Key Technology Investigation On The Strengthen Of Process Productivity In Synthesis Of Typical Chiral Drug Building Blocks

Optically active chiral compounds are important intermediates for many drugs. Biocatalytic preparations are important methods for synthesis of chiral compounds with high optical purity. But, many biocatalytic processes were not industrialized due to low productivity and high cost caused by the low expression, bad stability and difficult reuse of enzyme, or unreasonablely setted process parameters. In this paper, three bioreaction processes with the potential of industrial applicability were taken as the typical cases and the specific productivities of the used biocatalysts were markedly enhanced through different strategies, including gene heterogeneous expression, enzyme immobilization and process re-optimization based on the key technical bottleneck problems to facilitate the industrial application.Aimed to the issue of the low specific productivity resulted by the bad enzyme expression in the wild microorganism, enzymatic synthesis of (S)-2,2-dimethyl-cyclopropanecarboxylic acid (an important precursor of cilastatin) was taken as a case. The catalytic efficiency of the enzyme preparation was markedly enhanced through improvement of the expression level of the Rhodococcus sp. ECU1013 esterase by the strategy of gene cloning and heterogeneous overexpression. The specific productivity of the recombinant RhEstl increased 46-folds. The substrate loading increased from 62.5 mM to 500 mM and the reaction was significantly shortened from 120 h to 16 h. A decagram-scale preparation of (S)-DmCpCa was performed with an isolated yield of 30.2%(maximum conversion of 50%) and 97.4% ee.Aimed to the issue of the low usage efficiency resulted by the low stability and difficult reuse of the free enzyme preparation, enzymatic synthesis of (2R,3S)-(-)-3-(4’-methoxyphenyl)glycidic acid methyl ester (key precursor for synthesis of cardiovascular pharmacetical diltiazem) was taken as a case. Stability and the reuse of the Serratia marcescens ECU1010 extracellular lipase was markedly enhanced by cross-linking immobilization, which greatly improved the production efficiency of the enzyme. Compared with the free enzyme, the stability of the immobilized enzyme increase 13.4-folds and the specific productivity increased 9.8-folds. A deca-kilogran scale product preparation was performed with a cost fallen of 88.7%.Aimed to the issue of the low catalytic efficiency caused by the unreasonable setting of the process parameters, enzymatic synthesis of (S)-4-chloro-3-hydroxybutanoate (an important intermediate of blockbuster drug atorvastatin) was taken as a case. The specific productivity of the catalyst was efficiently improved by adjustment of process parameter and re-optimization of the reaction condition. After process re-optimization, the specific productivity of the whole cells biocatalyst increased by 78.7%. A kilo-gram scale of product preparation was performed with 85.4% yield and 99.9% ee.