Pepsin resistance improved phytase mutant and coding gene and application thereof
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
[0039] Embodiment 1: Obtaining of mutant genes
[0040] The gene sequence (SEQ ID NO.2) of phytase YkAPPA derived from Yersinia kristeenii (Yersinia kristeensenii) was transformed, the mutation was introduced by means of Overlap PCR, and the mutation was sequenced to obtain the mutant gene. The method uses the pEASY-T3-YkAPPA recombinant plasmid containing the wild phytase gene YkAPPA as a template, introduces mutations through two rounds of PCR reactions, and the required mutant genes are connected to the carrier pEASY-T3 carrier and confirmed by DNA sequencing.
[0041] Eight primers were used for the mutation: YkAPPA-F, YkAPPA-R, YkAPPA-L99A-F, YkAPPA-L99A-R, YkAPPA-L162G-F, YkAPPA-L162G-R, YkAPPA-E230G-F, and YkAPPA-E230G-R.
[0042] The primer sequences are as follows:
[0043] YkAPPA-F: 5'-cgcgaattcgcaccgcttgcagcacaatctac-3'
[0044] YkAPPA-R: 5'-gatgcggccgcttaaatggcaggctggctcG-3'
[0045] YkAPPA-L99A-F: 5'-tccgcagctatggg gcg ttaccggcggggtg-3'
[0046] YkAPPA-L99A-...
Embodiment 2
[0052] Example 2: Expression and purification of phytase in Escherichia coli before and after transformation
[0053] Phytase YkAPPA mutants 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 sequence of the wild-type phytase and its mutants was inserted between the EcoRI and NotI restriction sites of the prokaryotic expression vector pET-22b(+), and induced by T7lac in Escherichia coli BL21(DE3) cells. The agent was IPTG (isopropyl-β-D-galactoside) with a final concentration of 1 mM, and induced culture at 24° C. and 220 rpm in a shaker for 5 h. The crude enzyme liquid was chromatographically purified by nickel-nitrilotriacetic acid (Ni-NTA) column and diethylaminoethyl (DEAE) column. Detected by 10% SDS-PAGE electrophoresis, the surface molecular weight of...
Embodiment 3
[0054] Example 3: Pepsin resistance of mutant phytases
[0055] The pepsin resistance of phytase was measured by detecting the remaining phytase activity and phytase protein after treatment with different concentrations of pepsin for 2 hours. The mass ratio of pepsin to phytase is 1 / 1000, 1 / 500, 1 / 200, 1 / 100, 1 / 40 and 1 / 20.
[0056] The ferrous sulfate molybdenum blue method was used to determine the remaining phytase activity after 2 h treatment with different concentrations of pepsin. Add 50 μL enzyme solution of appropriate concentration to 950 μL 1.5mmol / L sodium phytate substrate (prepared with pH 4.5 and 0.25M NaAc-HAc buffer), react in a 37°C water bath for 30min, add 1mL 10% trichloro Acetic acid was used to terminate the reaction, and finally 2 mL of color developing solution (1% ammonium molybdate tetrahydrate, 3.2% concentrated sulfuric acid, 7.32% ferrous sulfate) was added for color development. Absorbance values were measured at 700 nm as a measure of the amo...
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