A modified polymer microsphere, preparation method and application thereof in the field of immobilized enzymes
A technology of immobilized enzymes and polymers, applied in the direction of immobilized enzymes, biochemical equipment and methods, carrier binding/immobilized peptides, etc., can solve the problems of reducing the catalytic activity of immobilized enzymes, and achieve simple post-processing methods and simple operations , the effect of relative activity improvement
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Embodiment 1
[0037] A histidine-tagged fragment was introduced into the green fluorescent protein, and E. coli heterologously expressed the green fluorescent protein eGFP. Using the plasmid p1300-1 Ganoderma lucidum as a template to design primers, the upstream restriction site BamHI, the downstream restriction site SalI, carry out PCR reaction, and the obtained product is recovered by gel. The plasmid pet28a was extracted, the digested plasmid was recovered by gel, the purified fragment and vector were ligated overnight at 16°C, and the ligated product was transformed into E. coli DE3 competent cells. Purification to obtain histidine-tagged green fluorescent protein.
[0038] 1.3g of styrene (distillation under reduced pressure), 1.225g of maleic anhydride, 0.026g of divinylbenzene, 0.05g of azobisisobutyronitrile (recrystallization), 35ml of isoamyl acetate were added to a 100ml round-bottomed flask, and nitrogen was blown After sweeping for 30 minutes, react in a water bath at 75°C for...
Embodiment 2
[0042] The activated recombinant strain GLpmi-QCDC was cultured overnight, and 1% of the inoculum was inoculated into fresh sterile LB medium containing 30 mg / L kanamycin. When the OD600 reached 0.6-0.8, IPTG was added to make the final concentration 0.5 mM, 25 Cultivated at 150r·min-1 for 10h. The bacterial solution was centrifuged at 12000r·min-1 for 5min, resuspended in 1×PBS (pH=7.4), and then sonicated in ice-water mixture. The supernatant was centrifuged at 4°C and 12000rpm for 20min to obtain phosphomannose isomerase. The crude enzyme solution was purified by Co column to obtain purified phosphomannose isomerase.
[0043] 1.3g of styrene (distilled under reduced pressure), 1.225g of maleic anhydride, 0.026g of divinylbenzene, 0.0253g of azobisisobutyronitrile (recrystallized), 35ml of isoamyl acetate were added to a 100ml round-bottomed flask, and nitrogen was blown After sweeping for 30 minutes, react in a water bath at 75°C for 90 minutes; remove the supernatant by c...
Embodiment 3
[0047] Purified phosphomannose isomerase was obtained according to the method of Example 2.
[0048] 1.3g of styrene (distillation under reduced pressure), 1.225g of maleic anhydride, 0.026g of divinylbenzene, 0.05g of azobisisobutyronitrile (recrystallization), 35ml of isoamyl acetate were added to a 100ml round-bottomed flask, and nitrogen was blown After sweeping for 30 minutes, react in a water bath at 75°C for 90 minutes; remove the supernatant by centrifugation, wash the solid product with isoamyl acetate, centrifuge twice, and then wash it with petroleum ether for 6 times, remove the supernatant, and dry it to obtain unchanged Sexual polymer microspheres.
[0049] Dissolve 0.404g of the product prepared by the above method, 450μl lutidine, triethylamine (freshly distilled) in ultra-dry dimethylformamide, and the reactants are incubated at room temperature for 8-12h under nitrogen protection ;Add 0.238g NiCl 6 ·6H 2 O; the mixture was stirred at room temperature for 5...
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