CN116555219B - Phytase mutant with improved thermal stability and preparation method and application thereof - Google Patents
Phytase mutant with improved thermal stability and preparation method and application thereof Download PDFInfo
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- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 185
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- 239000012634 fragment Substances 0.000 claims abstract description 44
- 238000010353 genetic engineering Methods 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 6
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- 238000012408 PCR amplification Methods 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 22
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- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 16
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- 241000894006 Bacteria Species 0.000 claims description 4
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- 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 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 2
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- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
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- 101000688187 Escherichia coli (strain K12) Phytase AppA Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
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- 229920002684 Sepharose Polymers 0.000 description 1
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- UNTBPXHCXVWYOI-UHFFFAOYSA-O azanium;oxido(dioxo)vanadium Chemical compound [NH4+].[O-][V](=O)=O UNTBPXHCXVWYOI-UHFFFAOYSA-O 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03008—3-Phytase (3.1.3.8)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention discloses a phytase mutant with improved thermal stability, and a preparation method and application thereof, belonging to the fields of genetic engineering and enzyme engineering. The amino acid sequence of the phytase mutant is shown as SEQ ID NO. 1. The gene sequence of the coding phytase mutant is shown as SEQ ID NO. 2. The invention adopts the USERecDNA recombination technology to replace the structural fragments Val 23-Thr 67 in the phytase YIAPPA with the structural fragments Val 21-Thr 65 in the phytase RPhyXT52, and replace the structural fragments Leu 381-Ile 424 in the phytase YIAPPA with the structural fragments Leu 379-Asp 423 in the phytase rPhyXT52, thereby obtaining the phytase mutant Yr-Mut with obviously improved thermal stability and basically unchanged phytase activity, and having good application prospect in the field of animal feed additives.
Description
Technical Field
The invention relates to the fields of genetic engineering and enzyme engineering, in particular to a phytase mutant with improved thermal stability, a preparation method and application thereof.
Background
Approximately 50 to 80% of the phosphorus in animal feed is present in the form of phytic acid. The phytic acid can not only be utilized by monogastric animals, but also be combined with metal cations, amino acids, proteins, starch and other substances to inhibit the absorption of the substances and influence the production performance of the animals, and meanwhile, the phosphorus phytate which is not digested and absorbed by the animals is discharged into soil and water through excrement to cause serious environmental pollution.
Phytase is also known as phytase and can degrade phytic acid to inorganic phosphorus and phosphoinositides. The phytase is added into the feed, so that the utilization rate of the phytic acid phosphorus can be effectively improved, the waste of phosphorus resources is reduced, the digestion and absorption of nutrient substances such as amino acid, protein, mineral elements and the like can be improved, and meanwhile, the environmental pollution caused by the phytic acid can be reduced by reducing the level of the phytic acid phosphorus in the excreta of the monogastric animals. Because of these advantages, phytases have received extensive attention in academia and commercial industries in many countries around the world. The phytase as a common additive in animal feed has wide application prospect and huge commercial value in the world. By 2024, the global phytase market size is expected to increase from dollars 3.8 billion in 2019 to dollars 5.9 billion.
Some of the enzymatic properties of natural phytase are not suitable for the requirements of livestock and poultry cultivation and feed processing, and the popularization and application of phytase in the feed industry are generally limited by the enzymatic properties. Among them, the poor thermal stability of natural phytase has been a key problem limiting the application of phytase in the feed industry. In the production of animal feed, a short high-temperature granulation process is involved, above 80 ℃ and even around 85-95 ℃, which can irreversibly inactivate the phytase. Therefore, searching and developing phytase with better heat resistance and searching better method for improving the heat stability of phytase are still hot spots for research of scholars at home and abroad.
The phytase YIAPPA from Yersinia intermedia is the phytase with highest phytase activity known at present, and the enzyme activity is up to 3960U/mg (37 ℃ C., pH 4.5), which is 40 times of the enzyme activity of the phytase Aspergillus nigerPhyA which is the most widely used at present. The enzymatic properties of YiAPPA make it of great application potential in the feed industry, but the enzymatic properties of YiAPPA are difficult to fully meet the requirements of the feed industry. Taking the thermal stability of YIAPPA as an example, the half-life of YIAPPA at 80 ℃ is only 15min, and the requirements of the feed industry are still difficult to achieve.
Disclosure of Invention
The invention aims to provide a phytase mutant with improved thermal stability, a preparation method and application thereof, and aims to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides phytase Yr-Mut with improved thermostability, and the amino acid sequence of the phytase Yr-Mut is shown as SEQ ID NO. 1.
The invention also provides a gene for encoding the phytase Yr-Mut, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The invention also provides a preparation method of the phytase Yr-Mut, which adopts the USERec DNA recombination technology to replace a structural fragment Y-F2 (Val 23-Thr 67) in the phytase YIAPPA with a structural fragment (Val 21-Thr 65) in the phytase RPhyXT52, and replace a structural fragment Y-F10 (Leu 381-Ile 424) in the phytase YIAPPA with a structural fragment r-F10 (Leu 379-Asp 423) in the phytase RPhyXT52, so as to construct the phytase Yr-Mut;
the amino acid sequence of the structural fragment Y-F2 is shown as SEQ ID NO.33, the amino acid sequence of the structural fragment r-F2 is shown as SEQ ID NO.31, the amino acid sequence of the structural fragment Y-F10 is shown as SEQ ID NO.34, and the amino acid sequence of the structural fragment r-F10 is shown as SEQ ID NO. 32.
Further, the method comprises the following steps:
(1) Cloning the gene sequence of phytase YIAPPA into a plasmid pBE-S to construct a recombinant plasmid pBE-S-YIAppa, wherein the gene sequence of phytase YIAPPA is shown as SEQ ID NO. 4;
(2) Cloning the gene sequence of phytase rPhyXT52 into a plasmid pBE-S to construct a recombinant plasmid pBE-S-rphixt 52, wherein the gene sequence of phytase rPhyXT52 is shown as SEQ ID NO. 6;
(3) Respectively taking a recombinant plasmid pBE-S-yiapp a and a recombinant plasmid pBE-S-rphyxt52 as templates, adopting a USERec DNA recombination technology to replace a structural fragment Y-F2 with a structural fragment r-F2, and replacing a structural fragment Y-F10 with a structural fragment r-F10 to construct a gene sequence of phytase Yr-Mut;
(4) Cloning the gene sequence of phytase Yr-Mut into plasmid pBE-S to construct expression vector pBE-S-yrmut of phytase mutant, the gene sequence of phytase Yr-Mut is shown in SEQ ID NO. 2;
(5) Converting an expression vector pBE-S-yrmut of the phytase mutant into bacillus subtilis to obtain bacillus subtilis genetic engineering bacteria, and fermenting and culturing the bacillus subtilis genetic engineering bacteria to obtain phytase Yr-Mut.
