CN108315310B - Phytase mutant with improved pepsin resistance as well as coding gene and application thereof - Google Patents

Phytase mutant with improved pepsin resistance as well as coding gene and application thereof Download PDF

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CN108315310B
CN108315310B CN201710027073.3A CN201710027073A CN108315310B CN 108315310 B CN108315310 B CN 108315310B CN 201710027073 A CN201710027073 A CN 201710027073A CN 108315310 B CN108315310 B CN 108315310B
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姚斌
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to the field of genetic engineering, in particular to a phytase mutant with improved pepsin resistance, and a coding gene and application thereof. The amino acid sequence of the phytase shown as SEQ ID NO.1 has the advantages that the 99 th leucine is mutated into alanine, the 162 th leucine is mutated into glycine, and the 230 th glutamic acid is mutated into glycine. Compared with the wild type, the phytase mutant of the invention has obviously improved pepsin resistance, acid resistance and thermal stability, and has huge application potential in the feed industry.

Description

Phytase mutant with improved pepsin resistance as well as coding gene and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to a phytase mutant with improved pepsin resistance, and a coding gene and application thereof.
Background
Phytase, also known as phytate hydrolase, hydrolyzes the phosphomonoesters of phytic acid to release inorganic phosphorus. The phytase added into the feed for monogastric animals can effectively improve the utilization efficiency of phytate phosphorus and reduce the environmental pollution caused by phosphorus emission in animal culture areas.
Most of the phytases obtained in nature at present have poor gastric protein resistance and are difficult to meet the requirements of feed industry. According to the action characteristics of the pepsin, the pepsin resistance of the phytase is improved by utilizing the site-directed mutagenesis technology, so that the improvement of the enzymology property and the excavation of new enzyme resources are facilitated, and the potential application value in the feed industry is realized.
Disclosure of Invention
The invention aims to provide a phytase mutant with improved pepsin resistance.
Another object of the present invention is to provide a gene encoding the above phytase mutant with improved pepsin resistance.
Another object of the present invention is to provide a recombinant vector comprising the above-mentioned phytase mutant gene with improved pepsin resistance.
Another object of the present invention is to provide a recombinant strain comprising the above-mentioned gene for a pepsin resistance-improving phytase mutant.
Another object of the present invention is to provide the use of the above-mentioned pepsin resistance improving phytase.
The present invention performs site-directed mutagenesis of a Yersinia kluyveri (Yersinia kristensenii) derived phytase YkAPPA gene, the mature protein of which has an amino acid sequence shown in SEQ ID NO.1 and is encoded by a nucleotide sequence shown in SEQ ID NO. 2.
SEQ ID NO.1
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTPRGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHTVEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.2
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatgggttgttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccataccgtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaagaagggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcgagccagcctgccatatttaa
According to the mutant of phytase YkAPPA with improved pepsin resistance, the amino acid sequence of the mutant is shown as SEQ ID NO.1, wherein the 99 th leucine of the phytase is mutated into alanine, the 162 th leucine is mutated into glycine, the mutation is further carried out, and the 230 th glutamic acid is mutated into glycine.
According to a specific embodiment of the present invention, 3 phytase mutants with improved pepsin resistance were obtained by site-directed mutagenesis, named YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G, respectively, namely: YkAPPA-L99A mutation of leucine at position 99 into valine; YkAPPA-L99A/L162G is characterized in that the 99 th leucine is mutated into valine and the 162 th leucine is mutated into glycine; YkAPPA-L99A/L162G/E230G mutated leucine 99 to valine, leucine 162 to glycine and glutamic acid 230 to glycine.
