Study On Production Of NADP?H? By Immobilized NAD Kinase On Fe₃O₄ Submicron Particles

The great potential of superparamagnetic submicron particles has been attracted wide attention and research in the fields of biology and medicine.In this paper,the superparamagnetic Fe3O4 submicron particles were prepared via a solvothermal method.After epoxy chloropropane modification,the epoxide-modified Fe3O4 submicron particles were connected by a simple and efficient connection of iminodiacetic acid(IDA),and chelated separately with two valence metal ions(Ni2+,Co2+,Zn2+,Cu2+ and Mg2+)and obtained the metal chelating affinity medium of the immobilized enzyme;MgFe2O4 submicron particles were synthesized via one step solvothermal method as an immobilized medium of enzyme.A series of characterizations have been carried out to confirm Fe3O4 which successfully modifies the upper IDA and chelate metal ions;MgFe2O4 is a spherical particle of about 440 nm,and is an anti-spinel structure.NAD(H)and NADP(H)are important coenzymes,especially NADPH plays an important role in anabolism.The NADP synthesis pathway is catalyzed by NAD phosphorylation and based on NAD kinase(NADK)as the sole enzyme.However,because the product NADP has a strong inhibitory effect on NADK,it will restrict its production in cells.In this study,His-Tagged NADK from genetic engineering E.coli MG 1655 with plasmid pET-28a was obtained and then immobilized onto metal chelated Fe3O4 submicron particle(Fe3O4-IDA-Ni)or MgFe2O4 and used for catalytic NAD production NADP in vitro.This study verified the specificity and stability of Fe3O4-IDA-Ni and MgFe2O4 for His-Tagged NADK immobilization.It is determined that the amount of Fe3O4-IDA-Ni immobilized NADK is fixed at 5 min to 140 mg/g medium at 25 ?,and the fixed 12 h can reach 720 mg/g medium;MgFe2O4 immobilized NADK is fixed 5 min up to 100 mg/g medium at 25 ?,and 12 h can reach 670 mg/g medium.The analytical method of NAD(H)and NADP(H)was established by high performance liquid chromatography(HPLC).The separation results were obtained within 7 min under the condition of simple pump constant flow rate and simple operation.The specific activity of immobilized NADK was determined.The immobilized NADK of Fe3O4-IDA-Ni was 1.8 U·g-1,and MgFe2O4 immobilized NADK was 1.4 U·g-1.The catalytic reaction conditions of the immobilized NADK were optimized,such as amount of immobilized NADK,reaction time,temperature,and NAD concentration on the production NADP.The response surface method was used to optimize the reaction conditions.The reaction time,reaction temperature and substrate concentration were selected by the Box-Behnken(BBD)center combination as the investigation factors.The three-factor/three-level experiment was designed and fitted,and obtained the optimized result.Immobilized NADK by Fe3O4-IDA-Ni(0.05 g)was added to 1 mL PBS buffer(0.1 mmol/L,pH=7.4)containing 0.9 mmol/L NAD at 50 ?for 13 h.Under the condition,the conversion rate of NAD reached 91.7%and retained the 86.3%after 5 batches.