New-source D-psicose-3-epimerase gene and application method thereof
A technology of epimerase and psicose, applied in the fields of genetic engineering and enzyme engineering, to achieve good substrate conversion rate, safe method and improved yield
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Embodiment 1
[0027] Example 1 Construction of Bacillus Secrecy Expression System
[0028] According to D-allows-3-differential interstitial use of genetic design, the primer:
[0029] Upstream primers: 5'-Tactaggatccatgaaacgaatctactacgcctatgggaaaagcaat-3;
[0030] Downstream primers: 5'-caccctctagettaatccagcatgtagcgctggaatgtgcgcgttttgcgtcgaggtcg-3 '.
[0031] The above primers were used as a synthetic plasmid containing Clostridia bacterium isolate INTA.CYC.091CONTIG-100_981DPEASE gene, cloned out of the two ends containing the target gene SEQ ID NO.1 containing BAMH I and XBA I restriction enzyme somewhere.
[0032] The PCR system of D-Alovate-3-differentially heterozyme gene is: Taq buffer (Mg 2+ PLUS) 10 μl, DNTPMixTure (2.5 mm) 4 μL, a forward primer (10 μm) 1 μL, reverse primer (10 μm) 1 μL, Template DNA 1 μL, TAQ DNAPOLYMERASE (1.25 U / μL) 1 μL, double steamed to 50 μl. The PCR amplification conditions were: 3 min at 98 ° C; 30 cycles were carried out (98 ° C 10s, 60 ° C 15S, 68 ° C for...
Embodiment 2
[0034]Example 2 recombinant D-Alovate-3-decomposed fermentation production
[0035] Single bacteria collasses with recombinant plasmids were collected from the deposition plate, and the LB medium containing ampicillin containing ampicillin (1% trypsin, 0.5% yeast extract, 1% sodium chloride, pH 7.0), 37 ° C, 200 rpm under shaking flask culture overnight; transferred to 50 mL according to 4% inoculation, TB medium containing ampicillin (1.2% trypsin, 2.4% yeast extract, 0.4% glycerin, 17mm kH) 2 PO 4 72mm K 2 HPO 4 In pH 6.0), 25 ~ 37 ° C, 200 rpm was shake the flask culture centered; 30 ml, OD 6 was centrifuged at 4 ° C, 10000 rpm, was centrifuged at 10 min, and the supernatant was discarded, 50 mM, pH 7.5 Tris-HCl washing the surface of the pellets 3 times, and Binding Buffer (50 mM Tris-HCl, 500 mMNAcl, pH 7.5) was used, then 1 mg / ml of lysozyme, 37 ° C water bath was allowed for 30 min, in 200W, open 1s, under 2S procedures, placed on ice is more than 15 min, and finally cent...
Embodiment 3
[0037] Example 3 Separation and purification of recombinant D-Alovate-3-differentially isomerase
[0038] After the fermentation crude enzyme solution was treated with a 0.45 μm aqueous film, the nickel column was balanced by 5 column volumes (500 mM NaCl, 50 mM Tris-HCl, pH 7.5); the flow rate was 1 mL / min, and then used After the A liquid was rebalance, the partial hybrid is washed away with a low concentration of imidazole eluent 1 (500 mM NaCl, pH 7.5), followed by 60% eluent 2 (500 mm NaCl) , 50 mM Tris-HCl, 500 mM imidazole, pH 7.5) washing the target protein, collecting the corresponding eluent according to the eluting peak, placed in the dialysis bag, 50 mM, pH 7.5 Tris-HCl under 4 ° C, overnight dialysis , Then pass through SDS-PAGE protein electrophoresis ( figure 2 Application of enzyme activity assay. According to reports, the calculated molecular weight of recombinant D-Alovate-3-differentially isomerase is 32.3 kDa, which is consistent with the SDS-PAGE estimation ...
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