Further, in the step (1), the cloning is performed by using the gene sequence of phytase YIAPPA as a template, and Y-F with the sequence shown as SEQ ID NO.27 and Y-R with the sequence shown as SEQ ID NO.28 as primers.
Further, in the step (2), the cloning is performed by using the gene sequence of phytase rPhyXT52 as a template, R-F with the sequence shown as SEQ ID NO.29 and R-R with the sequence shown as SEQ ID NO.30 as primers.
Further, in step (5), the bacillus subtilis is a Bacillus subtilis RIK1285 competent cell.
Further, in the step (5), the conditions of the fermentation culture were 37℃and 180rpm, and the culture was performed for 30 hours.
The invention also provides application of the phytase Yr-Mut in preparing feed additives.
The invention also provides application of the phytase Yr-Mut in preparing livestock and poultry feed.
The invention discloses the following technical effects:
the invention replaces the structural fragments in the phytase YIAPPA with the corresponding structural fragments in the phytase rPhyXT52 one by one, and constructs mutants related to the phytase YIAPPA and rPhyXT52. Mutants with improved thermostability were selected by comparing the phytase activity and thermostability of each mutant. On the basis, beneficial mutations are further combined, a combined mutant is constructed, and the enzymatic properties of the combined mutant are verified, so that the phytase mutant Yr-Mut with remarkably improved thermal stability and basically unchanged phytase activity is obtained.
The phytase mutant Yr-Mut provided by the invention has the advantages that the residual enzyme activity of heat preservation treatment for 20min at 80 ℃ is improved from 34.81% of control (before mutation) to 78.62%, and the residual enzyme activity is improved by 1.26 times; the residual enzyme activity of the incubation at 100℃for 10min was increased from 9.54% to 81.42% of the control (pre-mutation), by a factor of 7.53. The phytase mutant Yr-Mut has the optimal reaction pH of 4.5, the relative enzyme activity of more than 80 percent in the pH range of 1.0-10.0, the optimal reaction temperature is 55 ℃, and the phytase specific activity is 3892.58U/mg. In addition, the results of in vitro simulated digestion experiments of the phytase YIAPPA and the mutant Yr-Mut show that the release amount of inorganic phosphorus of the phytase mutant Yr-Mut experimental group is 5.61 times that of the phytase YIAPPA experimental group. The phytase mutant Yr-Mut has good application prospect in the field of animal feed additives.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of phytase mutant construction;
FIG. 2 shows the optimal reaction pH for the phytases YIAPPA and Yr-Mut;
FIG. 3 shows the pH stability of the phytases YIAPPA and Yr-Mut;
FIG. 4 shows the optimum reaction temperatures for the phytases YIAPPA and Yr-Mut;
FIG. 5 shows the thermostability of the phytases YIAPPA and Yr-Mut at 80 ℃;
FIG. 6 shows the thermostability of the phytases YIAPPA and Yr-Mut at 100 ℃;
FIG. 7 shows the protease resistance of the phytases YIAPPA and Yr-Mut.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Phytase rPhyXT52 is the best known phytase with the best thermostability and still leaves 93% of the enzyme activity after 10min incubation at 100 ℃. The phytase YIAPPA and rPhyXT52 belong to HAP phytase, and have similar molecular structures and the same catalytic mechanism, and the amino acid sequence similarity of the phytase YIAPPA and rPhyXT52 is 49%. The invention aims to introduce protein molecular structural elements related to thermal stability into the phytase YIAPPA by comparing and analyzing the protein molecular structure and enzymatic properties of the phytase YIAPPA and rPhyXT52, and to carry out molecular modification for improving the thermal stability of the phytase YIAPPA.
Experimental conditions:
1. strain and vector
Coli Escherichia coli HST, bacillus subtilis Bacillus subtilis RIK1285, and bacillus subtilis expression vector pBE-S were all purchased from baori doctor technology (beijing) inc.
2. Enzymes and other biochemical reagents
KOD DNA polymerase and KOD-Plus-neo DNA polymerase are purchased from Toyobo Co., ltd., DNA restriction enzyme from Fermentase Co., PCR product purification kit, plasmid extraction kit E.Z.N.A. from Omega Bio-tek Co., USER enzyme, T4 DNA ligase from New England Biolabs Co., chelating Sepharose TM Fast Flow is purchased from GE Healthcare corporation, the United states, the Bradford method protein concentration determination kit is purchased from Shanghai Biotechnology Inc., pepsin and trypsin are purchased from Beijing Soilebao technologies Inc., gene synthesis is completed by Shanghai Boyi technologies Inc., polymerase chain reaction primer synthesis and sequencing is completed by Shanghai Biotechnology Inc., and other chemical reagents are domestic or imported analytical pure.
3. Culture medium
LB medium (g/L): tryptone 10, yeast extract 5, naCl 10, pH 7.0. The screening medium was LB medium containing 50. Mu.g/mL ampicillin.
The molecular cloning techniques and protein detection techniques used in the present invention are conventional in the art. The techniques not described in detail in the examples below were all performed according to the relevant portions of the following experimental manuals. Green M R, sambrook J.molecular cloning: a laboratory manual [ M ]. New York: cold Spring Harbor Laboratory Press,2012.
EXAMPLE 1 construction of Phytase expression vector
1) Synthesis of the Gene yiappa
According to GenBank ID ABI95370.1 of phytase YIAPPA from Yersinia intermedia, searching to obtain its gene sequence, and delivering to Shanghai Bo Yi Biotechnology Co., ltd for total gene synthesis of phytase YIAPPA, as shown in SEQ ID No. 4.