Thus, according to an embodiment of the present invention, the phytase mutant YkAPPA-L99A with improved pepsin resistance, wherein the leucine amino acid at position 99 is mutated to alanine, the amino acid sequence of which is shown in SEQ ID NO.3
SEQ ID NO.3
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTPRGAGLVTLMGGFYGDYFRSYGALPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHTVEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI
The phytase mutant YkAPPA-L99A/L162G with improved pepsin resistance according to the specific embodiment of the invention, wherein the leucine amino acid at the 99 th position is mutated into alanine, the leucine amino acid at the 162 th position is mutated into glycine, and the amino acid sequence of the phytase mutant is shown as SEQ ID NO.4
SEQ ID NO.4
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTPRGAGLVTLMGGFYGDYFRSYGALPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHTVEAGVCKGDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The phytase mutant YkAPPA-L99A/L162G/E230G with the improved pepsin resistance according to the embodiment of the invention, wherein the 99 th leucine amino acid is mutated into alanine, the 162 th leucine amino acid is mutated into glycine, the 230 th glutamic acid is mutated into glycine, and the amino acid sequence is shown as SEQ ID NO.5
SEQ ID NO.5
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTPRGAGLVTLMGGFYGDYFRSYGALPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHTVEAGVCKGDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKGGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The invention also provides gene sequences for coding the phytase mutants with improved pepsin resistance, namely YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G, wherein the nucleotide sequences are respectively shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
SEQ ID NO.6Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatggggcgttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccataccgtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaagaagggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcgagccagcctgccatatttaa
SEQ ID NO.7
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatggggcgttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccataccgtcgaggcgggggtatgtaaaggcgacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaagaagggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcgagccagcctgccatatttaa
SEQ ID NO.8
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatggggcgttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccataccgtcgaggcgggggtatgtaaaggcgacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaaggcgggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcgagccagcctgccatatttaa
The above-described cDNA molecule encoding the mutant of phytase YkAPPA with improved pepsin resistance is inserted between the restriction sites of the vector in the proper orientation and correct reading frame, so that the nucleotide sequence thereof is operably linked to the expression control sequence. The preferred vector of the invention is pET-22b (+), the modified phytase gene is inserted between restriction sites of EcoRI and NotI on plasmid pET-22b (+), and the nucleotide sequence is positioned at the downstream of and controlled by the T7 lac promoter, thus obtaining the recombinant bacterial expression plasmid of each mutant. A preferred host bacterium of the present invention is BL21(DE 3).
Compared with the wild type, the pepsin resistance of the three phytase mutants of the invention, YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G, is obviously improved, especially the pepsin resistance, acid resistance and thermal stability of YkAPPA-L99A/L162G/E230G are improved at the same time, and the phytase mutants have huge application potential in the feed industry.
Drawings
FIG. 1 is a graph of the resistance of phytase to pepsin treatment at different concentrations before and after modification according to an embodiment of the present invention;
FIG. 2 is a graph showing enzyme activity curves of phytase before and after modification at different pH values according to an embodiment of the present invention;
FIG. 3 is a graph showing the enzyme activity of phytase before and after modification at 60 ℃ for various periods of time according to an embodiment of the present invention.
Detailed Description
Experimental Material
The prokaryotic expression vector pET-22b (+) was purchased from Novagen. Escherichia coli Trans1-T1 and BL21(DE3) cells were purchased from Tiangen and used as host bacteria for plasmid amplification and prokaryotic expression, respectively. DNA purification kits were purchased from TaKaRa. Pfu DNA polymerase, restriction enzyme, T4 DNA ligase were purchased from Tiangen. Sodium phytate and pepsin (p0685) were purchased from Sigma. The nucleotide primers were synthesized by Shanghai Jun Biotechnology Ltd.
Example 1: obtaining of mutant Gene
A gene sequence (SEQ ID NO.2) of phytase YkAPPA derived from Yersinia klebsiella (Yersinia kristeennii) was modified, a mutation was introduced by means of Overlap PCR, and sequencing was performed to obtain a mutant gene. The method uses pEASY-T3-YkAPPA recombinant plasmid containing wild phytase gene YkAPPA as a template, introduces mutation through two rounds of PCR reactions, and connects the needed mutant gene to a vector pEASY-T3 which is verified by DNA sequencing.
Eight primers were used for the mutations: YkAPPA-F, YkAPPA-R, YkAPPA-L99A-F, YkAPPA-L99A-R, YkAPPA-L162G-F, YkAPPA-L162G-R, YkAPPA-E230G-F and YkAPPA-E230G-R.
The primer sequences are as follows:
YkAPPA-F：5’-cgcgaattcgcaccgcttgcagcacaatctac-3’
YkAPPA-R：5’-gatgcggccgcttaaatatggcaggctggctcG-3’
YkAPPA-L99A-F：5’-tccgcagctatggggcgttaccggcggggtg-3’
YkAPPA-L99A-R：5’-caccccgccggtaacgccccatagctgcgga-3’
YkAPPA-L162G-F：5’-cgggggtatgtaaaggcgacccagagaaaac-3’
YkAPPA-L162G-R：5’-gttttctctgggtcgcctttacatacccccg-3’
YkAPPA-E230G-F：5’-tcgaggtaaataaaggcgggacaaaagtctc-3’
YkAPPA-E230G-R：5’-gagacttttgtcccgcctttatttacctcga-3’
the primer is italicized to represent restriction enzyme sites EcoR I and Not I, and the underlined nucleotide sequence is the mutation site.
