▎ 摘　　要

NOVELTY - Method for in situ separation and immobilization of salt-tolerant oxidoreductases dependent on NADH and NADPH, involves stirring the water, potassium hydroxide and polyethyleneimine, adding uniformly dispersed GO solution, and heating content under reflux at 22-85 degrees C for 22-26 hours, centrifuging and drying content to obtain graphene oxide-polyethylenimine (PEI) complex material, formulating graphene oxide-PEI complex material into a complex material solution having a concentration of 0.05-1 mg/ml, and mixing complex material solution with crude enzyme liquid to obtain GO-PEI-enzyme precipitation, resuspending the above GO-PEI-enzyme pellet with a second buffer, centrifuging and eluting NADH- and NADPH-dependent salt-tolerant oxidoreductase bound to graphene oxide-PEI complex, centrifuging and collecting supernatant to determine the protein concentration. USE - The method is useful for in situ separation and immobilization of salt-tolerant oxidoreductases dependent on NADH and NADPH. ADVANTAGE - The method utilizes PEI-grafted carbon nanomaterials to adsorb and elute crude enzyme liquids derived from marine strains at different salt concentrations, develops method for isolating salt-tolerant oxidoreductase from crude enzyme solution and immobilizing it in situ, can eliminate the interference of false negative and false positive, improves the screening efficiency of enzymes, realizes the effective construction of enzyme library, and fully exploit the enzyme resources of microorganisms. DETAILED DESCRIPTION - Method for in situ separation and immobilization of salt-tolerant oxidoreductases dependent on NADH and NADPH, involves (a) inoculating halophilic bacteria in 216 l medium with an inoculation amount of 1-10% for 12-48 hours to obtain a culture solution, (b) centrifuging above culture solution to obtain a precipitate, which is resuspended in trisaminomethane (tris)-hydrogen chloride buffer at pH=7.5-7.6 and washing the content several times with centrifugation, obtaining the cells, and then preparing the cells with the first buffer solution to prepare a cell liquid having a concentration of 25-5000 mg/L, and then breaking the cell liquid, centrifuging to remove cell debris, obtaining a supernatant, adjusting the pH of the supernatant to 9.5-11, that is, obtaining a crude enzyme solution, where the first buffer has a pH of 7.85-7.94, and contains 50-100 mM tris-hydrogen chloride, 0.05-0.14 mM EDTA, 0.05-0.14 mM DTT, and 0.14-0.16 M sodium chloride, (c) stirring the water, potassium hydroxide and polyethyleneimine, adding uniformly dispersed GO solution at a concentration of 4-10 mg/ml, and heating content under reflux at 22-85 degrees C for 22-26 hours, centrifuging and drying content, obtaining graphene oxide-PEI complex material, where ratio of water, potassium hydroxide, polyethyleneimine to GO solution is 5-100 ml: 20-200 mg: 150-300 mg: 4-10 ml, (d) formulating graphene oxide-polyethylenimine (PEI) complex material into a complex material solution having a concentration of 0.05-1 mg/ml, and mixing complex material solution with the above crude enzyme liquid in a volume ratio of 1:(1-4), producing a GO-PEI-enzyme solution with a final protein concentration of 0.25-2 mg/l, centrifuging at 10,000 rpm for 10-20 minutes to obtain GO-PEI-enzyme precipitation, (e) resuspending the above GO-PEI-enzyme pellet with a second buffer, shaking for 30-120 minutes, then centrifuging at 8000-10000 rpm for 10-20 minutes, obtaining GO-PEI-enzyme precipitation, the pH of the second buffer is 7.85-7.94, which contains 50-100 mM Tris-hydrogen chloride, 0.05-0.14 mM EDTA, 0.05-0.14 mM DTT, and 0.05-0.2 M sodium chloride, (f) shaking above-mentioned GO-PEI-enzyme precipitate and resuspending in a third buffer, and then centrifuging content at 5000-6000 rpm for 4-6 minutes at 3-4 degrees C, eluting NADH- and NADPH-dependent salt-tolerant oxidoreductase bound to the graphene oxide-PEI complex, centrifuging at 8000-10000 rpm for 8-12 minutes several times, obtaining supernatant and the precipitate and collecting supernatant to determine the protein concentration.