SEQ ID NO.4：
AATAGTTATGCGATTAGTGCCGCGCCGGTTGCCATACAACCCACGGGCTATACATTGGAGCGAGTGGTTATTTTGAGCCGCCATGGTGTTCGCTCGCCAACCAAACAAACACAGTTAATGAATGATGTTACCCCTGACACGTGGCCGCAATGGCCGGTCGCCGCAGGATACTTAACCCCCCGAGGTGCACAATTAGTGACATTGATGGGCGGATTCTATGGTGATTACTTCCGTAGCCAAGGGTTACTCGCAGCAGGGTGCCCAACTGACGCGGTTATTTATGCTCAGGCCGATGTTGATCAACGAACGCGTTTAACGGGGCAGGCATTCCTTGATGGAATAGCACCGGGGTGTGGACTGAAAGTACATTATCAGGCTGATTTGAAAAAAGTGGATCCGCTGTTTCATCCCGTCGACGCGGGGGTGTGTAAGTTAGATTCGACACAAACCCATAAGGCTGTTGAGGAGCGACTAGGTGGGCCATTAAGTGAACTGAGCAAACGCTATGCTAAGCCCTTTGCCCAGATGGGTGAGATTCTGAATTTTGCGGCATCTCCTTACTGTAAATCACTGCAACAGCAAGGGAAAACCTGTGATTTTGCCAACTTTGCAGCGAATAAGATCACGGTGAACAAGCCGGGGACAAAAGTCTCGCTCAGCGGACCACTGGCACTGTCATCAACCTTAGGTGAGATCTTTTTGCTACAAAATTCACAAGCGATGCCTGATGTTGCCTGGCATCGGTTAACGGGAGAAGATAATTGGATCTCGTTATTATCGTTGCACAATGCGCAATTTGATTTAATGGCAAAAACACCTTATATCGCTCGTCATAAGGGCACACCGTTGCTGCAACAGATCGAGACTGCCCTCGTCCTTCAGCGTGATGCTCAGGGGCAAACATTGCCATTATCACCTCAAACCAAAATTCTGTTCCTCGGGGGACATGATACAAACATCGCCAATATTGCTGGAATGTTGGGGGCTAACTGGCAATTACCACAGCAGCCCGATAATACCCCACCTGGGGGGGGATTGGTCTTCGAGCTATGGCAAAACCCAGATAATCATCAACGTTATGTCGCGGTGAAAATGTTCTATCAAACAATGGGCCAATTGCGAAATGCTGAGAAACTAGACCTGAAAAACAATCCGGCTGGTAGGGTCCCTGTTGCAATAGACGGT TGTGAAAATAGTGGTGATGACAAACTTTGTCAGCTTGATACCTTCCAAAAGAAAGTAGCTCAGGCGATTGAACCTGCTTGCCATATTTAA。
2) Construction of the expression vector pBE-S-yiappa
PCR primers Y-F, Y-R (see Table 1) were designed based on the gene sequence of YIAPPA, and PCR amplification was performed using the synthetic gene YIapp as a template and Y-F, Y-R as a primer. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃40sec,74℃2min,30 cycles; 74 ℃ for 10min. The amplified product was digested with NdeI and XbaI, ligated into vector pBE-S digested with the same double digestion, and recombinant plasmid pBE-S-yiapp was constructed.
TABLE 1 primers for construction of recombinant plasmids
Note that: the underlined parts are restriction enzyme cleavage sites.
3) Synthesis of the Gene rphyxt52
According to GenBank ID KM873028 of phytase rPhyXT52, searching to obtain the gene sequence, and delivering the gene sequence to Shanghai Boyi biotechnology limited company for complete gene synthesis of phytase rPhyXT52, as shown in SEQ ID NO. 6.
SEQ ID NO.6：
AATTCGGCTTCTGAAACCTCTGCTAACCCGGACCTGCAGAACCTGCAGCTGCAGCAGGCTGTTATCCTGTCTCGTCACGGTGTTCGTGCTCCGACCAAACAGTCTAAAGAAATGAAAGACCTGGCTGGTCAGGAATGGCCGAAATGGCCGGTTAAAGCTGGTAACCTGACCCCGCGTGGTCAGGAACTGGTTACCCTGATGGGTACCTACTACGGTGACTACTTCAAAAAACAGGGTCTGCTGGCTGCTGACCAGTGCCCGGCTGAAAACGAACTGTTCGGTTGGGGTGACACCGACCAGCGTACCCGTCTGACCACCCAGGCTCTGCTGAAAGGTATCGCTCCGCACTGCCACTTCACCGCTAAAAACCAGACCGACCTGAAAAAACCGGACCCGATCTTCCACCCGCTGAAAGCTGGTATCTGCACCCTGGACAAAGACACCGCTCTGAAAGCTATCGACAAAGCTGCTGGTGGTTCTCTGGCTGCTCTGGACCAGACCTACGCTCCGCAGATCAAACTGATGTCTCAGGTTATGGACTACCCGCAGTCTCCGTACTGCCTGCAGATGAAACAGACCGGTCAGACCTGCTCTGCTAACATCGACATCCCGTCTTCTGTTAAAATGAAAAAAAAAGGTACCGAAGCTACCCTGGAAGGTGGTATCGGTTACTCTTCTACCTTCGCTGAAAACTTCCTGCTG GAAGACGCTCAGGGTATGCAGAACGTTGCTTGGGGTCGTATCAAAGACCAGAAAACCTGGGACTCTCTGCTGGAACTGCACAACCTGCAGTTCCGTCTGATGTCTGGTACCCCGTACCTGGCTAAATCTAACGGTACCCCGGTTCTGCAGGTTATCGACTCTGCTCTGGGTGCTACCGCTCAGGTTTCTCCGGGTTTCACCCTGCCGGCTGGTAACAAAGTTCTGATCCTGGGTGGTCACGACACCAACATCGAAAACGTTGCTGGTTCTCTGGGTCTGTCTTGGACCCTGACCGACCAGCCGGACCAGACCCCGCCGGCTGGTGCTCTGATGTTCGAACGTTGGCAGGGTAAAACCACCCACAAACAGTACGTTTCTCTGAAAATGGTTTACCAGACCCAGGACCAGATGCGTTCTCAGCACAAACTGACCCTGAAACACCCGCCGATGTCTGTTGCTGTTTCTATCCCAGGTTGCGAAAACATCGGTGAAGACAAACTGTGCTCTCTGGACACCTTCCACCAGGTTATCGAAAAAGCTGAACTGCCGCAGTGCAAAATCGTCGACTAA。
4) Construction of expression vector pBE-S-rphixt 52
PCR primers R-F, R-R (see Table 1) were designed based on the gene sequence of rphyxt52, and PCR amplification was performed using the synthetic gene rphyxt52 as a template and R-F, R-R as a primer. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃40sec,74℃2min,30 cycles; 74 ℃ for 10min. The amplified product was digested with Nde I and Xba I, ligated into vector pBE-S digested with the same double digestion, and recombinant plasmid pBE-S-rphyxt52 was constructed.
