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

作者：Dressen Alana; Hilberath Thomas; Mackfeld Ursula; Rudat Jens; Pohl Martina^*

来源：Journal of Biotechnology, 2017, 258: 158-166.

DOI：10.1016/j.jbiotec.2017.04.035

• MIO enzyme
• Enzyme reactor
• (S)-2-Indolinecarboxylic acid
• Indolapril
• Perindopril

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chlonn-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 (R) 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 (R) 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 AtPAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula &anis in E. coil 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).

• 出版日期2017-9-20