Stabilization Study On Aldo-keto Reductase K1AKR And Carbonyl Reductase CR

Carbonyl reductase and aldo-keto reductase has excellent catalytic activity and stereoselectivity toward various aromatic ketones and aldehydes.t-Butyl 6-cyano-(3R,5R)-dihydroxylhexanoate[(3R,5R)-CDHHB]is the key intermediate of atorvastatin calcium,drug for atherosclerotic disease treatment.As highly effective biological catalysts,carbonyl reductase and aldo-keto reductase have great application prospect.however,they suffer from poor thermal stability,which weaken competitiveness.In this paper we analyze the inactivation mechanisms of aldo-keto reductase KlAKR through circular dichroism spectroscopy,fluorescence spectroscopy and UV-Vis spectroscopy.Results demonstrated that during the process of KlAKR inactivation,the number of?-helix decreased,while?-fold and random coil contents increased.Also,there are hydrophobic groups exposed,accompanied with protein aggregation.The change in KlAKR's secondary and tertiary structure result in the inactivation.The inactivation process of KlAKR obeys the first order deactivation kinetic model.we investigated the inactivation factor of KlAKR.Oxidation do not accelerate enzyme inactivation;mechanical agitation,substrate(5R)-CHOHB and product(3R,5R)-CDHHB are able to accelerate KlAKR inactivation.Among 12 test chemicals including saccharides,salts and polyols,16%glycerol,30%trehalose and 12%mannitol were found to be the best three additives.Adding the three substances at 30~°C,residue activity increase 26.4%,21.5%and 18.2%,respectively.Through a mechanism study of additives on KlAKR's stability,we found that additives can prevent the change in KlAKR's secondary and tertiary structure,maintain the natural structure.Based on the deactivation mechanism of KlAKR and stabilizers mechanism,we further investigate carbonyl reductase CR stability employing the orthogonal experimental design.The optimal compound stabilizer were composed of 20%glycerol,30%trehalose and 0.5 mol/L NaCl.The compound stabilizer enhanced the storage stability and the operation stability of the carbonyl reductase CR.After 25 d storage at 4~°C,residue activity of compound stabilizer addition was 27.9%higher than that of the control.Similarly,stabilizer enzyme increased CR's half-life in the presence of 20 mmol/L(5R)-CHOHB and 20 mmol/L(3R,5R)-CDHHB more than 2 times.