EXAMPLE 2 construction and screening of Phytase mutants
1) Construction of Phytase mutants
The amino acid sequence and the secondary structure of the phytase YIAPPA and the phytase rPhyXT52 are compared by CLUSTALW, the amino acid sequence of the phytase YIAPPA is shown as SEQ ID NO.3, and the amino acid sequence of the phytase rPhyXT52 is shown as SEQ ID NO. 5.
SEQ ID NO.3：
NSYAISAAPVAIQPTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDTWPQWPVAAGYLTPRGAQLVTLMGGFYGDYFRSQGLLAAGCPTDAVIYAQADVDQRTRLTGQAFLDGIAPGCGLKVHYQADLKKVDPLFHPVDAGVCKLDSTQTHKAVEERLGGPLSELSKRYAKPFAQMGEILNFAASPYCKSLQQQGKTCDFANFAANKITVNKPGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWHRLTGEDNWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIETALVLQRDAQGQTLPLSPQTKILFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMGQLRNAEKLDLKNNPAGRVPVAIDGCENSGDDKLCQLDTFQKKVAQAIEPACHI。
SEQ ID NO.5：
NSASETSANPDLQNLQLQQAVILSRHGVRAPTKQSKEMKDLAGQEWPKWPVKAGNLTPRGQELVTLMGTYYGDYFKKQGLLAADQCPAENELFGWGDTDQRTRLTTQALLKGIAPHCHFTAKNQTDLKKPDPIFHPLKAGICTLDKDTALKAIDKAAGGSLAALDQTYAPQIKLMSQVMDYPQSPYCLQMKQTGQTCSANIDIPSSVKMKKKGTEATLEGGIGYSSTFAENFLLEDAQGMQNVAWGRIKDQKTWDSLLELHNLQFRLMSGTPYLAKSNGTPVLQVIDSALGATAQVSPGFTLPAGNKVLILGGHDTNIENVAGSLGLSWTLTDQPDQTPPAGALMFERWQGKTTHKQYVSLKMVYQTQDQMRSQHKLTLKHPPMSVAVSIPGCENIGEDKLCSLDTFHQVIEKAELPQCKIVD。
YiAPPA, rPhyXT52 was divided into 10 structural fragments (FIG. 1) in combination with requirements of USERec DNA recombination technology and coding gene sequence information of YiAPPA, rPhyXT, wherein YIAPPA was divided into Y-F1 to Y-F10 and rPhyXT52 was divided into r-F1 to r-F10. The structural fragments in rPhyXT52 are respectively introduced into YIAPPA one by one to replace the original structural fragments in YIAPPA, so as to obtain a series of phytase mutants Yr-M1-Yr-M10. Primers were designed according to the usetec DNA recombination technique, combining the base sequences of the genes yiappa and rphyxt52 and the mutants to be constructed, as shown in table 2.
TABLE 2 primers used to construct phytase mutants
The construction steps of the phytase mutant Yr-M1 are as follows:
(1) The recombinant plasmid pBE-S-yiapp a is used as a template, and primers M1-YF and M1-YR are used for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Pl2. Mu.L of us-neo DNA polymerase; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃40sec,74℃2min,30 cycles; 74 ℃ for 10min. The recombinant plasmid pBE-S-rphyxt52 is used as a template, and primers M1-rF and M1-rR are used for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃10sec,74℃2min,30 cycles; 74 ℃ for 10min. And purifying the PCR amplified product by using a PCR product purification kit.
(2) Mixing the purified PCR amplified products, and treating the mixed PCR amplified products by using USER enzyme. The conditions for the USER enzyme treatment are: 37 ℃ for 20min;25 ℃ for 20min. After the reaction was completed, T4 ligase was added to the reaction system, and then the reaction system was treated at 25℃for 1 hour. Then adding DpnI enzyme to the reaction system to treat the residual template plasmid in the reaction system. And finally, purifying the DNA connecting fragment in the reaction system by adopting a PCR product purification kit.
(3) The DNA connecting fragment is subjected to double digestion by Nde I and Xba I, is connected to a vector pBE-S subjected to the same double digestion treatment, constructs a recombinant plasmid pBE-S-yrm1, converts competent cells of escherichia coli HST08, screens transformants by ampicillin resistance plates, and extracts the recombinant plasmid. The recombinant plasmid was sent to Shanghai Bioengineering Co., ltd for sequencing and aligned with the corresponding gene sequence, confirming the success of construction of the recombinant plasmid pBE-S-yrm. The construction method of the phytase mutant Yr-M10 is performed by referring to the construction method of the mutant Yr-M1.
The construction steps of the phytase mutant Yr-M2 are as follows:
(1) The recombinant plasmid pBE-S-yiapp a is used as a template, and primers M2-YF1 and M2-YR1 are adopted for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃10sec,74℃2min,30 cycles; 74 ℃ for 10min. Recombinant plasmid pBE-S-yiappa is used as template again, and primer is adoptedPCR amplification was performed on the M2-YF2 and M2-YR 2. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃30sec,74℃2min,30 cycles; 74 ℃ for 10min. The recombinant plasmid pBE-S-rphyxt52 is used as a template, and primers M2-rF and M2-rR are adopted for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃10sec,74℃2min,30 cycles; 74 ℃ for 10min. And purifying the PCR amplified product by using a PCR product purification kit.
