Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2) : A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids.. Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine

Abstract Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana ( At PAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated ( S )-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and immobilized on the cellulose carrier Avicel ® for the production of the hypertension drug precursor ( S )-2-chloro-phenylalanine using batch, fed-batch, as well as continuous membrane reactor and plug-flow reactor. For immobilization, a C-terminal fusion of the enzyme with a carbohydrate binding module (CBM) was produced, which selectively binds to Avicel ® directly from crude cell extracts, thus enabling a fast and cheap immobilization, stabilization and recycling of the enzyme. 1 g Avicel was loaded with 10 mg enzyme. Best results were obtained with whole cells using the continuous membrane reactor (47 g product /g DryCellWeight ) and using the immobilized enzyme in a repetitive fed-batch (274 g product /g immobilized enzyme ) or in a continuous plug-flow reactor (288 g product /g immobilize enzyme ). Therewith the productivity of At PAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula glutinis in E. coli as whole cell biocatalyst with a productivity of 0.14 g product /g WetCellWeight (ca. 0.7 g product /g DryCellWeight ) (de Lange et al., 2011; doi:10.1002/cctc.201000435).