A kind of xylosidase xyl_s and its coding gene and application
A technology of xylosidase and encoding gene, applied in the field of bioengineering, can solve the problem of not obtaining xylosidase reports and the like, and achieve the effect of improving the utilization rate
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Embodiment 1
[0025] Embodiment 1: the purification of glycosidase and the separation of active subunit thereof
[0026] 1. Cultivation of Cellulosimrobiumcellulans strain F16
[0027] Pick about 1 cm from the cultivated strain slant (xylan medium, containing 1.5% agar) 2 Square bacterial lawn, inoculated into 100ml aseptic wheat bran liquid medium (xylan medium composition: xylan 2%, yeast extract 0.2%, peptone 0.2%, K 2 HPO 4 0.1%), 30°C, 160rpm shake flask culture for 2 days.
[0028] 2. Separation and purification of xylosidase Xyl_S
[0029]10000g / min, after centrifugation for 2min, the supernatant collected is the crude enzyme solution. Proteins with β-xylosidase activity were tracked using p-nitrophenyl-β-D-xyloside (pNP-Xyl) as a specific chromogenic substrate. One enzyme unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol within 1 hour at 30°C, pH 7.5, with pNP-Xyl as the substrate. After ammonium sulfate precipitation in tur...
Embodiment 2
[0030] Embodiment 2: Cloning of xylosidase Xyl_S coding gene
[0031] With fibrosis fiber microbacteria (Cellulosimerbiumcellulans) bacterial strain F16 genome DNA as template, the coding gene 4929bp of PCR amplification xylosidase Xyl_S (see Figure 4 ), cloned into the pMD-T vector, and the sequencing verification results showed that it was consistent with the sequence shown in SEQ ID NO.1. The PCR reaction system and reaction conditions are shown in the table below:
[0032]
Embodiment 3
[0033] Embodiment 3: Recombinant expression of xylosidase Xyl_S in Escherichia coli
[0034] Using the vector obtained in Example 2 as a template, PCR amplified the 4929bp CDS region of the target gene, added NdeI / BamHI restriction sites on both sides of the fragment, added 6*His tags before the stop codon TGA, cloned into the pColdIV expression vector, and selected 2 The plasmids of positive clones were sequenced and verified, and the results showed that the sequences were correct. The corresponding recombinant vector is obtained. The PCR reaction system, reaction conditions and primers used are shown in the table below:
[0035]
[0036]
[0037] Take 1 μl of the above-mentioned recombinant vector and transfer it into Escherichia coli BL21 competent cells, use LB / antibiotic Amp (100 μg / ml) plate, coat with 50ul transformation solution, and culture at 37°C O / N. ControlpColdIV does the same.
[0038] Pick a single colony into 50ml LB / Amp (100μg / ml) medium, culture O / N...
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