(2) Mixing the purified PCR amplified products, and treating the mixed PCR amplified products by using USER enzyme. The conditions for the USER enzyme treatment are: 37 ℃ for 20min;25 ℃ for 20min. After the reaction was completed, T4 ligase was added to the reaction system, and then the reaction system was treated at 25℃for 1 hour. Then adding DpnI enzyme to the reaction system to treat the residual template plasmid in the reaction system. And finally, purifying the DNA connecting fragment in the reaction system by adopting a PCR product purification kit.
(3) The DNA connecting fragment is subjected to double digestion by Nde I and Xba I, is connected to a vector pBE-S subjected to the same double digestion treatment, constructs a recombinant plasmid pBE-S-yrm, converts competent cells of escherichia coli HST08, screens transformants by ampicillin resistance plates, and extracts the recombinant plasmid. The recombinant plasmid was sent to Shanghai Bioengineering Co., ltd for sequencing and aligned with the corresponding gene sequence, confirming the success of construction of the recombinant plasmid pBE-S-yrm. The construction methods of the phytase mutants Yr-M3, yr-M4, yr-M5, yr-M6, yr-M7, yr-M8 and Yr-M9 are carried out according to the construction method of the mutant Yr-M2.
2) Expression and purification of phytase YIAPPA and mutant thereof in bacillus subtilis
The expression vectors of phytase YIAPPA and mutants thereof are respectively transformed into competent cells of bacillus subtilis Bacillus subtilis RIK1285, and pBE-S is transformed as negative control Contr to obtain recombinant bacillus subtilis.
The seed culture conditions of the recombinant bacillus subtilis are as follows: LB liquid medium is adopted, and a 250mL triangular flask is used for culture, wherein the liquid loading amount of the medium is 25mL, the culture temperature is 37 ℃, the rotating speed is 180rpm, and the culture time is 24h. The fermentation culture conditions of the recombinant bacillus subtilis are as follows: LB liquid medium is adopted, and a 250mL triangular flask is used for culture, wherein the liquid loading amount of the medium is 25mL, the inoculation amount is 1%, the culture temperature is 37 ℃, the rotating speed is 180rpm, and the culture time is 30h.
By Ni 2+ Purifying the target protein in the fermentation supernatant by using an affinity chromatographic column, and eluting with 250mmol/L imidazole eluting buffer solution to obtain the purified recombinant phytase. The purity of the recombinant phytase was checked by SDS-PAGE and the concentration of the recombinant phytase was determined by the Bradford method.
3) Enzyme activity assay of recombinant phytase
Enzyme activity determination of recombinant phytase: mu.L of the enzyme solution was placed in a centrifuge tube, and 750. Mu.L of 0.25mol/L sodium acetate buffer (pH 4.5) was added thereto and mixed well. 2mL of 1.5mmol/L sodium phytate solution (0.25 mol/L sodium acetate buffer, pH 4.5) was added to the experimental tube, and 2mL of the color/end point mixture (ammonium molybdate/ammonium vanadate/nitric acid) was added to the control, and shaken well. After 30min of reaction at 37 ℃, 2mL of the color/end point mixture was added to the experimental group, 2mL of 1.5mmol/L sodium phytate solution was added to the control group, and the mixture was mixed uniformly, and the light absorption value was measured at 415 nm. The conversion formula of the inorganic phosphorus content in the reaction solution and the absorbance value of the reaction solution at 415nm is as follows: inorganic phosphorus (mmol/L) = 26.5510 ×OD 415nm +0.3113. The phytase activity unit (U) is defined as: the amount of phytase required to release 1. Mu. Mol/L inorganic phosphorus from 1.5mmol/L sodium phytate solution per minute at 37℃and pH 4.5 is one enzyme activity unit (U). The results of the enzyme activity measurement of the recombinant phytase are shown in Table 3.
TABLE 3 determination of enzyme Activity of recombinant phytase
4) Thermal stability of recombinant phytase at 80 DEG C
Thermal stability of recombinant phytase at 80 ℃): mu.L of the enzyme solution was placed in a centrifuge tube, and 750. Mu.L of 0.25mol/L sodium acetate buffer (pH 4.5) was added thereto and mixed well. After the enzyme solution is incubated at 80 ℃ for 15min, the enzyme activity of the sample is determined according to the method of measuring the enzyme activity of recombinant phytase, and the residual enzyme activity of the sample after incubation is calculated by taking the enzyme activity of the sample which is not incubated as 100%. The results of the remaining enzyme activity measurement after incubation of the recombinant phytase at 80℃for 20min are shown in Table 4. After the recombinant phytase is incubated at 80 ℃ for 20min, the residual enzyme activities of the phytase mutants Yr-M2 and Yr-M10 are obviously higher than that of YIAPPA. Compared with YIAPPA, the residual enzyme activity of the mutant Yr-M2 is improved by 0.80 times, and the residual enzyme activity of the mutant Yr-M10 is improved by 0.50 times.
Table 4 residual enzyme Activity after incubation of recombinant phytase at 80℃for 20min
5) Construction of recombinant phytase Yr-Mut and preliminary detection of enzymatic Properties thereof
In view of the measurement results of the enzyme activity and the thermal stability of the phytase mutant, the mutant Yr-M2 and the mutant Yr-M10 are subjected to further combination of structural fragments, namely, the structural fragments r-F2 and r-F10 in rPhyXT52 are simultaneously introduced into YIAPPA to replace the structural fragments Y-F2 and Y-F10 in YIAPPA, so as to construct the phytase mutant Yr-Mut. Wherein, the amino acid sequences of the structural fragments r-F2 and r-F10 are respectively shown as SEQ ID NO.31 and SEQ ID NO.32, and the amino acid sequences of the structural fragments Y-F2 and Y-F10 are respectively shown as SEQ ID NO.33 and SEQ ID NO. 34.