Example 2: expression and purification of phytase in escherichia coli before and after modification
Mutants of phytase YkAPPA, namely YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G, and wild enzymes thereof respectively code 441 amino acids and a stop codon, 23 amino acids at the N end are signal peptide sequences, and the theoretical molecular weight of mature protein is 48.6 kDa. The coding region sequence of the wild phytase and the mutant thereof is inserted between EcoRI and NotI restriction enzyme cutting sites of a prokaryotic expression vector pET-22b (+), is regulated and controlled by T7 lac in Escherichia coli BL21(DE3) cells, and an inducer is IPTG (isopropyl-beta-D-galactoside) with the final concentration of 1mM, and is subjected to induction culture for 5 hours in a shaking table with the temperature of 24 ℃ and the rpm of 220. The crude enzyme solution was purified by chromatography on a nickel-nitrilotriacetic acid (Ni-NTA) column and a Diethylaminoethyl (DEAE) column. The surface molecular weight of the mutant enzyme was identical to that of the wild enzyme, as determined by 10% SDS-PAGE electrophoresis, and was about 46 kDa.
Example 3: pepsin resistance of mutant phytases
And measuring the pepsin resistance of the phytase by detecting the enzyme activity and phytase protein of the phytase remained after the pepsin with different concentrations is treated for 2 hours. The mass ratio of pepsin to phytase is 1/1000, 1/500, 1/200, 1/100, 1/40 and 1/20.
And measuring the enzyme activity of the residual phytase after the pepsin with different concentrations is treated for 2 hours by using a ferrous sulfate molybdenum blue method. 50 μ L of an enzyme solution with an appropriate concentration was added to 950 μ L of 1.5mmol/L sodium phytate substrate (prepared with NaAc-HAc buffer solution of pH 4.5 and 0.25M), reacted in a water bath at 37 ℃ for 30min, 1mL of 10% trichloroacetic acid was added to terminate the reaction, and finally 2mL of a color developing solution (1% ammonium molybdate tetrahydrate, 3.2% concentrated sulfuric acid, 7.32 ferrous sulfate) was added to develop color. The absorbance value was measured at 700 nm to measure the amount of inorganic phosphate released. One unit of phytase activity is defined as the amount of enzyme required to release 1 micromole of phosphate per minute under the conditions of the assay. All reactions were repeated three times. The results (FIG. 1) show that the mutant phytases YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G keep more enzyme activity and show stronger pepsin resistance when being treated for 2 hours by pepsin concentrations of 1/1000-1/20 compared with the wild enzyme; when the concentration of pepsin is increased from 1/1000 to 1/20, YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G respectively keep 16%, 67% and 92% of enzyme activity, and YkAPPA only remains 1% of enzyme activity.
The remaining proteins of the phytase after 2h treatment with pepsin at different concentrations were separated by PAGE and the grey values of the protein bands were evaluated using Image J software. The pepsin hydrolysis constant of the phytase was calculated from the remaining proteins of the mutated phytase. The results (Table 1) show that the mutant phytase has stronger pepsin resistance, and the mutant phytases YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G have smaller hydrolysis rate and larger half-life than the wild enzyme.
TABLE 1 hydrolysis rate and half-life of phytase before and after modification under pepsin treatment
Figure GDA0003637699910000071
Example 4: pH and temperature response patterns of mutant phytases
The phytase before and after modification is subjected to enzymatic reaction for 30min at different pH (1-12) and 37 ℃, and the optimum pH is determined. The pH stability of the enzyme was investigated by treating the enzyme solution at pH 1-9 and 37 ℃ for 1h or at pH1-4 and 37 ℃ for 2 h. The buffers used were: 0.1mol/L glycine-hydrochloric acid buffer solution, pH 1-3; 0.1mol/L sodium acetate-acetic acid buffer solution, pH 3-6; 0.1mol/L Tris-hydrochloric acid buffer solution, pH 6-8; 0.1mol/L glycine-sodium hydroxide buffer, pH 8-12. The optimum pH of the mutant enzyme was similar to that of the wild type and was pH 4.5 (Table 2). Compared with wild type and mutant enzyme YkAPPA-L99A, mutant enzyme YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G have better acid stability after being treated for 1h at the pH value of 1-2; when the enzyme is treated for 1 hour at the pH value of 1, the mutant enzyme YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G can keep the enzyme activity of up to 94 percent, while the wild type enzyme YkAPPA-L99A and the mutant enzyme YkAPPA-L99A can only keep the enzyme activity of 64 percent (figure 2). Therefore, compared with the wild enzyme and the mutant enzyme YkAPPA-L99A, the mutant enzymes YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G have higher acid stability.
Treating the mutant enzyme and the wild enzyme at different temperatures (30-80 ℃) for 30min, and determining the optimal temperature. The thermal stability is measured by treating the enzyme solution at a certain temperature for 0, 2, 5, 10, 20, 30 and 60 min. The optimum temperature of the mutant phytase YkAPPA-L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G is consistent with that of the mutant enzyme and is 55 ℃. The mutant enzyme has better thermal stability than wild enzyme when treated for 30min at 60 ℃, and can keep 35% of enzyme activity, while the wild enzyme keeps 161% of enzyme activity. Therefore, the mutation of leucine at position 162 of phytase YkAPPA to valine improves the optimal temperature and thermal stability of phytase.
