Phytase mutants with improved pepsin resistance and their encoding genes and applications
A technology of pepsin and phytase, applied in the field of genetic engineering, can solve the problems of poor pepsin resistance, difficult to meet the requirements of the feed industry, etc.
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Embodiment 1
[0038] Example 1: Obtainment of mutant genes
[0039] The gene sequence (SEQ ID NO. 2) of the phytase YkAPPA derived from Yersinia kristeensenii was modified, and mutations were introduced by means of Overlap PCR and sequenced to obtain the mutant gene. The method uses the pEASY-T3-YkAPPA recombinant plasmid containing the wild phytase gene YkAPPA as a template, and introduces mutations through two rounds of PCR reactions. The desired mutant genes are connected to the vector pEASY-T3 and confirmed by DNA sequencing.
[0040] Eight primers were used for mutation: YkAPPA-F, YkAPPA-R, YkAPPA-L99A-F, YkAPPA-L99A-R, YkAPPA-L162G-F, YkAPPA-L162G-R, YkAPPA-E230G-F and YkAPPA-E230G-R.
[0041] The primer sequences are as follows:
[0042] YkAPPA-F: 5’-cgcgaattcgcaccgcttgcagcacaatctac-3’
[0043] YkAPPA-R: 5’-gatgcggccgcttaaatatggcaggctggctcG-3’
[0044] YkAPPA-L99A-F: 5’-tccgcagctatggggcgttaccggcggggtg-3’
[0045] YkAPPA-L99A-R: 5’-caccccgccggtaacgccccatagctgcgga-3’
[0046] YkAP...
Embodiment 2
[0051] Example 2: Expression and purification of phytase before and after transformation in Escherichia coli
[0052] The mutants of phytase YkAPPA YkAPPA-L99A, YkAPPA-L99A / L162G and YkAPPA-L99A / L162G / E230G and their wild enzymes encode 441 amino acids and a stop codon, respectively, and the N-terminal 23 amino acids are signal peptide sequences. The theoretical molecular weight of the protein is 48.6kDa. The coding region sequences of wild-type phytase and its mutants were inserted between the EcoRI and NotI restriction sites of the prokaryotic expression vector pET-22b(+), under the control of T7 lac, in Escherichia coli BL21(DE3) cells, The inducer was IPTG (isopropyl-β-D-galactoside) at a final concentration of 1 mM, and the culture was induced for 5 h at 24° C. and a shaker at 220 rpm. The crude enzyme solution was purified by chromatography on nickel-nitrilotriacetic acid (Ni-NTA) column and diethylaminoethyl (DEAE) column. Detected by 10% SDS-PAGE electrophoresis, the...
Embodiment 3
[0053] Example 3: Pepsin resistance of mutant phytases
[0054] The pepsin resistance of phytase was measured by detecting the remaining phytase enzyme activity and phytase protein after treatment with different concentrations of pepsin for 2 h. The mass ratio of pepsin to phytase was 1 / 1000, 1 / 500, 1 / 200, 1 / 100, 1 / 40 and 1 / 20.
[0055] The phytase activity remaining after 2h treatment with different concentrations of pepsin was determined by the ferrous sulfate molybdenum blue method. Add 50μL of enzyme solution of appropriate concentration to 950μL of 1.5mmol / L sodium phytate substrate (prepared with pH 4.5 and 0.25M NaAc-HAc buffer), react in a water bath at 37°C for 30min, add 1mL of 10% trichloride Acetic acid was used to terminate the reaction, and 2 mL of color developing solution (1% ammonium molybdate tetrahydrate, 3.2% concentrated sulfuric acid, 7.32 ferrous sulfate) was finally added for color development. The amount of released inorganic phosphate was measured b...
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