Study On The Biocatalysis Process For(4R,6S)-6-chloromethyl-2H-pyran-2,4-diol

2-deoxyribose-5-phosphate aldolase（DERA）could be used in catalyzing sequential aldol reactions for efficient production of the common chiral side chains of statin-type cholesterol-lowering drugs,the HMG-Co A reductase inhibitors.In this study,the reaction catalyzed by DERA was used to form（4R,6S）-6-chlorotetr-ahydro-2H-pyran-2,4-diol with installation of two stereogenic centers.The DERA S233E protein from Thermotoga maritima was used for rational design and directed evolution,E.coli BL21（DE3）was used as the host strain,and the mutant DERA S233E&R214H with 35%increased enzyme activity was screened.The optimal expression conditions of the mutant DERA S233E&R214H were explored.When the cells were cultured at 37℃until OD₆₀₀=0.7,the inducer IPTG solution with final concentration of 0.6m M was added,and the expression was induced at 28℃for 12h.The fermentation of recombinant was performed in 5L fermentor,230g wet cells were collected.The mutant DERA S233E&R214H was used to catalyze the condensation react-ion of acetaldehyde and chloroacetaldehyde at a concentration of 100g/L.The optim-um reaction conditions were as follows:the molar ratio of substrate acetaldehyde and chloroacetaldehyde was 2.2:1,the mass ratio of the wet cells and substrate was 1:1,the reaction p H was 7.0,the reaction temperature was 15℃,and the duration of substrate addition was 3h.Under above optimized condition,the yield of the biocatalysis reaction reached 88%.