Study On The Immobilization Of Isopropanol Dehydrogenase

NADH mainly exists in mitochondria,which is the core substance of metabolism.As a drug,NADH has the functions of scavenging free radicals,preventing cancer,treating cancer and treating neurodegenerative diseases.It is a kind of medicine,also is a kind of health food,have broad market.In this experiment,the immobilization of isopropyl alcohol dehydrogenase was carried out,and its immobilization process and catalytic reaction process were optimized to lay a foundation for its application.Extraction of crude enzyme liquid.The optimal fermentation conditions were selected,and the amount of the liquid in the bottle was 40mL,and the final concentration of the inducer was 0.8mM,and the inducer was added at the time of the bacteria OD600 to about 0.6,and the induction time was 12h.Under the ultrasonic condition of 300W,the concentration of suspension was 0.15g/mL,and the crushing time of 10min could maximize the activity of the enzyme.Immobilized carrier screening.The dehydrogenase or engineered bacteria were fixed by adsorption,embedding,crosslinking and whole cell immobilization.In all kinds of resin,gel particles and other 20 kinds of carriers,the enzyme activity retention rate of carrier ESR-3 was 68.3%after initial screening.Optimization of immobilization process.The immobilization process using the carrier selected by initial screening was optimized,and finally the retention rate of isopropanol dehydrogenase was 78.3%.Optimal conditions for immobilized isopropyl alcohol dehydrogenase were as the follows:immobilization time 2 h,buffer pH of 7,table speed 150 r/min,immobilized temperature 20?.The properties of immobilized isopropyl alcohol dehydrogenase.The mostsuitable reaction temperature of immobilized enzyme is 30?,while optimum reaction temperature of free enzyme 35?.The optimum reaction pH of the immobilized enzyme and the free enzyme was 9.The storage stability is good,and 89.7%of the enzymes are still active after 28 days.The activity of immobilized enzyme was reduced to half by the repeated use of enzymes 8 times,and the reaction scale was amplified by 7 times to 210mL,and after 10h of the reaction 345 mg of the product was obtained,and the conversion rate was 70.4%,which could be considered for the production application.