A kind of alginate lyase algb and its coding gene and application
A technology of alginate lyase and encoding gene, applied in the field of alginate lyase gene algb, can solve the problems of difficult control of degradation conditions, complicated operation, long time consumption and the like
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Embodiment 1
[0029] The cultivation of embodiment 1Vibrio alginolyticus 40B and the extraction of bacterial strain genome DNA
[0030] The strain used was Vibrio alginolyticus, and the monoclonal colony of the strain was picked and inoculated into 50ml liquid medium, and then cultured on a shaker with a temperature of 30°C and a rotation speed of 220rpmin for 24 hours, and then the culture medium was centrifuged Collect the bacteria and discard the supernatant medium.
[0031] The liquid medium formula used is (g / L): beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl5.0g, MgSO4 7H2O 0.5g, CaCl20.2g, KH2PO41.0g, FeSO4 7H2O 0.02g, The pH value is 7.0.
[0032]Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. Discard the supernatant, resuspend the bacteria with 180 μL of Digestion Solution, then add 20 μL of proteinase K, mix thoroughly, and incubate at 56°C for 30 min. Add 20 μL of RNaseA, mix thoroughly, and place at room tempera...
Embodiment 2
[0033] Example 2 Cloning and identification of the gene encoding alginate lyase Algb
[0034] The following amplification primers were designed according to the possible alginate lyase gene in the Vibrio alginolyticus40B genome: upstream primer (5'-CGCGGATCCATGCGCTCAGAAGTTCGTGA-3') and downstream primer (5'-CCGCTCGAGTTGATGAAGAGTGCTCAAAG-3'). Using the extracted strain genome as a template, PCR amplification was performed to obtain the full-length sequence of alginate lyase Algb gene. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band between 1.0kb and 2.0kb, which was excised from the agarose gel and purified using a DNA gel recovery kit.
[0035] Ligate the purified DNA fragment to the cloning vector pMD19-T, transform Escherichia coli DH5α competent cells, culture in LB solid medium (conta...
Embodiment 3
[0065] Example 3 Construction of alginate lyase Algb recombinant expression vector
[0066] The upstream primer (5'-CGCGGATCCATGCGCTCAGAAGTTCGTGA-3') and the downstream primer (5'-CCGCTCGAGTTGATGAAGAGTGCTCAAAG-3') were designed according to the full sequence of the alginate lyase gene, and the full-length sequence of the alginate lyase Algb gene was obtained by PCR amplification. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. The PCR product was digested with Bam HI and Xho I and recovered; the Escherichia coli expression vector pET21a(+) was also digested with Bam HI and Xho I and recovered, connected with the target gene obtained above and transformed into E. coli DH5α , to screen for ampicillin-resistant transformants. The positive transformant plasmid was extracted with a plasmid extraction kit, and Bam HI and Xho I were used for enz...
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