A method for reducing the sensitivity of pullulanase to cyclodextrin
A pullulanase and cyclodextrin technology, which is applied in the field of changing the interaction between cyclodextrin and pullulanase, can solve problems such as unprovided solutions, and achieves improved utilization and cyclodextrin yield, Sensitivity-reducing effect
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Embodiment 1
[0037] A method for reducing the sensitivity of pullulanase to cyclodextrin by rational mutation, said method comprising the steps of:
[0038] (1) Using the pullulanase (PDB: 2E8Z) from Bacillus subtilis str.168, which has a known crystal structure of the interaction between cyclodextrin and pullulanase, as a template, its NCBI The accession number is AF008220.1, and the pullulanase gene sequence AmyX was synthesized by chemical total synthesis. The plasmid used to construct the expression vector in E. coli is pET20b(+) with T7 promoter.
[0039] (2) The pET20b(+) plasmid and the plasmid containing the AmyX gene were double-digested with NcoI and BamI respectively, and the digested products were recovered by tapping and then ligated with T4 ligase, and the ligated products were transformed into E.coli DH5α competent cells, Cultivate at 37°C for 8-12 hours, pick the transformant and shake it in liquid LB medium containing 100 μg / mL ampicillin, extract the plasmid, and verify ...
Embodiment 2
[0065] A method for reducing the sensitivity of pullulanase to cyclodextrin by rational mutation, said method comprising the steps of:
[0066] (1) Using the pullulanase (PDB: 2E8Z) from Bacillus subtilis str.168, which has a known crystal structure of the interaction between cyclodextrin and pullulanase, as a template, its NCBI The accession number is AF008220.1, and the pullulanase gene sequence AmyX was synthesized by chemical total synthesis. The plasmid used to construct the expression vector in E. coli is pET20b(+) with T7 promoter.
[0067] (2) The pET20b(+) plasmid and the plasmid containing the AmyX gene were double-digested with NcoI and BamI respectively, and the digested products were recovered by tapping and then ligated with T4 ligase, and the ligated products were transformed into E.coli DH5α competent cells, Cultivate at 37°C for 8-12 hours, pick the transformant and shake it in liquid LB medium containing 100 μg / mL ampicillin, extract the plasmid, and verify ...
Embodiment 3
[0093] A method for reducing the sensitivity of pullulanase to cyclodextrin by rational mutation, said method comprising the steps of:
[0094] (1) Using the pullulanase (PDB: 2E8Z) from Bacillus subtilis str.168, which has a known crystal structure of the interaction between cyclodextrin and pullulanase, as a template, its NCBI The accession number is AF008220.1, and the pullulanase gene sequence AmyX was synthesized by chemical total synthesis. The plasmid used to construct the expression vector in E. coli is pET20b(+) with T7 promoter.
[0095] (2) The pET20b(+) plasmid and the plasmid containing the AmyX gene were double-digested with NcoI and BamI respectively, and the digested products were recovered by tapping and then ligated with T4 ligase, and the ligated products were transformed into E.coli DH5α competent cells, Cultivate at 37°C for 8-12 hours, pick the transformant and shake it in liquid LB medium containing 100 μg / mL ampicillin, extract the plasmid, and verify ...
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