A kind of phytase mutant and its carrier and application
A technology of mutants and phytase, applied in the direction of application and use of vectors to introduce foreign genetic material, enzymes, etc., can solve the problems of loss, poor heat resistance of EcAppA, etc., achieve the effect of reducing enzyme activity loss and good application prospects
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Embodiment 1
[0049] Embodiment 1 The acquisition of phytase mutant and the construction of expression vector
[0050] Two parental phytase sequences were synthesized, namely ExAPPA phytase sequence and CxAPPA phytase sequence. The two genes were synthesized by Synbio Technologies (http: / / www.synbio-tech.com.cn / ).
[0051] The sequence is as follows:
[0052] ExAPPA amino acid sequence:
[0053] QSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQPGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPVKMGVCAFNVQKTQEAILTRAEGNIERYTQRYDSAFRTLEQVLNFPQSKLCFNREKQNESCSLTQALPSELKVSADNVSITGAVSLASMLTEIFLLQEAQGMPEPGWGRITDSHQWNTLLSLHNAQFDLLQRTPEVARSRATPLLDMIMAALTPHPPQKQAYGVTLPTSVLFIAGHDTNLANLGGALGLNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVYQTLQQMRDKTPLSLNTPPGEVKLTLPGCEERNAQGMCSLAGFTQIVNEARIPACSL(SEQ ID NO.1)。
[0054] CxAPPA amino acid sequence:
[0055] EEQNGMKLERVVIVSRHGVRAPTKFTPIMKNVTPDQWPQWDVPLGWLTPRGCELVSELGQYQRLWFTSKGLLNNQTCPSPGQVAVIADTDQRTRKTGECFLAGLAPKCQIQVHYQKDEEKNDPLFNPLKTGV...
Embodiment 2
[0079] The optimal reaction temperature of embodiment 2 phytase mutants
[0080] The phytase-mutated enzyme solution was heat-treated at different temperatures (60°C-90°C) for 5 minutes, and the remaining phytase activity was measured. Using the untreated enzyme activity as a control, the remaining relative enzyme activity at this temperature was calculated. The results are shown in Table 3 below.
[0081] Enzyme activity retention rate at different temperatures of table 3 phytase mutants
[0082] Enzyme 60℃ 65℃ 70℃ 75℃ 80℃ 85℃ 90℃ ExAPPA 99.5 94.3 80.2 56.3 41.6 22.4 12.3 CxAPPA 99.4 97.6 85.5 64.8 52.5 34.6 24.6 ExCxAPPA1 99.2 99.5 94.6 76.5 67.6 45.6 34.5 ExCxAPPA2 87.2 80.3 73.3 69.5 73.4 73.6 67.5 ExCxAPPA3 99.5 99.5 92.6 91.5 82.3 62.6 45.5 ExCxAPPA4 100.4 93.8 97.5 90.7 89.6 73.8 64.5 ExCxAPPA5 102.8 102.4 104.1 100.6 98.1 86.4 76.3 ExCxAPPA6 98.5 93.7 91.1 78.9...
Embodiment 3
[0084] Thermostability under different pH environments of embodiment 3 phytase mutants
[0085] The enzyme activity of phytase was measured in buffer systems with different pH (2.5-7.0), and the relative enzyme activity at different pH values was measured at 37°C with the enzyme activity at pH 5.5 as a control. The results are shown in Table 4 below.
[0086] Thermostability under different pH environments of table 4 phytase mutants
[0087]
[0088] The test results showed that the six phytases obtained by screening had a wide pH range of more than 3 pH units, and within the pH range, the retention rate was at least 70%. Among them, the ExCxAPPA3 and ExCxAPPA4 mutants, especially at 85°C and 90°C pelleting conditions, have a smaller loss rate than wild phytase enzyme activity, indicating that ExCxAPPA3 and ExCxAPPA4 mutants can exert the best enzymatic hydrolysis effect in the gastrointestinal tract of monogastric animals .
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