A kind of glucoamylase derived from Staphylococcus niger
A technology of glucoamylase and glucose, which is applied in the field of genetic engineering and can solve problems that have not yet been reported.
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Embodiment 1
[0035]Example 1: Cloning of the glucoamylase gene
[0036] Genomic DNA was extracted from overnight cultures of P. niger using the Fungal Genomic DNA Extraction Kit (Omega).
[0037] Amplifying the glucoamylase gene in Staphylococcus niger with Genomic DNA as an amplification template, wherein the forward primer P573-F sequence used is (the sequence shown in the underline is the AflII restriction site);
[0038] 5′-AA CTTAAG ATCATGCTGACTTCGAACCTCGT-3′,
[0039] The reverse primer P573-R sequence is (the underlined sequence is the XbaI restriction site):
[0040] 5′-TGC TCTAGA TTATGCCCTGGACATGAC-3′
[0041] The gene was amplified from P. niger genomic DNA using Phusion DNA polymerase (Thermo scientific).
[0042] The above PCR product was purified using a gel purification kit (Fermentas). The purified PCR product was digested with restriction enzymes AflII and XbaI (Fermentas); meanwhile, the plasmid pGm was digested with restriction enzymes AflII and XbaI. The digeste...
Embodiment 2
[0045] Embodiment 2: PEG-mediated protoplast fusion transforms Aspergillus niger and verification
[0046] Pipette the spore suspension of Aspergillus niger GAP1 on the center of the CMA plate (9 cm petri dish), wait until the colony covers the entire petri dish, cut 1 / 4 size of the culture based on 200 mL of CMA liquid medium, at 30°C, 200 rpm Cultivate for 14-16 h.
[0047] Collect the mycelium with a sterile Miracloth filter cloth and wash it once with solution A, transfer the washed mycelium to 40 mL protoplastization solution under aseptic conditions, and incubate at 30°C and 200 rpm for 1~ After 2 h, the progress of protoplast transformation was detected by microscope observation.
[0048] Filter the above-mentioned warm bath liquid with a sterile Miracloth filter cloth, and the obtained filtrate is the protoplast solution. Divide the protoplast solution into two 50 mL sterile disposable centrifuge tubes, and dilute the volume of each tube to 45 mL with solution B, cen...
Embodiment 3
[0061] Embodiment 3: the fermentation of Aspergillus niger engineering bacterium and the expression of enzyme
[0062] Pick the positive transformant Aspergillus niger XUJ-1 obtained in Example 2, inoculate it in 30 mL TSB fermentation medium, and cultivate it at 30°C and 200 rpm for 5 days; the obtained fermentation broth is filtered with 8 layers of gauze, and the filtrate is in Centrifuge at 14,000 g for 10 min to collect the supernatant; run the supernatant on a 12% SDS-PAGE gel for electrophoresis. The result is as figure 2 Shown, swimming lane 1, swimming lane 2 show the expression situation of Aspergillus niger host protein; Swimming lane 3 shows the expression situation of Aspergillus niger XUJ-1 protein of the present invention, wherein the 95 kDa place indicated by the arrow has the target protein band, and expected It is consistent, indicating that the glucoamylase of the present invention is successfully expressed in Aspergillus niger, and its expression level is...
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