Pullulanase enzyme production gene, carrier containing same and application of carrier
A technology of pullulanase and enzyme gene, applied in the preparation of pullulanase, in the field of pullulanase enzyme production gene, can solve the restriction of pullulanase industrial application, pullulanase enzyme activity is not high , can not adapt to the acidic high temperature environment and other problems
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Embodiment 1
[0071] The pullulanase enzyme-producing gene provided by the present invention is compatible with Klebsiella mutabilis HN7 ( Klebsiella variicola Compared with the wild-type pullulanase gene of HN7), the N-terminus of the de-signal peptide lacks the gene sequence corresponding to 31 amino acids, and the specific sequence is shown in the sequence table.
[0072] Because the pullulanase enzyme production gene of the present invention and Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) is relatively close to the wild-type pullulanase gene, so the applicant uses Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) wild type as the basis, and the vector containing the pullulanase enzyme production gene was constructed at the same time, and the specific construction process is introduced as follows.
[0073] (1) Genomic DNA extraction Specifically, the Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) was inoculated into the seed medium, the bacteria were collected, and ...
Embodiment 2
[0135] The carrier containing the pullulanase enzyme-producing gene prepared in Example 1 is used in the preparation of pullulanase, and the specific steps are as follows:
[0136] (1) Heat shock transformation to construct pullulanase truncated mutant engineered bacteria ,Specifically,
[0137] The pET21a-pulA-N31 expression vector constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and after recovery, the competent cells were spread on LB plates containing ampicillin (Amp), Cultivate; in detail:
[0138] This example is a period of simple operation, directly add 10 μL of the ligation product to 200 μL of Escherichia coli BL21 (DE3) competent cells, gently pipette evenly, place on ice for 30 minutes, then heat shock at 42°C for 90 seconds, and then place on ice for 90 seconds. Spread the competent cells on LB plates with a final concentration of Amp of 0.1 mg / mL, and incubate at 37°C for 12-16 hours;
[0139] Aft...
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