Study On The Technique For Extract, Isolate And Purify A New Cytoprotective Agent-coenzyme NADH

Background and Objective:Life is a constant flow of energy,with which ensure the maintenance,gowth and reproduction of the organisms. Nicotinamide adenine dinucleotide reduced (NADH) is present in every living cell and is essential to cellular energy production. One mole of NADH produces three moles of ATP and 36 kilocalories. It plays a very important role in cell growth,cell differentiation and energy metabolism.NADH serves as not only a important biochemical cofactor in enzymatic catalysis,but also a potent cytoprotective agent. Medical science proved that free radicals are involved in more than 81 diseases,including heart & cardiovascular disease,cancer and the diseases we associate with aging (like Alzheimer's,Parkinson's and other neuro-degenerative disorders). On the basis of its biochemical properties,NADH is the most biological potent antioxidant found in the body and has the ability to protect the body from the damages of free radicals. Science is learning that NADH may have a direct positive involvement in enhancing the level of energy metabolism,repairing cellular damage,improving cellular stress ability,decreasing cytotoxicity caused by chemotherapy drugs,chemical toxic agents and radiation. It has a prospect of wide application as a remarkable natural substance,so there have significant social effects and economic returns to find a new technique of extraction,isolation and purification NADH in a inexpensive,large-scaled and high-yielding way.We want to find and optimize a technique which is simple,inexpensive and high-yielding,and it should be able to be scaled up to deal with large volumes in industrial operation. An available way can be provided by producing cytoprotective drugs contained NADH in cytoprotection treatment for clinical and military medical cytotoxicity. Methods and Results:l.The method to crude extraction of NADHSaccharomyces cerevisiae cells were treated by chemical permeabilization to release nicotinamide adenine dinucleotide reduced. Factors such as cells concentration,the volume of ether and the permeabilization time,which affect the cell disruption efficiency and NADH recovery were studied by Lm() orthogonal designed-test. The results showed that 0.10 g/ml Saccharomyces cerevisiae cells,4 fold of ether volume and 20min-permeabih'zation were the best choices. The NADH release kinetics of a chemical permeabilization process using ether was characterized. The overall yield of NADH obtained by chemical permeabilization is significantly higher than mechanical disintegration.2. Isolation and purification of NADH by affinity chromatography Yeast alcohol dehydrogenase(YADH) was linked to CNBr activedSepharose 4B to prepare the immobilised dehydrogenase affinity column. NADH was isolated and purified by affinity chromatography using YADH as an affinity ligand. The efficiency of coupling of YADH droped from 95% to 68.2% when the applied load was increased from 16mg/gCNBr-Sepharose 4B to60mg/gCNBr-Sepharose 4B. Activity relative to free enzyme fell from 25% to 9.5% NADH binding fell froml.48mg to l.lmg when the flow rate was increased from 0.5ml/min to 4.5ml/min.3. Isolation and purification of NADH by affinity ultrafiltrationThe crude yeast cell permeate contained low molecular weightmolecules like NADH,ATP,NAD and various other coenzymes. The NADH in crude cell permeate was separated by binding to YADH at PH 8.0,ionic strength 0.1mol/L. The NADH-YADH complex was separated from other molecules by ultrafiltration using membrane of which the molecular weight cut-off (MWCO) was 30000 and the binary complex was recovered as retentate. The NADH-YADH complex was cleaved by subjecting it to PH and ionic strength change . Ultrafiltration was carried out then to isolate NADH as ultrafiltrate. YADH was recovered as ultra-retentate with 95% recovery and 15% loss in activity. Conclusion:It was found that treating Saccharomyces cerevisiae cells by chemical permeabilization and purification by affinity ultrafiltration gives ni...