body obtained by the existing widely applied escherichia coli expression system has the problems of reduced enzyme activity, insufficient stability and the like. Therefore, there is still a need for lysyl-specific endonucleases that are better adapted to E.coli expression systems and have higher enzyme activities and stability of enzyme activities. In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides the lysyl specific endonuclease mutant, the recombinant genetically engineered bacterium and the construction method thereof through a large amount of researches, and the lysyl specific endonuclease mutant has remarkably improved enzyme activity and enzyme activity stability.
The first aspect of the present invention provides a lysyl-specific endonuclease mutant obtained by mutating amino acids at position 51 and/or 137 of a wild-type lysyl-specific endonuclease as follows: Y51D mutation and R137K mutation. The amino acid Sequence of the wild lysyl specific endonuclease is shown as Sequence ID in Genbank 1ARB_A, and specifically comprises the following steps:
GVSGSCNIDVVCPEGDGRRDIIRAVGAYSKSGTLACTGSLVNNTANDRKMYFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVKATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAEKRISNSTSPTSFVAWGGGAGTTHLNVQWQPSGGVTEPGSSGSPIYSPEKRVLGQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP。
the invention carries out intensive research analysis on lysyl specific endonuclease sequences, screens tens of mutation points and combinations thereof, and finally obtains the Y51D mutation, the R137K mutation and the combinations thereof. The mutant can improve the in vitro renaturation efficiency of the protein, improve the solubility of the protein, maintain the specificity and activity of the protease, and remarkably improve the enzyme activity and the enzyme activity stability of the protease.
Wherein the mutation has the meaning shown in Table 1:
TABLE 1
As an improvement of the technical scheme of the invention, the mutation of the lysyl specific endonuclease mutant is selected from the group consisting of:
(1) Y51D mutation;
(2) R137K mutation;
(3) Y51D mutation and R137K mutation.
As an improvement of the technical scheme of the invention, specific examples of mutants include:
(1) Mutant HSE-LCs, including Y51D mutations and R137K mutations;
(2) Mutant HSE-LC-1, comprising only the Y51D mutation;
(3) Mutant HSE-LC-2, comprising only the R137K mutation.
Experimental results prove that both the mutant HSE-LC and the mutant HSE-LC-2 can obviously improve the relative enzyme activity, and the mutant HSE-LC has better effect. The mutant HSE-LC and the mutant HSE-LC-1 can both obviously improve the enzyme activity stability, and the effect of the mutant HSE-LC is better. The comprehensive relative enzyme activity and enzyme activity stability experimental results show that the comprehensive performance of the mutant HSE-LC is optimal.
In a second aspect the invention provides a polynucleotide sequence encoding the recombinant fusion protein described above. Wherein, for example, the coding sequence is designed and optimized according to the amino acid sequence of the mutant HSE-LC, and the specific polynucleotide sequence is obtained as follows:
ggcgtgagcggcagctgcaacattgatgtggtgtgcccggaaggcgatggccgccgcgatattattcgcgcggtgggcgcgtatagcaaaagcggcaccctggcgtgcaccggcagcctggtgaacaacaccgcgaacgatcgcaaaatggattttctgaccgcgcatcattgcggcatgggcaccgcgagcaccgcggcgagcattgtggtgtattggaactatcagaacagcacctgccgcgcgccgaacaccccggcgagcggcgcgaacggcgatggcagcatgagccagacccagagcggcagcaccgtgaaagcgacctatgcgaccagcgattttaccctgctggaactgaacaacgcggcgaacccggcgtttaacctgttttgggcgggctgggatcgcaaagatcagaactatccgggcgcgattgcgattcatcatccgaacgtggcggaaaaacgcattagcaacagcaccagcccgaccagctttgtggcgtggggcggcggcgcgggcaccacccatctgaacgtgcagtggcagccgagcggcggcgtgaccgaaccgggcagcagcggcagcccgatttatagcccggaaaaacgcgtgctgggccagctgcatggcggcccgagcagctgcagcgcgaccggcaccaaccgcagcgatcagtatggccgcgtgtttaccagctggaccggcggcggcgcggcggcgagccgcctgagcgattggctggatccggcgagcaccggcgcgcagtttattgatggcctggatagcggcggcggcaccccg
in a third aspect the present invention provides a recombinant expression vector for expressing a lysyl-specific endonuclease mutant, the recombinant expression vector comprising the nucleotide sequence as described above.