TABLE 2 comparison of the influence of pH and temperature on the enzymatic Activity and stability of Phytase before and after modification
Figure GDA0003637699910000081
Sequence listing
<110> feed institute of Chinese academy of agricultural sciences
<120> phytase mutant with improved pepsin resistance, and coding gene and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 441
<212> PRT
<213> Yersinia kluyveri (Yersinia kristensenii)
<400> 1
Met Thr Ile Ala Lys Glu Tyr Leu Arg Leu Ser Ile Leu Thr Leu Val
1 5 10 15
Leu Ser Ser Phe Thr Leu Ser Ala Ala Pro Leu Ala Ala Gln Ser Thr
20 25 30
Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His Gly Val Arg
35 40 45
Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr Pro Asp Lys
50 55 60
Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr Leu Thr Pro Arg Gly Ala
65 70 75 80
Gly Leu Val Thr Leu Met Gly Gly Phe Tyr Gly Asp Tyr Phe Arg Ser
85 90 95
Tyr Gly Leu Leu Pro Ala Gly Cys Pro Ala Asp Glu Ser Ile Tyr Val
100 105 110
Gln Ala Asp Val Asp Gln Arg Thr Arg Leu Thr Gly Gln Ala Phe Leu
115 120 125
Asp Gly Ile Ala Pro Asp Cys Gly Leu Lys Val His Tyr Gln Ala Asp
130 135 140
Leu Lys Lys Ile Asp Pro Leu Phe His Thr Val Glu Ala Gly Val Cys
145 150 155 160
Lys Leu Asp Pro Glu Lys Thr His Gln Ala Val Glu Lys Arg Leu Gly
165 170 175
Gly Pro Leu Asn Glu Leu Ser Gln Arg Tyr Ala Lys Pro Phe Ala Leu
180 185 190
Met Gly Glu Val Leu Asn Phe Ser Ala Ser Pro Tyr Cys Asn Ser Leu
195 200 205
Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala Thr Phe Ala Ala Asn Glu
210 215 220
Ile Glu Val Asn Lys Glu Gly Thr Lys Val Ser Leu Ser Gly Pro Leu
225 230 235 240
Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln Asn Ser Gln
245 250 255
Ala Met Pro Asp Val Ala Trp Asn Arg Leu Ser Gly Glu Glu Asn Trp
260 265 270
Ile Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Met Ala Lys
275 280 285
Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu Gln Gln Ile
290 295 300
Asp Thr Ala Leu Val Leu Gln Arg Asp Ala Gln Gly Gln Thr Leu Pro
305 310 315 320
Leu Ser Pro Gln Thr Lys Leu Leu Phe Leu Gly Gly His Asp Thr Asn
325 330 335
Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Asn Trp Gln Leu Pro Gln
340 345 350
Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe Glu Leu Trp
355 360 365
Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys Met Phe Tyr
370 375 380
Gln Thr Met Glu Gln Leu Arg Asn Ala Asp Lys Leu Asp Leu Lys Asn
385 390 395 400
Asn Pro Ala Arg Ile Val Pro Ile Ala Ile Glu Gly Cys Glu Asn Glu
405 410 415
Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr Phe Gln Lys Lys Val Ala
420 425 430
Gln Val Ile Glu Pro Ala Cys His Ile
435 440
<210> 2
<211> 1326
<212> DNA
<213> Yersinia klystenii (Yersinia kristensenii)
<400> 2
atgacaatag caaaagaata tctgcggtta tccatactca ctttggtgct cagtagtttt 60
acgctaagtg ctgcaccgct tgcagcacaa tctaccggtt acactttgga gcgcgtggtg 120
attttgagcc gccacggtgt tcgttccccg acgaaacaaa cacagttaat gaatgatgtt 180
acaccggaca aatggccaca atggccagta aaagcgggct atttaacgcc gcgaggggca 240
ggattagtca ctttaatggg cgggttctat ggtgattatt tccgcagcta tgggttgtta 300
ccggcggggt gcccggcaga cgaatccatc tatgtgcaag ctgatgttga ccaacgtacc 360
cgcttaaccg ggcaggcatt tctggacggt atagccccgg attgcggcct gaaagtacat 420
tatcaagctg atttgaaaaa aattgaccca ttgttccata ccgtcgaggc gggggtatgt 480
aaattggacc cagagaaaac tcatcaggct gttgaaaaac gcttgggtgg gccattaaat 540
gaactgagtc aacgctatgc caagcccttt gccctgatgg gcgaggtgct gaatttttcg 600
gcctcacctt attgcaactc actgcaacag aaaggaaaaa cctgtgattt tgcgactttt 660
gcagcaaatg aaatcgaggt aaataaagaa gggacaaaag tctcactgag tgggccattg 720
gcgctatcat cgacattagg tgaaattttc ctattacaaa attcacaggc catgccagat 780
