Novel coding gene of Type-I pullulanase, and recombinant expression and application thereof
A technology of pullulanase coding and pullulanase, applied in the construction of pullulanase coding gene recombinant plasmid and the preparation of recombinant pullulanase, pullulanase coding of pullulanase-producing strains of Bacillus inflata In the field of gene cloning and expression, it can solve problems such as cloning the pullulanase gene
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Embodiment 1
[0052] Example 1: The full-length gene pulB of TumebacillusflagellatesGST4 pullulanase was obtained by PCR method.
[0053] According to the genome sequencing results of TumebacillusflagellatesGST4 (derived from the information obtained after the whole genome sequencing of the enzyme-producing strain), through genetic informatics analysis, the PCR primers PulB-F and PulB-R were designed as follows:
[0054] PulB-F: 5'–CGC GGATCC CAATCAGGAAGCTATTTTTCA-3' (forward primer, the underline is the restriction enzyme cutting site BamHI), that is, SEQ ID NO:3.
[0055] PulB-R: 5'–ACC AAGCTT CACCGTTCCGCCGCTCA-3' (reverse primer, the underline is the restriction enzyme cutting site HindIII), ie SEQ ID NO:4.
[0056] Using the total genomic DNA of T.flagellatesGST4 as a template, PCR amplification was carried out with the above primers. The composition of the PCR reaction system and the PCR reaction procedure are as follows: 50 μL reaction liquid contains: 10 μL of 5×PSPCR buffer, 20...
Embodiment 2
[0058] Embodiment 2: Construction of recombinant plasmid and Escherichia coli BL21 / pET-pulB genetically engineered bacteria
[0059] Using the plasmid pMDT-pulB as a template, the following are primers:
[0060] PulB-F: 5'–CGC GGATCC CAATCAGGAAGCTATTTTTCA-3' (forward primer, the underline is the restriction enzyme cutting site BamHI), that is, SEQ ID NO:3.
[0061] PulB-R: 5'–ACC AAGCTT CACCGTTCCGCCGCTCA-3' (reverse primer, the underline is the restriction enzyme cutting site HindIII), ie SEQ ID NO:4.
[0062] Perform PCR amplification. The composition of the PCR reaction system and the PCR reaction procedure are as follows: 50 μL reaction solution contains: 10 μL of 5×PSPCR buffer, 200 μM of dNTPs, 10 ng of total DNA template, 3.2 pmol of each primer, and 5 units of PrimeSTAR DNA polymerase. The amplification program was: pre-denaturation at 95°C for 2min; 98°C for 10sec; 53.6°C for 15sec; 72°C for 2min, a total of 30 cycles of extension; 72°C for 10min. detected by aga...
Embodiment 3
[0068] Embodiment 3: recombinant pullulanase expression and purification
[0069] Expression: The prokaryotic expression plasmid pET-pulB was transformed into Escherichia coli BL21(DE3) according to the method of "Molecular Cloning Experiment Guide", spread on LB+Amp plates, and cultured upside down at 37°C until transformants appeared. Pick 5 positive transformants from the transformants with a toothpick, and inoculate them into 5mL LA (LB culture solution + 100ug / ml ampicillin) culture solution together with the negative control of the recipient bacteria containing empty plasmid, and culture overnight at 37°C and 220rpm. The next day, take 1.0mL overnight bacteria into 100mL LA, culture at 37°C, 220rpm, when the bacterial density (OD 600 ) to 0.4-0.6, add IPTG with a final concentration of 1 mM, and continue culturing at 30° C. and 220 rpm for 12 hours. Collect the supernatant by centrifugation, put the supernatant in a dialysis bag, apply PEG8000 externally, and place it i...
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