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Optically pure chiral intermediates demand growth as medicine, agriculture, food , and fine chemical industry , have been fully aware of the importance of hand of . Enzymatic biocatalytic synthesis of chiral compounds become a hot research at home and abroad in recent years because of its unique advantages . Purification and enzymatic properties of the in-depth study of the transformation process in the enzyme reaction mechanisms , has important practical and theoretical value . Morgan 's screened in this laboratory bacteria (Morganella morganii) J-8 strain , the substrate 1 - phenyl-2 - the methylamino acetone (MAK) , specificity converted to d- pseudoephedrine . The study by Morganella morganii while J - 8 as the original strain , the asymmetry of its production carbonyl reductase enzyme production conditions , purification and enzymatic properties study results are as follows : optimization of enzyme production conditions to determine the enzyme production compared The appropriate medium: glucose 3% , peptone 2.5% , yeast extract 0.5 % , K2HPO4 on 0.3% , MgSO4 · 7H2O 0.2 % , 0.2% NaCl . The optimum conditions for enzyme production medium initial pH 7.5 , 24 h for the the best pick various age ; 30 ° C , 150 rpm shaking culture for 36 h . The basic nature of the crude enzyme was studied, the optimum temperature and optimum pH of the enzyme was 30 ° C and 7.5 , respectively . The stability of the enzyme is preferably in the range of pH 5 to 8 , 35 ° C or less stable . Sonicating after crushing , by ammonium sulfate precipitation , and the four-step purification of a Superose Phenyl hydrophobic column chromatography , DEAD anion column chromatography and non - denaturing gel electrophoresis SDS-PAGE electrophoresis of pure carbonyl asymmetric reductase . Subunit molecular weight of 42.5 kD. HPLC protein column analysis of the molecular weight of the enzyme , and a molecular weight of about 84.1 kD, and preliminary view is that the enzyme is a dimer protein . A preliminary study the enzymatic properties of the resulting partially purified enzyme NADH as a coenzyme for the reaction crude enzymatic activity with respect to NADPH as a coenzyme , MAK as substrate , the enzymatic reaction the optimum pH value of 6.5 , the optimum temperature of 30 ° C . Enzyme below 35 ℃ , pH 6 to 8 range , more stable. Vitality metal chelator and heavy metal ions to be enzyme inhibition produced of metal ions , Mg2 and Mn2 certain enzyme activation . Pure enzyme matrix-assisted laser desorption ionization - flight mass spectrometry , retrieved from meaningful match protein leucine dehydrogenase .
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