gtcgcctgga accgtctcag cggtgaagaa aattggattt cattattgtc actgcataat 840
gcacagttcg atttgatggc caaaacccct tatatcgccc ggcataaagg aactccgttg 900
ttgcaacaaa ttgatacggc attagtgttg caacgtgatg ctcaggggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc gggggacatg acaccaatat tgccaatatt 1020
gcgggtatgt taggggccaa ttggcaatta ccgcagcaac ctgataatac cccgccaggc 1080
ggagggctag tctttgagct atggcagaat ccggataacc atcaacgcta tgtggcggtg 1140
aaaatgttct atcaaacgat ggagcagttg cgcaatgcag ataagttaga tttgaaaaac 1200
aacccggcaa gaattgttcc cattgctatt gaagggtgtg aaaacgaggg tgataacaaa 1260
ctttgtcagc ttgaaacgtt ccaaaagaaa gtcgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 3
<211> 441
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Thr Ile Ala Lys Glu Tyr Leu Arg Leu Ser Ile Leu Thr Leu Val
1 5 10 15
Leu Ser Ser Phe Thr Leu Ser Ala Ala Pro Leu Ala Ala Gln Ser Thr
20 25 30
Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His Gly Val Arg
35 40 45
Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr Pro Asp Lys
50 55 60
Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr Leu Thr Pro Arg Gly Ala
65 70 75 80
Gly Leu Val Thr Leu Met Gly Gly Phe Tyr Gly Asp Tyr Phe Arg Ser
85 90 95
Tyr Gly Ala Leu Pro Ala Gly Cys Pro Ala Asp Glu Ser Ile Tyr Val
100 105 110
Gln Ala Asp Val Asp Gln Arg Thr Arg Leu Thr Gly Gln Ala Phe Leu
115 120 125
Asp Gly Ile Ala Pro Asp Cys Gly Leu Lys Val His Tyr Gln Ala Asp
130 135 140
Leu Lys Lys Ile Asp Pro Leu Phe His Thr Val Glu Ala Gly Val Cys
145 150 155 160
Lys Leu Asp Pro Glu Lys Thr His Gln Ala Val Glu Lys Arg Leu Gly
165 170 175
Gly Pro Leu Asn Glu Leu Ser Gln Arg Tyr Ala Lys Pro Phe Ala Leu
180 185 190
Met Gly Glu Val Leu Asn Phe Ser Ala Ser Pro Tyr Cys Asn Ser Leu
195 200 205
Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala Thr Phe Ala Ala Asn Glu
210 215 220
Ile Glu Val Asn Lys Glu Gly Thr Lys Val Ser Leu Ser Gly Pro Leu
225 230 235 240
Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln Asn Ser Gln
245 250 255
Ala Met Pro Asp Val Ala Trp Asn Arg Leu Ser Gly Glu Glu Asn Trp
260 265 270
Ile Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Met Ala Lys
275 280 285
Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu Gln Gln Ile
290 295 300
Asp Thr Ala Leu Val Leu Gln Arg Asp Ala Gln Gly Gln Thr Leu Pro
305 310 315 320
Leu Ser Pro Gln Thr Lys Leu Leu Phe Leu Gly Gly His Asp Thr Asn
325 330 335
Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Asn Trp Gln Leu Pro Gln
340 345 350
Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe Glu Leu Trp
355 360 365
Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys Met Phe Tyr
370 375 380
Gln Thr Met Glu Gln Leu Arg Asn Ala Asp Lys Leu Asp Leu Lys Asn
385 390 395 400
Asn Pro Ala Arg Ile Val Pro Ile Ala Ile Glu Gly Cys Glu Asn Glu
405 410 415
Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr Phe Gln Lys Lys Val Ala
420 425 430
Gln Val Ile Glu Pro Ala Cys His Ile
435 440
<210> 4
<211> 441
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Thr Ile Ala Lys Glu Tyr Leu Arg Leu Ser Ile Leu Thr Leu Val
1 5 10 15
Leu Ser Ser Phe Thr Leu Ser Ala Ala Pro Leu Ala Ala Gln Ser Thr
20 25 30
Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His Gly Val Arg
35 40 45
Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr Pro Asp Lys
50 55 60
Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr Leu Thr Pro Arg Gly Ala
65 70 75 80
Gly Leu Val Thr Leu Met Gly Gly Phe Tyr Gly Asp Tyr Phe Arg Ser
85 90 95
Tyr Gly Ala Leu Pro Ala Gly Cys Pro Ala Asp Glu Ser Ile Tyr Val
100 105 110
Gln Ala Asp Val Asp Gln Arg Thr Arg Leu Thr Gly Gln Ala Phe Leu
115 120 125
Asp Gly Ile Ala Pro Asp Cys Gly Leu Lys Val His Tyr Gln Ala Asp