The fourth aspect of the present invention provides a recombinant genetically engineered bacterium comprising the recombinant expression vector described above. The recombinant genetically engineered bacterium is prepared by the following method:
(1) Synthesizing the polynucleotide sequence;
(2) Inserting the polynucleotide into an expression vector, and constructing to obtain a recombinant expression vector;
(3) And (3) introducing the recombinant expression vector into host bacteria to obtain recombinant genetically engineered bacteria for expressing lysyl specific endonuclease mutants.
Among them, the host bacterium is selected from E.coli, and E.coli expression strain BL21 (DE 3) is further preferred. Specifically, the expression vector of the embodiment of the invention preferably uses a plasmid vector, and specifically a plasmid pET-32a (+); and preferably, the recombinant plasmid is constructed by inserting the polynucleotide of the present invention through NcoI and XhoI cleavage sites. The fourth aspect of the invention provides a construction method of recombinant genetically engineered bacteria expressing lysyl specific endonuclease mutants, comprising the following steps:
(1) Synthesizing the polynucleotide sequence;
(2) Inserting the polynucleotide into an expression vector, and constructing to obtain a recombinant expression vector;
(3) And (3) introducing the recombinant expression vector into host bacteria to obtain recombinant genetically engineered bacteria for expressing lysyl specific endonuclease mutants.
Among them, the host bacterium is selected from E.coli, and E.coli expression strain BL21 (DE 3) is further preferred. Specifically, the expression vector of the embodiment of the invention preferably uses a plasmid vector, and specifically a plasmid pET-32a (+); and preferably, the recombinant plasmid is constructed by inserting the polynucleotide of the present invention through NcoI and XhoI cleavage sites.
Compared with the prior art, the technical scheme provided by the invention has the following advantages:
the lysyl specific endonuclease mutant has higher enzyme activity and enzyme activity stability compared with a wild type.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
Example 1: preparation of lysyl-specific endonucleases
1. According to the amino acid sequence of lysyl specific endonuclease, designing and synthesizing its coding polynucleotide sequence, such as mutant HSE-LC, which is mutant enzyme obtained by respectively making Y51D and R137K mutations on amino acids 51 and 137 of wild type lysyl specific endonuclease, the amino acid sequence is specifically shown as follows:
GVSGSCNIDVVCPEGDGRRDIIRAVGAYSKSGTLACTGSLVNNTANDRKMDFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVKATYATSDFTLLELNNAANPAFNLFWAGWDRKDQNYPGAIAIHHPNVAEKRISNSTSPTSFVAWGGGAGTTHLNVQWQPSGGVTEPGSSGSPIYSPEKRVLGQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP；
the coding sequence is designed and optimized according to the amino acid sequence, and the coding nucleotide sequence is as follows:
ggcgtgagcggcagctgcaacattgatgtggtgtgcccggaaggcgatggccgccgcgatattattcgcgcggtgggcgcgtatagcaaaagcggcaccctggcgtgcaccggcagcctggtgaacaacaccgcgaacgatcgcaaaatggattttctgaccgcgcatcattgcggcatgggcaccgcgagcaccgcggcgagcattgtggtgtattggaactatcagaacagcacctgccgcgcgccgaacaccccggcgagcggcgcgaacggcgatggcagcatgagccagacccagagcggcagcaccgtgaaagcgacctatgcgaccagcgattttaccctgctggaactgaacaacgcggcgaacccggcgtttaacctgttttgggcgggctgggatcgcaaagatcagaactatccgggcgcgattgcgattcatcatccgaacgtggcggaaaaacgcattagcaacagcaccagcccgaccagctttgtggcgtggggcggcggcgcgggcaccacccatctgaacgtgcagtggcagccgagcggcggcgtgaccgaaccgggcagcagcggcagcccgatttatagcccggaaaaacgcgtgctgggccagctgcatggcggcccgagcagctgcagcgcgaccggcaccaaccgcagcgatcagtatggccgcgtgtttaccagctggaccggcggcggcgcggcggcgagccgcctgagcgattggctggatccggcgagcaccggcgcgcagtttattgatggcctggatagcggcggcggcaccccg。
2. construction of recombinant expression vector and construction of engineering bacteria
The coding genes are synthesized and respectively inserted between NcoI and XhoI sites of pET-32a (+) plasmid to construct recombinant plasmid; the recombinant plasmid is transformed into escherichia coli Escherichia coli BL (DE 3), LB solid culture medium (containing 30 mug/mL kanamycin) is coated on the transformation liquid, and the engineering bacteria containing the target gene recombinant plasmid are obtained by resistance screening.
