Improve the Efficiency of Candida parapsilosis Catalyzed Asymmetric Synthesis of S-CHBE by Releasing Inhibition of Substrate and Product
|Keywords||asymmetric reduction (S)-4-chloro-3- hydroxybutyrate ethyl inhibition and toxicity of substrate (product) in-situ extraction with aqueous/organic biphasic system in-vitro product removal|
Due to the high regio-, stereo-, enantioselectivity, unattainable 100% of the theoretical yield, low cost substrates and mild reaction conditions, asymmetric synthesis optically active alcohols, which were generally important and interesting building blocks for pharmaceuticals, agrochemicals, and fine chemicals, by biocatalytic reduction had been widely researched in recent years. However, the catalytic efficiency and productivity usually couldn’t meet the requirement of industrial manufacture for lack of efficient biocatalysts, affecting by environmental factors and substrate (product) inhibition and toxicity. In this paper, asymmetric reduction synthesis of S-CHBE, an important precursor of statins, from COBE catalyzed by whole-cell microbial was introduced as model reaction. Based on well selected wild-type microorganism with high stereoselectivity and reduction capacity, we focused on finding the bottlenecks of whole-cell catalyzed asymmetric reduction and the affecting mechanisms of substrate (product) to key reductases and coenzyme circulatory system. To industrial productivity and efficiency as the goal, a variety of reaction engineering strategies were conducted and the platform technology for preparing optically pure S-CHBE by biocatalytic reduction was expected to be established. Main results were as follows:(1) Through product-oriented screening, Candida parapsilosis CCTCC M 203011was selected as catalyst from laboratory preserved strains. It could catalyze the asymmetric reduction of COBE for synthesis of S-CHBE with more than 97% in e.e. value. And its optimal reaction conditions were as follows: reaction temperature was 30℃, initial pH was 6.5, glucose was chosen as co-substrate and the usage was nglucose/nCOBE=1.(2) The key bottlenecks of microbial whole-cell catalyzed asymmetric reduction process were analyzed systematically. It was found that in addition to environmental factors, such as temperature and pH, the substrate and product affected the catalytic efficiency by different mechanisms: the substrate was toxicity to enzyme and cell, especially to whole-cell coenzyme regeneration systems. When the substrate was 40 g/L, the key reductase activity and cell metabolic activity were reduced to 33% and 8% respectively, it suggested that the key reductase was severely inactivated and the cell almost had no coenzyme regenerating ability. And the product mainly showed inhibitory effect to enzymatic reaction. When the product concentration increased to 40 g/L, the initial reaction rate dropped to 13 mmol/L?h, reduced by 84% than not add the product.(3) Concerned with these different mechanisms, a series reaction engineering strategies were conducted. Substrate feeding strategy was rational designed. In order to control the substrate was below 4 g/L in aqueous phase, the next batch would be added when the last was almost completely converted to product. And the aqueous-organic biphase system could ensure both the substrate and product concentration remain at low levels in aqueous. Meanwhile by increasing the usage of cell, the productivity was greatly enhanced, the substrate concentration increased to 240 g/L with 95.8% in yield.(4) Designed and established a continuous reactor combined with membrane cell retention technology, the cell concentration in reactor remained at a high level so that the space-time yield could be enhanced. And optimized the operating conditions: substrate was kept at 4 g/L in feeding fluid, feeding strategy was 62.5 mL/h during 0-4 h, 45.5 mL/h during 4-15 h of, and 41.7 mL/h after 15 h. Reaction for 16 h, the product was accumulated to 308 g/L with 97.2% in yield, and given a relative high space time yield of 19.26 g/L·h (116 mmol/L·h).(5) Macroporous resin NKA-II was selected for adsorbing product in the outside loop through studied the Static adsorption ability and Dynamic adsorption performance of resins. During reaction process, the recovery was 99.7%. Then, through extraction with ethyl acetate and reduced pressure distillation, 2.28 g product was obtained with 75.1% in isolated yield. The chemical purity and e.e. value were 97.4%, 96.7% respectively, and the specific rotation [α]23=-19.1°.