130 135 140
Leu Lys Lys Ile Asp Pro Leu Phe His Thr Val Glu Ala Gly Val Cys
145 150 155 160
Lys Gly Asp Pro Glu Lys Thr His Gln Ala Val Glu Lys Arg Leu Gly
165 170 175
Gly Pro Leu Asn Glu Leu Ser Gln Arg Tyr Ala Lys Pro Phe Ala Leu
180 185 190
Met Gly Glu Val Leu Asn Phe Ser Ala Ser Pro Tyr Cys Asn Ser Leu
195 200 205
Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala Thr Phe Ala Ala Asn Glu
210 215 220
Ile Glu Val Asn Lys Glu Gly Thr Lys Val Ser Leu Ser Gly Pro Leu
225 230 235 240
Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln Asn Ser Gln
245 250 255
Ala Met Pro Asp Val Ala Trp Asn Arg Leu Ser Gly Glu Glu Asn Trp
260 265 270
Ile Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Met Ala Lys
275 280 285
Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu Gln Gln Ile
290 295 300
Asp Thr Ala Leu Val Leu Gln Arg Asp Ala Gln Gly Gln Thr Leu Pro
305 310 315 320
Leu Ser Pro Gln Thr Lys Leu Leu Phe Leu Gly Gly His Asp Thr Asn
325 330 335
Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Asn Trp Gln Leu Pro Gln
340 345 350
Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe Glu Leu Trp
355 360 365
Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys Met Phe Tyr
370 375 380
Gln Thr Met Glu Gln Leu Arg Asn Ala Asp Lys Leu Asp Leu Lys Asn
385 390 395 400
Asn Pro Ala Arg Ile Val Pro Ile Ala Ile Glu Gly Cys Glu Asn Glu
405 410 415
Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr Phe Gln Lys Lys Val Ala
420 425 430
Gln Val Ile Glu Pro Ala Cys His Ile
435 440
<210> 5
<211> 441
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Thr Ile Ala Lys Glu Tyr Leu Arg Leu Ser Ile Leu Thr Leu Val
1 5 10 15
Leu Ser Ser Phe Thr Leu Ser Ala Ala Pro Leu Ala Ala Gln Ser Thr
20 25 30
Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His Gly Val Arg
35 40 45
Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr Pro Asp Lys
50 55 60
Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr Leu Thr Pro Arg Gly Ala
65 70 75 80
Gly Leu Val Thr Leu Met Gly Gly Phe Tyr Gly Asp Tyr Phe Arg Ser
85 90 95
Tyr Gly Ala Leu Pro Ala Gly Cys Pro Ala Asp Glu Ser Ile Tyr Val
100 105 110
Gln Ala Asp Val Asp Gln Arg Thr Arg Leu Thr Gly Gln Ala Phe Leu
115 120 125
Asp Gly Ile Ala Pro Asp Cys Gly Leu Lys Val His Tyr Gln Ala Asp
130 135 140
Leu Lys Lys Ile Asp Pro Leu Phe His Thr Val Glu Ala Gly Val Cys
145 150 155 160
Lys Gly Asp Pro Glu Lys Thr His Gln Ala Val Glu Lys Arg Leu Gly
165 170 175
Gly Pro Leu Asn Glu Leu Ser Gln Arg Tyr Ala Lys Pro Phe Ala Leu
180 185 190
Met Gly Glu Val Leu Asn Phe Ser Ala Ser Pro Tyr Cys Asn Ser Leu
195 200 205
Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala Thr Phe Ala Ala Asn Glu
210 215 220
Ile Glu Val Asn Lys Gly Gly Thr Lys Val Ser Leu Ser Gly Pro Leu
225 230 235 240
Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln Asn Ser Gln
245 250 255
Ala Met Pro Asp Val Ala Trp Asn Arg Leu Ser Gly Glu Glu Asn Trp
260 265 270
Ile Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Met Ala Lys
275 280 285
Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu Gln Gln Ile
290 295 300
Asp Thr Ala Leu Val Leu Gln Arg Asp Ala Gln Gly Gln Thr Leu Pro
305 310 315 320
Leu Ser Pro Gln Thr Lys Leu Leu Phe Leu Gly Gly His Asp Thr Asn
325 330 335
Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Asn Trp Gln Leu Pro Gln
340 345 350
Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe Glu Leu Trp
355 360 365
Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys Met Phe Tyr
370 375 380
Gln Thr Met Glu Gln Leu Arg Asn Ala Asp Lys Leu Asp Leu Lys Asn
385 390 395 400
Asn Pro Ala Arg Ile Val Pro Ile Ala Ile Glu Gly Cys Glu Asn Glu