Sequencing and verification prove that the sequence in the obtained engineering bacteria is consistent with the designed sequence.
The gene synthesis and sequencing services were all performed by the biotechnology company Jin Weizhi, su.
3. Inducible expression of a protein of interest
(1) The preparation method of the culture medium comprises the following steps:
LB liquid medium, trypton 10 g/L, yeast extract 5 g/L, naCl 10 g/L.
LB solid medium, trypton 10 g/L, yeast extract 5 g/L, naCl 10 g/L and agar 20 g/L.
(2) Expression of the protein of interest
And inoculating the preserved recombinant engineering bacteria into an LB liquid culture medium, culturing at 30 ℃ and 220 rpm overnight, measuring OD600, transferring into a fresh LB culture medium of 20 mL, culturing until OD600 = 0.6-4.0, adding IPTG (final concentration is 0.1-1.0 mM), culturing at 16-30 ℃ and 220 rpm for 12-24 hours in an induction way, and collecting supernatant to measure the enzyme activity.
The coding sequences for the wild-type lysyl-specific endonucleases, mutant HSE-LC-1 and mutant HSE-LC-2 were prepared separately according to the above procedure, and the enzymes were prepared according to the above procedure.
Example 2: enzyme activity detection
Preparing a substrate solution, wherein the substrate solution comprises the following formula: 180 mmol/L Tris-HCl,0.25 mmol/L Bz-Lys-pNA, pH 9.2.
1450. Mu.L of the substrate solution was heated in a constant temperature water bath at 30℃for 5 minutes, then 50. Mu.L of the supernatant (dilution of an appropriate multiple) obtained in the step (2) of example 1 was added thereto, the reaction was carried out in a water bath at 30℃for 5 minutes, 500. Mu.L of 45% (V/V) acetic acid solution was immediately added thereto to terminate the reaction, and after 3-fold dilution with purified water, the absorbance of the reaction solution was measured at 405. 405 nm wavelength.
The enzyme activity is defined as: the amount of enzyme catalyzing the substrate to 1. Mu. Mol of p-nitroaniline per minute at 30℃is defined as 1U. The experimental results of calculating the relative enzyme activities, which are defined as 100% relative enzyme activities by the wild-type enzyme activities, are shown in Table 2:
TABLE 2
As can be seen from the experimental data in Table 2, the activity of the mutant HSE-LC-1 containing the Y51D mutation was slightly decreased, but the activity of the mutant HSE-LC-2 containing the R137K mutation was significantly increased, compared with the wild-type lysyl-specific endonuclease. Meanwhile, experiments prove that the mutant HSE-LC containing both the Y51D mutation and the R137K mutation has the enzyme activity which is more than 3 times that of a wild type, and the enzyme activity is obviously improved.
Example 3: stability study
The supernatant obtained in step (2) of example 1 was collected, left at room temperature, sampled at 0, 24 and 72 hours, and the enzyme activity was measured as in example 2, defined as 100% relative enzyme activity per se 0 h, and the enzyme activity stability was calculated as shown in Table 3.
TABLE 3 Table 3
As is clear from the experimental data in Table 3, the mutant HSE-LC-1 comprising the Y51D mutation had an increased enzyme activity stability as compared with the wild-type lysyl-specific endonuclease, but the mutant HSE-LC-2 comprising the R137K mutation had an essentially unchanged enzyme activity stability. However, the mutant HSE-LC combining the mutations of the two has improved enzyme activity stability.
It is known from the contents of examples 2 and 3 of the present invention that the Y51D mutation can bring about improvement of enzyme activity stability, but has limited influence on enzyme activity, while the R137K mutation has remarkable improvement on enzyme activity, but has no influence on enzyme activity stability basically; however, when the two are combined, the enzyme activity and the enzyme activity stability are obviously improved, and the influence of the Y51D mutation on the structural stability of the wild type lysyl specific endonuclease is seen to further improve the enzyme activity, so that the combination of the two mutations obtains obvious enzyme activity and enzyme activity stability improvement.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (12)
1. A lysyl-specific endonuclease mutant, characterized in that the lysyl-specific endonuclease mutant is obtained by mutating the amino acid at position 51 and/or 137 of a wild-type lysyl-specific endonuclease as follows: Y51D mutation and/or R137K mutation;
the amino acid Sequence of the wild type lysyl specific endonuclease is shown as Sequence ID in Genbank 1 ARB_A.