SEQ ID NO.31：
VILSRHGVRSPTKQTQLMNDVTPDTWPQWPVAAGYLTPRGAQLVT。
SEQ ID NO.32
LKNNPAGRVPVAIDGCENSGDDKLCQLDTFQKKVAQAIEPACHI。
SEQ ID NO.33
VILSRHGVRAPTKQSKEMKDLAGQEWPKWPVKAGNLTPRGQELVT。
SEQ ID NO.34
LKHPPMSVAVSIPGCENIGEDKLCSLDTFHQVIEKAELPQCKIVD。
The construction steps of the phytase mutant Yr-Mut are as follows: (1) The recombinant plasmid pBE-S-yrm2 is used as a template, and primers M10-YF and M10-YR are adopted for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃40sec,74℃2min,30 cycles; 74 ℃ for 10min. The recombinant plasmid pBE-S-rphyxt52 is used as a template, and primers M10-rF and M10-rR are used for PCR amplification. The PCR amplification system is as follows: 10 Xbuffer I5. Mu.L; dNTP 5. Mu.L; mgSO (MgSO) 4 5. Mu.L; 2. Mu.L of each primer; template 1. Mu.L; KOD-Plus-neo DNA polymerase 2. Mu.L; ddH 2 O28. Mu.L. The PCR amplification conditions were: 98 ℃ for 5min;98℃20sec,60℃10sec,74℃2min,30 cycles; 74 ℃ for 10min. And purifying the PCR amplified product by using a PCR product purification kit. (2) Mixing the purified PCR amplified products, and treating the mixed PCR amplified products by using USER enzyme. The conditions for the USER enzyme treatment are: 37 ℃ for 20min;25 ℃ for 20min. After the reaction was completed, T4 ligase was added to the reaction system, and then the reaction system was treated at 25℃for 1 hour. Then adding DpnI enzyme to the reaction system to treat the residual template plasmid in the reaction system. And finally, purifying the DNA connecting fragment in the reaction system by adopting a PCR product purification kit. (3) The DNA connecting fragment is subjected to double digestion by Nde I and Xba I, is connected to a vector pBE-S subjected to the same double digestion treatment, constructs a recombinant plasmid pBE-S-yrmut, converts competent cells of escherichia coli HST08, screens transformants by ampicillin resistance plates, and extracts the recombinant plasmid. The recombinant plasmid was sent to Shanghai Bioengineering Co., ltd for sequencing and aligned with the corresponding gene sequence, confirming the success of construction of the recombinant plasmid pBE-S-yrmut. The gene sequence of the phytase mutant Yr-Mut is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ IDNO. 1.
SEQ ID NO.1：
NSYAISAAPVAIQPTGYTLERVVILSRHGVRAPTKQSKEMKDLAGQEWPKWPVKAGNLTPRGQELVTLMGGFYGDYFRSQGLLAAGCPTDAVIYAQADVDQRTRLTGQAFLDGIAPGCGLKVHYQADLKKVDPLFHPVDAGVCKLDSTQTHKAVEERLGGPLSELSKRYAKPFAQMGEILNFAASPYCKSLQQQGKTCDFANFAANKITVNKPGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWHRLTGEDNWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIETALVLQRDAQGQTLPLSPQTKILFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMGQLRNAEKLDLKHPPMSVAVSIPGCENIGEDKLCSLDTFHQVIEKAELPQCKIVD。
SEQ ID NO.2：
AATAGTTATGCGATTAGTGCCGCGCCGGTTGCCATACAACCCACGGGCTATACATTGGAGCGAGTGGTTATTTTGTCTCGTCACGGTGTTCGTGCTCCGACCAAACAGTCTAAAGAAATGAAAGACCTGGCTGGTCAGGAATGGCCGAAATGGCCGGTTAAAGCTGGTAACCTGACCCCGCGTGGTCAGGAATTAGTGACATTGATGGGCGGATTCTATGGTGATTACTTCCGTAGCCAAGGGTTACTCGCAGCAGGGTGCCCAACTGACGCGGTTATTTATGCTCAGGCCGATGTTGATCAACGAACGCGTTTAACGGGGCAGGCATTCCTTGATGGAATAGCACCGGGGTGTGGACTGAAAGTACATTATCAGGCTGATTTGAAAAAAGTGGATCCGCTGTTTCATCCCGTCGACGCGGGGGTGTGTAAGTTAGATTCGACACAAACCCATAAGGCTGTTGAGGAGCGACTAGGTGGGCCATTAAGTGAACTGAGCAAACGCTATGCTAAGCCCTTTGCCCAGATGGGTGAGATTCTGAATTTTGCGGCATCTCCTTACTGTAAATCACTGCAACAGCAAGGGAAAACCTGTGATTTTGCCAACTTTGCAGCGAATAAGATCACGGTGAACAAGCCGGGGACAAAAGTCTCGCTCAGCGGACCACTGGCACTGTCATCAACCTTAGGTGAGATCTTTTTGCTACAAAATTCACAAGCGATGCCTGATGTTGCCTGGCATCGGTTAACGGGAGAAGATAATTGGATCTCGTTATTATCGTTGCACAATGCGCAATTTGATTTAATGGCAAAAACACCTTATATCGCTCGTCATAAGGGCACACCGTTGCTGCAACAGATCGAGACTGCCCTCGTCCTTCAGCGTGATGCTCAGGGGCAAACATTGCCATTATCACCTCAAACCAAAATTCTGTTCCTCGGGGGACATGATACAAACATCGCCAATATTGCTGGAATGTTGGGGGCTAACTGGCAATTACCACAGCAGCCCGATAATACCCCACCTGGGGGGGGATTGGTCTTCGAGCTATGGCAAAACCCAGATAATCATCAACGTTATGTCGCGGTGAAAATGTTCTATCAAACAATGGGCCAATTGCGAAATGCTGAGAAACTAGACCTGAAACACCCGCCGATGTCTGTTGCTGTTTCTATCCCAGGTT GCGAAAACATCGGTGAAGACAAACTGTGCTCTCTGGACACCTTCCACCAGGTTATCGAAAAAGCTGAACTGCCGCAGTGCAAAATCGTCGACTAA。
Transforming the expression vector of the mutant Yr-Mut into bacillus subtilis Bacillus subtilis RIK1285 competent cells to obtain recombinant bacillus subtilisBacillus subtilis. Fermenting and culturing recombinant bacillus subtilis by adopting Ni 2+ The protein of interest in the fermentation supernatant was purified by affinity chromatography, the purity of the recombinant phytase was checked by SDS-PAGE, and the concentration of the recombinant phytase was determined by the Bradford method. The results of the enzyme activity and the thermal stability of the phytase mutant Yr-Mut are shown in tables 3 and 4, the specific activity of the mutant Yr-Mut is 3892.58U/mg, and the residual enzyme activity of the phytase mutant Yr-Mut subjected to heat preservation at 80 ℃ for 20min is improved from 34.81% of that of a control (before mutation) to 78.62%, and the residual enzyme activity is improved by 1.26 times.