405 410 415
Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr Phe Gln Lys Lys Val Ala
420 425 430
Gln Val Ile Glu Pro Ala Cys His Ile
435 440
<210> 6
<211> 1326
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgacaatag caaaagaata tctgcggtta tccatactca ctttggtgct cagtagtttt 60
acgctaagtg ctgcaccgct tgcagcacaa tctaccggtt acactttgga gcgcgtggtg 120
attttgagcc gccacggtgt tcgttccccg acgaaacaaa cacagttaat gaatgatgtt 180
acaccggaca aatggccaca atggccagta aaagcgggct atttaacgcc gcgaggggca 240
ggattagtca ctttaatggg cgggttctat ggtgattatt tccgcagcta tggggcgtta 300
ccggcggggt gcccggcaga cgaatccatc tatgtgcaag ctgatgttga ccaacgtacc 360
cgcttaaccg ggcaggcatt tctggacggt atagccccgg attgcggcct gaaagtacat 420
tatcaagctg atttgaaaaa aattgaccca ttgttccata ccgtcgaggc gggggtatgt 480
aaattggacc cagagaaaac tcatcaggct gttgaaaaac gcttgggtgg gccattaaat 540
gaactgagtc aacgctatgc caagcccttt gccctgatgg gcgaggtgct gaatttttcg 600
gcctcacctt attgcaactc actgcaacag aaaggaaaaa cctgtgattt tgcgactttt 660
gcagcaaatg aaatcgaggt aaataaagaa gggacaaaag tctcactgag tgggccattg 720
gcgctatcat cgacattagg tgaaattttc ctattacaaa attcacaggc catgccagat 780
gtcgcctgga accgtctcag cggtgaagaa aattggattt cattattgtc actgcataat 840
gcacagttcg atttgatggc caaaacccct tatatcgccc ggcataaagg aactccgttg 900
ttgcaacaaa ttgatacggc attagtgttg caacgtgatg ctcaggggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc gggggacatg acaccaatat tgccaatatt 1020
gcgggtatgt taggggccaa ttggcaatta ccgcagcaac ctgataatac cccgccaggc 1080
ggagggctag tctttgagct atggcagaat ccggataacc atcaacgcta tgtggcggtg 1140
aaaatgttct atcaaacgat ggagcagttg cgcaatgcag ataagttaga tttgaaaaac 1200
aacccggcaa gaattgttcc cattgctatt gaagggtgtg aaaacgaggg tgataacaaa 1260
ctttgtcagc ttgaaacgtt ccaaaagaaa gtcgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 7
<211> 1326
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgacaatag caaaagaata tctgcggtta tccatactca ctttggtgct cagtagtttt 60
acgctaagtg ctgcaccgct tgcagcacaa tctaccggtt acactttgga gcgcgtggtg 120
attttgagcc gccacggtgt tcgttccccg acgaaacaaa cacagttaat gaatgatgtt 180
acaccggaca aatggccaca atggccagta aaagcgggct atttaacgcc gcgaggggca 240
ggattagtca ctttaatggg cgggttctat ggtgattatt tccgcagcta tggggcgtta 300
ccggcggggt gcccggcaga cgaatccatc tatgtgcaag ctgatgttga ccaacgtacc 360
cgcttaaccg ggcaggcatt tctggacggt atagccccgg attgcggcct gaaagtacat 420
tatcaagctg atttgaaaaa aattgaccca ttgttccata ccgtcgaggc gggggtatgt 480
aaaggcgacc cagagaaaac tcatcaggct gttgaaaaac gcttgggtgg gccattaaat 540
gaactgagtc aacgctatgc caagcccttt gccctgatgg gcgaggtgct gaatttttcg 600
gcctcacctt attgcaactc actgcaacag aaaggaaaaa cctgtgattt tgcgactttt 660
gcagcaaatg aaatcgaggt aaataaagaa gggacaaaag tctcactgag tgggccattg 720
gcgctatcat cgacattagg tgaaattttc ctattacaaa attcacaggc catgccagat 780
gtcgcctgga accgtctcag cggtgaagaa aattggattt cattattgtc actgcataat 840
gcacagttcg atttgatggc caaaacccct tatatcgccc ggcataaagg aactccgttg 900
ttgcaacaaa ttgatacggc attagtgttg caacgtgatg ctcaggggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc gggggacatg acaccaatat tgccaatatt 1020
gcgggtatgt taggggccaa ttggcaatta ccgcagcaac ctgataatac cccgccaggc 1080
ggagggctag tctttgagct atggcagaat ccggataacc atcaacgcta tgtggcggtg 1140
aaaatgttct atcaaacgat ggagcagttg cgcaatgcag ataagttaga tttgaaaaac 1200
aacccggcaa gaattgttcc cattgctatt gaagggtgtg aaaacgaggg tgataacaaa 1260
ctttgtcagc ttgaaacgtt ccaaaagaaa gtcgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 8
<211> 1326
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgacaatag caaaagaata tctgcggtta tccatactca ctttggtgct cagtagtttt 60
acgctaagtg ctgcaccgct tgcagcacaa tctaccggtt acactttgga gcgcgtggtg 120