2. The lysyl-specific endonuclease mutant according to claim 1, wherein the mutation of the lysyl-specific endonuclease mutant is selected from the group consisting of:
(1) Y51D mutation;
(2) R137K mutation; or (b)
(3) Y51D mutation and R137K mutation.
3. Polynucleotide sequence encoding a lysyl-specific endonuclease mutant according to any one of claims 1-2.
4. A recombinant expression vector for expressing a lysyl-specific endonuclease mutant, wherein said recombinant expression vector comprises the polynucleotide sequence of claim 3.
5. A recombinant genetically engineered bacterium comprising the recombinant expression vector of claim 4.
6. The recombinant genetically engineered bacterium of claim 5, wherein the recombinant genetically engineered bacterium is constructed by:
(1) Synthesizing the polynucleotide sequence of claim 3;
(2) Inserting the polynucleotide into an expression vector to construct a recombinant expression vector;
(3) And introducing the recombinant expression vector into host bacteria to obtain the recombinant genetically engineered bacteria for expressing the lysyl specific endonuclease mutant.
7. The recombinant genetically engineered bacterium of claim 6, wherein the host bacterium is selected from the group consisting of escherichia coli expression strain BL21 (DE 3).
8. The recombinant genetically engineered bacterium of claim 6, wherein the expression vector is selected from the group consisting of plasmid pET-32a (+).
9. The construction method of the recombinant genetically engineered bacterium for expressing lysyl specific endonuclease mutant is characterized by comprising the following steps:
(1) Synthesizing the polynucleotide sequence of claim 3;
(2) Inserting the polynucleotide into an expression vector to construct a recombinant expression vector;
(3) And introducing the recombinant expression vector into host bacteria to obtain the recombinant genetically engineered bacteria for expressing the lysyl specific endonuclease mutant.
10. The method of claim 9, wherein the host bacterium is selected from the group consisting of E.coli expression strain BL21 (DE 3).
11. The method of construction according to claim 9, characterized in that the expression vector is selected from the group consisting of plasmid pET-32a (+).
12. The method of construction according to claim 11, wherein the polynucleotide is inserted between the NcoI and XhoI sites of pET-32a (+) plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211681726.7A CN115717137B (en) | 2022-12-27 | 2022-12-27 | Lysyl specific endonuclease mutant and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211681726.7A CN115717137B (en) | 2022-12-27 | 2022-12-27 | Lysyl specific endonuclease mutant and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115717137A CN115717137A (en) | 2023-02-28 |
| CN115717137B true CN115717137B (en) | 2024-01-26 |
Family
ID=85257820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211681726.7A Active CN115717137B (en) | 2022-12-27 | 2022-12-27 | Lysyl specific endonuclease mutant and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115717137B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116355101B (en) * | 2023-03-09 | 2024-05-03 | 北京惠之衡生物科技有限公司 | Preparation method of insulin aspart |
| CN116284452A (en) * | 2023-03-09 | 2023-06-23 | 北京惠之衡生物科技有限公司 | Preparation method of recombinant human insulin |
| CN116425884B (en) * | 2023-03-09 | 2024-04-26 | 北京惠之衡生物科技有限公司 | De-glu-insulin purifying and preparing process |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997033984A1 (en) * | 1996-03-12 | 1997-09-18 | Novo Nordisk A/S | Novel achromobacter lyticus protease variants |
| CN103509775A (en) * | 2012-06-15 | 2014-01-15 | 上海抗体药物国家工程研究中心有限公司 | Achromobacter protease I variant |
| WO2017005213A1 (en) * | 2015-07-09 | 2017-01-12 | 广东东阳光药业有限公司 | Preparation method of recombined lysine-specific enzyme |
| CN112824527A (en) * | 2019-11-20 | 2021-05-21 | 珠海联邦制药股份有限公司 | Artificially designed lysyl endonuclease, coding sequence and fermentation method |
-
2022
- 2022-12-27 CN CN202211681726.7A patent/CN115717137B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997033984A1 (en) * | 1996-03-12 | 1997-09-18 | Novo Nordisk A/S | Novel achromobacter lyticus protease variants |
