Method for screening beta-glucosidase from fosmid library by using esculin and 4-MUG
A glucosidase and glucoside technology, which is applied in microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problems of slow DNA sequence and amino acid sequence of β-glucosidase.
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Embodiment 1
[0065] Take 10g of soil samples collected in Panjin City, Liaoning, and apply MoBio's PowerSoil TM DNA Isolation Kit, strictly follow the instructions to extract soil DNA. Take 50ml DNA (0.5μg / μl), according to Epicentre's CopyControl TM Instructions for the use of Fosmin LibraryProduction Kit, fill in the ends of the DNA fragment, after low-melting point agarose gel electrophoresis, cut and separate the DNA bands containing 20-60kb bases, recover the DNA by sol method, ligate, and package , transfected into EPI300 host cells, spread on a plate containing chloramphenicol, and cultured at 37°C for 24 hours. Pick a single clone for preservation, and elute the colony preservation pool library on the plate, which is named No. 16 Fosmid library.
[0066] The construction method of No. 18 Fosmid library was the same as above, and the soil samples were collected from Hohhot, Inner Mongolia.
Embodiment 2
[0068] The final concentration composition of the basal medium (primary screening medium) containing aescin: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar powder 15g / L, aescin 0.5g / L, Ferric citrate 2g / L, chloramphenicol 15mg / L, solvent is water.
[0069] Basal medium (re-screening medium) final concentration composition: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar powder 15g / L, chloramphenicol 15mg / L, solvent is water.
[0070] Prepare 4-MUG low melting point agarose solution: the final concentration of 4-methylumbelliferone-β-D-glucoside (4-MUG) is 0.4g / L, low melting point agarose (Agarose LM GQT, gel temperature (24~28°C), the final concentration is 5g / L, and the solvent is water.
[0071] From the No. 16 Fosmid library of embodiment 1, get the bacterium solution 0.01ml that a tube pool preserves, dilute 10 respectively with sterile water -3 、10 -4 、10 -5 、10 -6 times, to obtain 4 parts of diluted bacterial solution, absorb 0.2mL of d...
Embodiment 3
[0072] Example 3 Application of β-glucosidase HU-Y1 in the preparation of resveratrol:
[0073] The β-glucosidase HU-Y1 gene obtained in Example 2 was connected to the pET 28a expression vector, and then transformed into E. Coil BL21. The obtained E. Coil BL21 / pET 28a / Y1 was cultured overnight on a shaker in LB liquid medium containing 100 mL of 50 μg / mL kanamycin. Pour 10ml of the overnight cultured bacterial solution into 1L 50μg / mL kanamycin LB liquid medium and cultivate until the bacterial solution OD 600When it reaches 0.6-0.8, add IPTG to a final concentration of 1.0 mmol / L. After induction at 28°C for 12 hours, the cells are collected by centrifugation, resuspended with 25 ml of phosphate buffer pH 7.4, and repeatedly frozen and thawed to break the cells. The supernatant was collected by centrifugation, and the supernatant containing β-glucosidase HU-Y1 protein was filtered through a 0.22 μm cellulose acetate filter membrane, and then purified by nickel column affinit...
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