Example 3 enzymatic Property verification of Phytase mutant Yr-Mut
Determination of optimal reaction pH of recombinant phytase: the enzyme activity of the sample was measured under different pH (1.0 to 8.0) conditions according to the method of "3" enzyme activity measurement of recombinant phytase in example 2, and the optimum reaction pH was determined by plotting the relative enzyme activity against pH. The buffers used were as follows: 0.25mol/L glycine-hydrochloric acid buffer solution, and the pH value is 1.0-3.5; 0.25mol/L sodium acetate-acetic acid buffer solution, pH 3.5-6.0; 0.25mol/L Tris-hydrochloric acid buffer solution, pH 6.0-8.5. The results of the pH optimum reaction measurements for the phytase YIAPPA and the phytase mutant Yr-Mut are shown in FIG. 2.
Determination of the pH stability of recombinant phytase: the enzyme solution was diluted with buffers of different pH (1.0 to 12.0) to be treated at 37℃for 2 hours under different pH conditions, then diluted with an optimum pH buffer, and the enzyme activity of the sample was measured according to the method of "3) enzyme activity measurement of recombinant phytase" in example 2. The remaining enzyme activity of the treated sample was calculated with the enzyme activity of the untreated sample being 100%, and the pH stability was determined by plotting the remaining enzyme activity against pH. The buffers used were as follows: 0.25mol/L glycine-hydrochloric acid buffer solution, and the pH value is 1.0-3.5; 0.25mol/L sodium acetate-acetic acid buffer solution, pH 3.5-6.0; 0.25 mol/LTris-hydrochloric acid buffer solution, pH 6.0-8.5; 0.25mol/L glycine-sodium hydroxide buffer solution, and the pH value is 8.5-12.0. The pH stability measurements of the phytase YIAPPA and the phytase mutant Yr-Mut are shown in FIG. 3.
Determination of optimal reaction temperature of recombinant phytase: the enzyme activity of the sample was measured according to the method of "3) enzyme activity measurement of recombinant phytase" in example 2, and the enzyme activities of the samples were measured under different temperature conditions by reacting at 30 to 90℃for 30 minutes, respectively, and the optimum reaction temperature was determined by plotting the relative enzyme activities versus temperature. The results of the determination of the optimum reaction temperatures for the phytase YIAPPA and the phytase mutant Yr-Mut are shown in FIG. 4.
Measurement of thermostability of recombinant phytase: the enzyme solution was incubated at 80℃or 100℃and a part of the sample was taken out at a time gradient, and the enzyme activity of the sample was measured according to the method of "3) enzyme activity measurement of recombinant phytase" in example 2. The enzyme activity of the untreated enzyme solution was defined as 100% and the heat stability of the enzyme was evaluated by plotting the percentage of the remaining enzyme activity against time. The results of the thermal stability measurements of recombinant phytases YIAPPA and Yr-Mut at 80deg.C are shown in FIG. 5, and the results of the thermal stability measurements of phytases YIAPPA and phytase mutants Yr-Mut at 100deg.C are shown in FIG. 6.
Protease resistance assay of phytase: pepsin and trypsin were formulated at 0.1mg/mL with 0.25mol/L Gly-HCl buffer (pH 2.0) and 0.25mol/L Tris-HCl buffer (pH 7.0), respectively. Pepsin and trypsin are respectively added into phytase according to the mass ratio of protease to phytase of 1:10, after the temperature is kept for 2 hours at 37 ℃, protease inhibitor is added into the mixture to stop the reaction, then the protease-treated sample is diluted 100 times by an optimal pH buffer solution (0.25 mol/L sodium acetate buffer solution, pH 4.5), and then the enzyme activity of the sample is measured according to the method of measuring the enzyme activity of recombinant phytase of '3' in the example 2, and the residual enzyme activity of the treated sample is calculated by taking the enzyme activity of untreated enzyme solution as 100%. The results of the protease resistance assays for phytase YIAPPA and phytase mutant Yr-Mut are shown in FIG. 7.
The measurement results of the above enzymatic properties show that: the phytase mutant Yr-Mut has an optimal reaction temperature of 55 ℃, an optimal reaction pH of 4.5, a relative enzyme activity of more than 80% within the pH range of 1.0-10.0 and a specific activity of 3892.58U/mg. The optimal reaction temperature, optimal reaction pH and pH stability, specific activity and protease resistance of the phytase mutant Yr-Mut are basically consistent with those of the phytase YIAPPA. In addition, the thermal stability of the phytase mutant Yr-Mut is obviously improved. The residual enzyme activity of the phytase mutant Yr-Mut, which is treated at 80 ℃ for 20min in a heat preservation way, is improved from 34.81% of a control (before mutation) to 78.62%, and the residual enzyme activity is improved by 1.26 times; the residual enzyme activity of the incubation at 100℃for 10min was increased from 9.54% to 81.42% of the control (pre-mutation), by a factor of 7.53.
Example 4 in vitro simulated digestion experiments
The phytase mutant Yr-Mut and phytase YIAPPA are diluted by 0.25mol/L sodium acetate buffer (pH 4.5), and the recombinant phytase is prepared into diluted enzyme solution. 0.2g of corn-based feed (corn: soybean meal=7:3, m: m) was taken, 2mL of diluted enzyme solution (final concentration of 2.5U/g feed) was added, and placed in a 10mL centrifuge tube. The corn-based feed/enzyme mixture was treated at 95 ℃ for 5min to simulate the high temperature pelletization process of animal feed. The corn-based feed/enzyme mixture without 95℃treatment was also used as a control.
The in vitro simulated digestion experiment comprises the following specific steps: first, the corn-based feed/enzyme mixture is treated at 40 ℃ for 30min; then 100. Mu.L of a 10mg/mL pepsin solution was added thereto, the pH was adjusted to 2.9 by adding 1.5mol/LHCl, and the reaction mixture was treated at 40℃for 45 minutes; then 100. Mu.L of 1mol/L NaHCO containing 3.7mg/mL trypsin was added thereto 3 The solution, reaction mixture was treated at 40℃for 2h. After the reaction was completed, the reaction mixture was centrifuged at 12000rpm for 15min, and the supernatant was taken for measurement of inorganic phosphorus content, and the inorganic phosphorus content of the buffer-treated group to which pepsin and trypsin were not added was used as a control.
The results of in vitro simulated digestion experiments of phytase YIAPPA and mutant Yr-Mut are shown in Table 5. Under the condition that the corn-based feed/enzyme mixture is not subjected to high-temperature treatment, the inorganic phosphorus release amount of the phytase mutant Yr-Mut experimental group is slightly lower than that of the phytase YIAPPA experimental group; under the condition of high temperature treatment of the corn-based feed/enzyme mixture, the inorganic phosphorus release amount of the phytase mutant Yr-Mut experimental group is 5.61 times that of the phytase YIAPPA experimental group.
TABLE 5 in vitro simulated digestion test results
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The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The phytase Yr-Mut with improved heat stability is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the phytase Yr-Mut of claim 1, characterized in that the nucleotide sequence is shown in SEQ ID No. 2.
3. The method for preparing the phytase Yr-Mut according to claim 1, wherein the structural fragment Y-F2 in the phytase YIAPPA is replaced by the structural fragment r-F2 in the phytase rPhyXT52, and the structural fragment Y-F10 in the phytase YIAPPA is replaced by the structural fragment r-F10 in the phytase rPhyXT52 by using a USERec DNA recombination technology, so as to construct the phytase Yr-Mut;
the amino acid sequence of the structural fragment Y-F2 is shown as SEQ ID NO.33, the amino acid sequence of the structural fragment r-F2 is shown as SEQ ID NO.31, the amino acid sequence of the structural fragment Y-F10 is shown as SEQ ID NO.34, and the amino acid sequence of the structural fragment r-F10 is shown as SEQ ID NO. 32;
the initial complete gene sequence of the phytase YIAPPA is shown as SEQ ID NO. 4;
the initial complete gene sequence of the phytase rPhyXT52 is shown in SEQ ID NO. 6.
4. A method of preparation according to claim 3, comprising the steps of:
(1) Cloning the gene sequence of phytase YIAPPA into a plasmid pBE-S to construct a recombinant plasmid pBE-S-YIAppa, wherein the gene sequence of phytase YIAPPA is shown as SEQ ID NO. 4;
(2) Cloning the gene sequence of phytase rPhyXT52 into a plasmid pBE-S to construct a recombinant plasmid pBE-S-rphixt 52, wherein the gene sequence of phytase rPhyXT52 is shown as SEQ ID NO. 6;
(3) Respectively taking a recombinant plasmid pBE-S-yiapp a and a recombinant plasmid pBE-S-rphyxt52 as templates, adopting a USERec DNA recombination technology to replace a structural fragment Y-F2 with a structural fragment r-F2, and replacing a structural fragment Y-F10 with a structural fragment r-F10 to construct a gene sequence of phytase Yr-Mut;
(4) Cloning the gene sequence of phytase Yr-Mut into plasmid pBE-S to construct expression vector pBE-S-yrmut of phytase mutant, the gene sequence of phytase Yr-Mut is shown in SEQ ID NO. 2;
(5) Converting an expression vector pBE-S-yrmut of the phytase mutant into bacillus subtilis to obtain bacillus subtilis genetic engineering bacteria, and fermenting and culturing the bacillus subtilis genetic engineering bacteria to obtain phytase Yr-Mut.
5. The method according to claim 4, wherein in the step (1), the cloning is performed by PCR amplification using the gene sequence of phytase YIAPPA as a template and Y-F having the sequence shown in SEQ ID NO.27 and Y-R having the sequence shown in SEQ ID NO.28 as primers, to obtain an amplified product, which is digested with Nde I and Xba I and ligated to the vector pBE-S having the same double digestion.
6. The preparation method according to claim 4, wherein in the step (2), the cloning is performed by using the gene sequence of phytase rPhyXT52 as a template and R-F having the sequence shown in SEQ ID NO.29 and R-R having the sequence shown in SEQ ID NO.30 as primers, and an amplified product is obtained, and the amplified product is digested with Nde I and Xba I and ligated to vector pBE-S having the same double digestion.
7. The method of claim 4, wherein in step (5), the bacillus subtilis is a Bacillus subtilis RIK1285 competent cell.
8. The method according to claim 4, wherein in the step (5), the fermentation culture is carried out at 37℃and 180rpm for 30 hours.
9. Use of the phytase Yr-Mut of claim 1 in the preparation of a feed additive.
10. The use of the phytase Yr-Mut of claim 1 in the preparation of livestock feed.
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| WO2007128160A1 (en) * | 2006-04-30 | 2007-11-15 | Feed Research Institute Chinese Academy Of Agricultural Sciences | Cloning and expression of a novel phytase |
| CN102943083A (en) * | 2012-11-27 | 2013-02-27 | 青岛根源生物技术集团有限公司 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
| CN106047836A (en) * | 2016-06-15 | 2016-10-26 | 昆明爱科特生物科技有限公司 | Phytase mutant and preparation method and application thereof |
| CN114164193A (en) * | 2021-12-02 | 2022-03-11 | 江西省科学院微生物研究所 | Phytase mutant with improved thermal stability and protease resistance as well as preparation method and application thereof |
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| WO2007128160A1 (en) * | 2006-04-30 | 2007-11-15 | Feed Research Institute Chinese Academy Of Agricultural Sciences | Cloning and expression of a novel phytase |
| CN102943083A (en) * | 2012-11-27 | 2013-02-27 | 青岛根源生物技术集团有限公司 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
| CN106047836A (en) * | 2016-06-15 | 2016-10-26 | 昆明爱科特生物科技有限公司 | Phytase mutant and preparation method and application thereof |
| CN114164193A (en) * | 2021-12-02 | 2022-03-11 | 江西省科学院微生物研究所 | Phytase mutant with improved thermal stability and protease resistance as well as preparation method and application thereof |
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