| CN103509775A (en) * | 2012-06-15 | 2014-01-15 | 上海抗体药物国家工程研究中心有限公司 | Achromobacter protease I variant |
| WO2017005213A1 (en) * | 2015-07-09 | 2017-01-12 | 广东东阳光药业有限公司 | Preparation method of recombined lysine-specific enzyme |
| CN112824527A (en) * | 2019-11-20 | 2021-05-21 | 珠海联邦制药股份有限公司 | Artificially designed lysyl endonuclease, coding sequence and fermentation method |
Non-Patent Citations (3)
| Title |
|---|
| Histidine 2 10 Mutant of a Trypsin-Type Achromobacter Protease I Shows Broad Optimum pH Range;KENTARO SHIRAKI 等;JOURNALO F BIOSCIENCAEN DB IOENGINEERING;第93卷(第3期);331-333 * |
| Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0: Protonation states and hydration structure in the free-form;Yuki Ohnishi 等;Biochimica et Biophysica Acta;第1834卷;1642-1647 * |
| 无色杆菌蛋白酶工突变体的结构与稳定性研究;张红缨 等;生物化学杂志;第13卷;73-78 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115717137A (en) | 2023-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115717137B (en) | Lysyl specific endonuclease mutant and preparation method and application thereof | |
| CA2756752C (en) | Improved type milk-clotting protease derived from a microorganism | |
| CN113862233B (en) | Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application | |
| CN108841851B (en) | Method for expressing glutamine transaminase by using food-source-safe host | |
| JP4815219B2 (en) | DNA containing an alkalophilic cyclodextran synthase gene, recombinant DNA, and method for producing an alkalophilic cyclodextran synthase | |
| CN108410845B (en) | D, D-carboxypeptidase DacA mutant with improved catalytic efficiency and preparation method thereof | |
| CN115896050A (en) | End transformation combined point mutation of 7 alpha-hydroxysteroid dehydrogenase and efficient synthesis of ursodeoxycholic acid intermediate | |
| CN112301014B (en) | Esterase mutant with improved thermal stability and application thereof | |
| WO2008047936A1 (en) | LYTIC ENZYME INHIBITOR, LYSIS INHIBITOR, INHIBITOR OF DEGRADATION OF POLY-γ-GLUTAMIC ACID, AND METHOD FOR PRODUCTION OF POLY-γ-GLUTAMIC ACID | |
| JP2020522999A (en) | Glucose oxidase CnGODA and its gene and use | |
| JP5289801B2 (en) | Protein with uricase activity | |
| JP5247112B2 (en) | Bacterial enzyme inhibitor, lysis inhibitor, poly-gamma-glutamic acid degradation inhibitor, and method for producing poly-gamma-glutamic acid | |
| CN115261363A (en) | Method for determining RNA deaminase activity of APOBEC3A and APOBEC3A variant with high RNA activity | |
| CN113046376A (en) | Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof | |
| KR900007648B1 (en) | Stable creatine amidihony-drolase mutants | |
| JP2001292772A (en) | Nitrile hydratase gene and amidase gene derived from bacterium belonging to the genus rhodococcus | |
| CN114480345B (en) | MazF mutant, recombinant vector, recombinant engineering bacterium and application thereof | |
| CN112680425B (en) | Alcohol dehydrogenase mutant and application thereof | |
| CN113073107B (en) | Mannase gene AbMan5, recombinant expression plasmid, recombinant expression strain, mannase and application thereof | |
| JP2729045B2 (en) | Sarcosine oxidase and method for producing the same | |
| KR102235833B1 (en) | Novel triolein selective lipase gene Lip-1307 derived from soil metagenome and use thereof | |
| JPH10174585A (en) | Stable creatine amidinohydrolase | |
| CN113913414B (en) | Double-base enzyme Kex2 mutant with high stability and high catalytic efficiency | |
| JP2005168487A (en) | Modified sacrosine oxidase, modified sacrosine oxidase gene, and method for producing the modified sacrosine oxidase | |
| JP2008022766A (en) | Method for increasing specific activity of uricase and modified uricase having improved specific activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 100025 21 floor, 2 building, 2000 business center, Eight Mile Village, Chaoyang District, Beijing. Applicant after: Beijing huizhiheng Biotechnology Co.,Ltd. Applicant after: Jilin Huisheng Biopharmaceutical Co.,Ltd. Address before: 100025 21 floor, 2 building, 2000 business center, Eight Mile Village, Chaoyang District, Beijing. Applicant before: Beijing huizhiheng Biotechnology Co.,Ltd. Applicant before: Jilin Huisheng biopharmaceutical Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |