Aspergillus niger, its α-l-rhamnosidase preparation method, plasmid vector and recombinant bacteria
A technology of rhamnosidase and Aspergillus niger, applied in the field of bioengineering, can solve the problem of low enzyme production and achieve good industrialization prospects
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Embodiment 1
[0030] Screening and identification of strains that hydrolyze naringin:
[0031] Plate selection medium: MgSO 4 ·7H 2 O 3-5.0g / L, K 2 HPO 4 5-10.0g / L, KCl 2-5.0g / L, FeSO 4 ·7H 2 O0.5-0.1g / L, 20g agar, 2-10g / L sucrose, 2.5-5g / L naringin, 1L distilled water, natural pH, steam sterilization at 121°C for 20-30min.
[0032] Enzyme production fermentation medium: MgSO 4 ·7H 2 O 0.5-2g / L, KH 2 PO 4 1.5-2.5g / L, (NH 4 ) 2 SO 4 2-4.0g / L, ZnSO 4 ·7H 2 O 0.05-0.1g / L, CaCl 2 0.05-0.1g / L, yeast extract 0.5-1g / L, naringin 5-8g / L, sucrose 10-20g / L, peptone 1-2g / L, pH 6.0-7.0, 115℃ for 20min
[0033] Break the rotten citrus peels in a shaker flask filled with glass beads, then dilute the plate screening medium, culture at 25-28°C for 3-5 days, and add 70-90% diethylene oxide to the medium Diol solution, using diethylene glycol to react with naringin to form a yellow color, does not react with the naringin hydrolyzate citronin to produce color, and a transparent circle is prod...
Embodiment 2
[0035] Identification of the WDQ-1 strain:
[0036] Morphological identification: the strain WDQ-1 was grown on the PDA plate, on the first day: yellow bulges were produced in the middle, and white fluff was produced around the bulges, with neat edges. Day 2: White spores start to appear on the yellow bumps, and folds form around the center of the bumps. Day 3: The spores around the bumps start to turn gray, forming a circle of gray spores, and the spores in the furrows formed by the folds also start to turn gray. Day 4: The white spores around the bulge start to turn gray, and the gray spores in the middle start to turn brown. Day 5: Multiple concentric circles are produced, and the outer spores start to turn brown. Day 6: The ring of spores closest to the outer ring of white down begins to turn brown. Day 7: The overall color is brown, the middle bulge is the darkest, and the color becomes lighter when the bulge goes out, and the spores that reach the outermost white fluf...
Embodiment 3
[0043] Cloning of the α-L-rhamnosidase gene:
[0044] Aspergillus niger strain WDQ-1 was inoculated in PDA liquid medium and cultured at 25-28°C for 1-3 days, the mycelium was collected, ground into white powder with liquid nitrogen, total RNA was extracted, and then reverse-transcribed into cDNA. PCR amplification was carried out using cDNA as template and F0 and R3 as primers. The PCR reaction conditions are: 90-95°C pre-denaturation for 3-5min, 94°C denaturation for 30-45sec, 55°C annealing for 30-45sec, 70-72°C extension for 2.5min, 30 cycles, 70-72°C total extension for 5-10min .
[0045] After the PCR reaction was completed, the PCR products were detected by agarose gel electrophoresis, and the results were as follows: figure 1 As shown, the α-L-rhamnosidase gene rha was recovered by cutting the gel. The primers are:
[0046] F0: 5'-ATGTGGTCTTCCTGGCTGCTG-3'
[0047] R3: 5'-CTAATTATTACTCAACTTCCACTTTTCCACCCTGC-3'
[0048] Ligate the α-L-rhamnosidase gene rha obtained...
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