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

- The biocatalytic production of (S)-2-Cl-Phe was tested in different reactor concepts.
- AtPAL2 was studied as whole cell biocatalst and immobilized enzyme on Avicel®.
- Productivities of 47 gproduct/gDryCellWeight to 288 gproduct/gimmobilize enzyme were reached.

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) 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 gproduct/gDryCellWeight) and using the immobilized enzyme in a repetitive fed-batch (274 gproduct/gimmobilized enzyme) or in a continuous plug-flow reactor (288 gproduct/gimmobilize enzyme). Therewith the productivity of AtPAL2 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 gproduct/gWetCellWeight (ca. 0.7 gproduct/gDryCellWeight) (de Lange et al., 2011; doi:10.1002/cctc.201000435).