attttgagcc gccacggtgt tcgttccccg acgaaacaaa cacagttaat gaatgatgtt 180
acaccggaca aatggccaca atggccagta aaagcgggct atttaacgcc gcgaggggca 240
ggattagtca ctttaatggg cgggttctat ggtgattatt tccgcagcta tggggcgtta 300
ccggcggggt gcccggcaga cgaatccatc tatgtgcaag ctgatgttga ccaacgtacc 360
cgcttaaccg ggcaggcatt tctggacggt atagccccgg attgcggcct gaaagtacat 420
tatcaagctg atttgaaaaa aattgaccca ttgttccata ccgtcgaggc gggggtatgt 480
aaaggcgacc cagagaaaac tcatcaggct gttgaaaaac gcttgggtgg gccattaaat 540
gaactgagtc aacgctatgc caagcccttt gccctgatgg gcgaggtgct gaatttttcg 600
gcctcacctt attgcaactc actgcaacag aaaggaaaaa cctgtgattt tgcgactttt 660
gcagcaaatg aaatcgaggt aaataaaggc gggacaaaag tctcactgag tgggccattg 720
gcgctatcat cgacattagg tgaaattttc ctattacaaa attcacaggc catgccagat 780
gtcgcctgga accgtctcag cggtgaagaa aattggattt cattattgtc actgcataat 840
gcacagttcg atttgatggc caaaacccct tatatcgccc ggcataaagg aactccgttg 900
ttgcaacaaa ttgatacggc attagtgttg caacgtgatg ctcaggggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc gggggacatg acaccaatat tgccaatatt 1020
gcgggtatgt taggggccaa ttggcaatta ccgcagcaac ctgataatac cccgccaggc 1080
ggagggctag tctttgagct atggcagaat ccggataacc atcaacgcta tgtggcggtg 1140
aaaatgttct atcaaacgat ggagcagttg cgcaatgcag ataagttaga tttgaaaaac 1200
aacccggcaa gaattgttcc cattgctatt gaagggtgtg aaaacgaggg tgataacaaa 1260
ctttgtcagc ttgaaacgtt ccaaaagaaa gtcgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326

Claims (8)

1. A phytase mutant with improved pepsin resistance, which is characterized in that the 99 th leucine of the phytase with an amino acid sequence shown as SEQ ID NO.1 is mutated into alanine.
2. The phytase mutant with the improved pepsin resistance is characterized in that the amino acid sequence of the phytase shown as SEQ ID NO.1 is that the 99 th leucine of the phytase is mutated into alanine, and the 162 th leucine of the phytase is mutated into glycine.
3. The phytase mutant with the improved pepsin resistance is characterized in that the 99 th leucine of the phytase with the amino acid sequence shown as SEQ ID NO.1 is mutated into alanine, the 162 th leucine is mutated into glycine, and the 230 th glutamic acid is mutated into glycine.
4. A phytase mutant gene encoding a phytase mutant with improved pepsin resistance according to claim 1, 2 or 3.
5. The phytase mutant gene according to claim 4, wherein the nucleotide sequence of the gene is shown as SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
6. A recombinant vector comprising the phytase mutant gene of claim 4.
7. A recombinant strain comprising the phytase mutant gene of claim 4.
8. A method for preparing a phytase mutant with improved pepsin resistance, comprising the following steps:
1) transforming a host cell with the recombinant vector of claim 6 to obtain a recombinant strain;
2) culturing the recombinant strain, and inducing the expression of the recombinant phytase mutant; and
3) recovering and purifying the expressed phytase mutant.
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CN106011101A (en) * 2016-07-06 2016-10-12 中国农业科学院饲料研究所 Phytase mutants YkAPPA-L162V and encoding gene and application thereof
CN106011102A (en) * 2016-07-06 2016-10-12 中国农业科学院饲料研究所 Phytase mutants YkAPPA-L396V and YeAPPA-L396V and encoding gene and application thereof

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CN106011101A (en) * 2016-07-06 2016-10-12 中国农业科学院饲料研究所 Phytase mutants YkAPPA-L162V and encoding gene and application thereof
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Inventor after: Yang Peilong

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