Robust, site-specifically immobilized phenylalanine ammonia-lyases for the enantioselective ammonia addition of cinnamic acids.

Autor: Boros K; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro., Moisă ME; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro., Nagy CL; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro., Paizs C; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro., Toşa MI; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro., Bencze LC; Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University Arany János Str. 11 RO-400028 Cluj-Napoca Romania cslbencze@chem.ubbcluj.ro.
Jazyk: angličtina
Zdroj: Catalysis science & technology [Catal Sci Technol] 2021 Jun 29; Vol. 11 (16), pp. 5553-5563. Date of Electronic Publication: 2021 Jun 29 (Print Publication: 2021).
DOI: 10.1039/d1cy00195g
Abstrakt: Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans -cinnamic acid, while in the presence of high ammonia concentration, the synthetically attractive reverse reaction occurs. Although they have been intensively studied, the wider application of PALs for the large scale synthesis of non-natural amino acids is still rather limited, mainly due to the decreased operational stability of PALs under the high ammonia concentration conditions of ammonia addition. Herein, we describe the development of a highly stable and active immobilized PAL-biocatalyst obtained through site-specific covalent immobilization onto single-walled carbon nanotubes (SWCNTs), employing maleimide/thiol coupling of engineered enzymes containing surficial Cys residues. The immobilization method afforded robust biocatalysts (by strong covalent attachment to the support) and allowed modulation of enzymatic activity (by proper selection of binding site, controlling the orientation of the enzyme attached to the support). The novel biocatalysts were investigated in PAL-catalyzed reactions, focusing on the synthetically challenging ammonia addition reaction. The optimization of the immobilization (enzyme load) and reaction conditions (substrate : biocatalyst ratio, ammonia source, reaction temperature) involving the best performing biocatalyst SWCNT NH 2 -SS- Pc PAL was performed. The biocatalyst, under the optimal reaction conditions, showed high catalytic efficiency, providing excellent conversion ( c ∼90% in 10 h) of cinnamic acid into l-Phe, and more importantly, possesses high operational stability, maintaining its high efficiency over >7 reaction cycles. Moreover, the site-specifically immobilized Pc PAL L134A/S614C and Pc PAL I460V/S614C variants were successfully applied in the synthesis of several l-phenylalanine analogues of high synthetic value, providing perspectives for the efficient replacement of classical synthetic methods for l-phenylalanines with a mild, selective and eco-friendly enzymatic alternative.
Competing Interests: There are no conflicts to declare.
(This journal is © The Royal Society of Chemistry.)
Databáze: MEDLINE
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