Microbial Conversion Of Simvastatin And Study On The Properties Of Its Product

Cardiovascular disease has been among the gravest diseases with morbidity and mortality both ranking the first overtaking that of tumours, posing severe threat to mankind. In China, along with various infectious diseases being placed under control and improving of people's life, these has been an increasing number of people suffering with cardiovascular disease as a result of ever-enlarging pool of the senior citizens. Hypercholesterolemia is known to be one of the prime risk factors for cardiovascular disease such as arteriosclerosis and coronary heart disease. 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, is a group of very active antihypercholesterolemic agents that function by limiting cholesterol biosynthesis by competitively inhibiting the HMG-CoA reductase, the rate limiting enzyme in cholesterol synthsis. At present, HMG-CoA reductase inhibitors appear on the market include lovastatin, pravastatin, simvastatin, fluvasatatin, atorvastatin, rosuvastatin and pitavastatin, called by a joint name Statins. Comparing with other cholesterol-lowering agents, Statins, with remarkable cholesterol-lowering effect, high security and less side effect, have been widely used in the therapeutic treatment of hypercholesterolemy.3, 5-dihydroxy-heptanoic acid portion, the basic chemical structure of statins, is crucial for the inhibiting activity. More studies have since been made by chemical methods or microbial conversion with no change in its active groups to discover new inhibitors or derivatives of already known compounds to obtain more potent inhibitors of cholesterol biosynthesis. Simvastatin was hard for chemical method to modify its structure and we could see the superiority of microbial conversion to chemical method, that was higher conversion, simpler procedures and less pollution. Microbial conversion of simvastatin have not been reported nationally.Many microorganisms and their enzyme preparations are able to perform site directed and stereospecific modifications of organic molecules that may be difficult ortedious to obtain through ordinary chemical methods.The goal of this thesis was first to transform simvastatin by Nocardia autotrophica sp. canberrica ATCC 35203 and monitor products by TLC analyses. The bioconverted product was purified and structure was further identified as 6-hydroxymethyl-simvastatin (elemental composition C₂₅H₄₀O₇, with parent ion M+ 453. 1) on the basis of spectral data (MS and ¹H NMR). It was found that under certain conditions, 6-hydroxymethyl-simvastatin was present in two different forms:as a lactone and as the corresponding open acid (β-hydroxy acid), and that the two forms can convert to each other, related with pH. Lactone form can be hydrolyzed to open-acid form easily under alkali conditions while open-acid form can be converted to the lactone form at acidic pH, but not efficiently. Under normal conditions, two forms exist simultaneously and keep a dynamic balance. It is possible to get only open-acid form by controling conditions.Secondly, To obtain the optimal fermentation conditions, the conversion conditions, which affected hydroxylation such as different kinds of solubilizers, cell age, time, substrate, temperature, pH and air flow were investigated. The results showed that the productivity was maximized when strain Nocardia autotrophica sp. canberrica was incubated in 10 mL culture medium (pH 8.0) in 150 mL shake flask at 28℃, with induction of simvastatin (20 μg/mL) at the beginning, and then after 36 hours of growth, simvastatin treated with acetone was added to a final concentration of 800 ～1 000 μg/mL and the incubation was continued in the medium (pH 8.0) for 48 ～ 60 hours. High concentration simvastatin could be bioconverted efficiently through fed-batch fermentation.Finally, we investigated the kinetics of inhibition of HMG-CoA reductase. It was indicated that 6-hydroxymethyl-simvastatin was a highly potent inhibitor of HMG-CoA reductase and behaved in a competitive manner with respect to HMG-CoA. in vitro, the K_i for simvastatin was found to be 11. 96 nmol/L, and the K_i for 6-hydroxymethyl-simvastatin was 6. 43 nmol/L, thus the bioconversion product, 6-hydroxymethyl-simvastatin, was more than two times as active as the simvastatin as an inhibitor of HMG-CoA reductase. Nevertheless, the apparent potency of 6-hydroxymethyl-simvastatin reported here in in vitro, indicate the potential of 6-hydroxymethyl-simvastatin as a therapeutic agent for the treatment of hypercholesterolemia.