We report an immobilized enzyme-catalyzed batch and continuous-flow synthesis of optically pure ethyl(R)-pantothenate((R)-PaOEt),the direct precursor of d-pantothenic acid.Firstly,a ketoreductase mutant designated as ...We report an immobilized enzyme-catalyzed batch and continuous-flow synthesis of optically pure ethyl(R)-pantothenate((R)-PaOEt),the direct precursor of d-pantothenic acid.Firstly,a ketoreductase mutant designated as M2,carrying two-point mutations of F97L and M242F relative to the wild-type SSCR,was constructed by site-directed mutagenesis,exhibited simultaneously improved activity toward ethyl 2′-ketopantothenate(K-PaOEt)and isopropanol,and could effectively catalyze the stereoselective reduction of K-PaOEt to(R)-PaOEt by using isopropanol as the sacrificial co-substrate to regenerate NADPH.After screening six commercially available carriers,an amino resin LXTE-700 was identified as the best solid support for the immobilization of M2 via the glutaraldehyde activation method.Upon optimization of the immobilization process and reaction conditions,the fabricated immobilized enzyme M2@amino resin demonstrated excellent recyclability and reusability,with the complete conversion of K-PaOEt to(R)-PaOEt being still realized after 12 cycles of reuse.Finally,M2@amino resin-catalyzed synthesis of(R)-PaOEt was successfully implemented in continuous-flow,accomplishing a 6.3 times higher space-time yield than that with the batch synthesis(529.2 versus 84 g L^(-1) d^(-1)).Our developed flow biocatalysis system also features an outstanding operational stability,as evidenced by the 100%conversion rate achieved after 15 consecutive days of operation.展开更多
基金the Science and Technology R&D Major Project of Jiangxi Province(No.20244AFI92001)the National Natural Science Foundation of China(Nos.22071033 and 21801047)for the financial supports.
文摘We report an immobilized enzyme-catalyzed batch and continuous-flow synthesis of optically pure ethyl(R)-pantothenate((R)-PaOEt),the direct precursor of d-pantothenic acid.Firstly,a ketoreductase mutant designated as M2,carrying two-point mutations of F97L and M242F relative to the wild-type SSCR,was constructed by site-directed mutagenesis,exhibited simultaneously improved activity toward ethyl 2′-ketopantothenate(K-PaOEt)and isopropanol,and could effectively catalyze the stereoselective reduction of K-PaOEt to(R)-PaOEt by using isopropanol as the sacrificial co-substrate to regenerate NADPH.After screening six commercially available carriers,an amino resin LXTE-700 was identified as the best solid support for the immobilization of M2 via the glutaraldehyde activation method.Upon optimization of the immobilization process and reaction conditions,the fabricated immobilized enzyme M2@amino resin demonstrated excellent recyclability and reusability,with the complete conversion of K-PaOEt to(R)-PaOEt being still realized after 12 cycles of reuse.Finally,M2@amino resin-catalyzed synthesis of(R)-PaOEt was successfully implemented in continuous-flow,accomplishing a 6.3 times higher space-time yield than that with the batch synthesis(529.2 versus 84 g L^(-1) d^(-1)).Our developed flow biocatalysis system also features an outstanding operational stability,as evidenced by the 100%conversion rate achieved after 15 consecutive days of operation.
