Enhanced Simvastatin Production From Lovastatin By Immobilization Of LovD

At present,among the diseases that seriously harm human health in many countries around the world,the incidence and mortality of cardiovascular and cerebrovascular diseases have ranked first,and even surpassed high-risk diseases such as cancer.Hyperlipidemia is a branch of cardiovascular and cerebrovascular diseases.The main indicator of hyperlipidemia is total cholesterol,which directly affects the incidence and mortality of coronary heart disease,myocardial infarction and other diseases.An effective way to treat hyperlipidemia is to suppress the excessive synthesis of cholesterol.Therefore,lipid-lowering drugs have become the focus of modern drug research.The pharmacological action of statins is as a competitive inhibitor to inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoA reductase),thereby reducing cholesterol biosynthesis.Among them,the inhibitory activity of simvastatin on HMG-CoA reductase in vivo is 4 times that of lovastatin,which can effectively reduce the blood lipid level in patients with primary hyperlipidemia.In this study,lovastatin was dissolved in the aqueous phase at a pH of 11 and then NaOH was added to the lovastatin aqueous solution with a final concentration of 1 M.So,the lovastatin aqueous solution was alkali-hydrolyzed to monacolin J with a conversion rate of 99%.LovD was used for enzymatic hydrolysis to synthesize simvastatin from monacolin J.The LovD enzyme was produced by E.coli engineered strain BS01/pBS-lovD.The specific enzyme activity of LovD crude enzyme solution was 1.2 U/mg,while the specific activity of LovD enzyme purified by Ni column reached 25 U/mg.The final titer of simvastatin by crude enzyme solution and LovD enzyme were 0.81 g/L and 0.94 g/L,respectively,while the yield were 0.87 g/g and 0.96 g/g,respectively.The immobilization process of LovD enzyme was further studied to be suitable for industrial mass production.The lysine-modified cross-linked chitosan microspheres were prepared as immobilized carriers.According to scanning electron microscopy,the particle diameter was 400-1200 ?m and the pore size was 30-200 ?m.The specific enzyme activity of the immobilized LovD enzyme was 4.6 U/mg.The immobilized enzyme prepared with lysine-modified cross-linked chitosan microspheres catalyzes the synthesis of simvastatin with a yield of 0.84 g/g and a final titer of 0.76 g/L.At the same time,amino magnetic nanoparticles were prepared as an immobilized carrier.According to scanning electron microscopy,the particle diameter was 400-900 ?m and the pore size was 35-150 ?m.The specific enzyme activity of the immobilized LovD enzyme was 4.1 U/mg.The immobilized enzyme prepared from amino magnetic nanoparticles catalyzed the synthesis of simvastatin with a final titer of 0.70 g/L and a yield of 0.80 g/g.The yields of immobilized enzyme catalyzed obtained by the two immobilization methods decreased with the increase in the number of recycling cycles.By the sixth time,the yields decreased to 0.62 g/g and 0.59 g/g,respectively.Although the immobilized enzyme method reduces the enzyme activity of LovD,it can be used repeatedly,which reduces the production cost and is more conducive to industrial mass production.