Novel feruloyl esterase, coding gene and application thereof
A ferulic acid esterase and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of non-cultivation, limited application, limited development and utilization of new ferulic acid esterase, etc., to achieve easy digestion and absorption , high enzyme activity, the effect of improving feed utilization
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[0051] Example 1: Screening of ferulic acid esterase gene in soil metagenomic library
[0052] 1. Preliminary screening by plate method
[0053] To prepare the FAE screening medium, add 1.5% (v / v) methyl ferulate (dissolved in dimethylformamide, 10% w / v) to the LB liquid medium. Add ampicillin with a final concentration of 100 mg / L at a temperature (50-60 °C), shake vigorously to make the methyl ferulate evenly distributed, and immediately pour the plate on the ultra-clean table. Cosmid library bacterial solution was coated on each screening plate, cultured at 37°C for 1-2 days, and the formation of a transparent circle around the colony was observed ( figure 1 ).
[0054] 2. Rescreening
[0055] After the primary screening, the clones were inoculated into LB liquid medium, cultured overnight at 37°C, and centrifuged at 12,000 g for 8 min to collect the bacteria. The cells were washed three times with sterile water and resuspended in deionized water. The bacterial suspen...
Example Embodiment
[0060] Example 2: Cloning of ferulic acid esterase gene
[0061] 1. PCR amplification
[0062] Using the primer fae-f (5'-CATG CCATGG GCATGCGTGCAGGGGGAG-3') and fae-r (5'-CCC AAGCTT CCGGCCGCTCAGCCAGT-3') to amplify the ferulic acid esterase gene fae-xuan, and the underlines of the upstream and downstream primers indicate the restriction sites of NcoI and HindIII, respectively. PCR reaction system (25 μL): 9.5 μL of ultrapure water, 12.5 μL of Mix, 1 μL of upstream and downstream primers, and 1 μL of positive subcloned plasmid DNA. PCR reaction conditions: 94°C for 4 min; 35 cycles of 94°C for 30s, 60°C for 45s, 72°C for 1 min; 72°C for 10 min. The PCR product was electrophoresed and recovered by gel tapping to obtain a purified PCR product. 2. Enzyme digestion
[0063] The purified and recovered PCR product was subjected to double digestion, and the digestion time was 3h. The digestion system was as follows: NcoI 5 μL, HindIII 5 μL, 10×K Buffer 10 μL, 0.1% BSA 10 μL, PC...
Example Embodiment
[0069] Example 3: Heterologous expression and purification of ferulic acid esterase FAE-Xuan
[0070] 1. Conversion
[0071] Take 10 μL of the pET28a-fae-xuan plasmid obtained in Example 2 and add it to 100 μL of Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 min, heat shock in a water bath at 42°C for 90 s, and add 900 μL LB liquid culture after ice bathing for 2 min. base, 150rpm, 37°C shaking culture for 45min. The culture was centrifuged at 2500 g for 5 min, 600 μL of supernatant was removed, the bacteria were resuspended with the remaining medium and spread on LB plates containing kanamycin, and single colonies were picked after overnight culture at 37°C. Thus, Escherichia coli BL21(DE3) containing pET28a-fae-xuan was obtained.
[0072] 2. Express
[0073] The recombinant bacteria were inoculated into 5 mL LB liquid medium containing kanamycin, and cultured at 37° C. and 180 rpm for 12 h. Inoculate 1.5 mL of bacterial liquid into 150 mL of fresh L...
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