EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE AMMONIA-LYASE FROM PETROSELINUM CRISPUM

Norbert Artur DIMA; Alina FILIP; László Csaba BENCZE; Márk OLÁH; Péter SÁTORHELYI; Beáta G. VÉRTESSY; László POPPE; Csaba PAIZS

Authors

Norbert Artur DIMA Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca, Romania.
Alina FILIP Research Center for Enzymology and Applied Biocatalysis, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca, Romania. https://orcid.org/0000-0001-6820-9204 (unauthenticated)
László Csaba BENCZE Biocatalysis and Biotransformations Research Center, Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering, Cluj-Napoca, Romania. https://orcid.org/0000-0003-0956-9749 (unauthenticated)
Márk OLÁH Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Hungary. https://orcid.org/0000-0003-4628-6887 (unauthenticated)
Péter SÁTORHELYI Fermentia Ltd, Budapest, Hungary.
Beáta G. VÉRTESSY Department of Biotechnology and Food Sciences, Budapest University of Technology and Economics; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary. https://orcid.org/0000-0002-1288-2982 (unauthenticated)
László POPPE Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Hungary. https://orcid.org/0000-0002-8358-1378 (unauthenticated)
Csaba PAIZS Faculty of Chemistry and Chemical Engineering, Hungarian Line of Study, Babes-Bolyai University, Cluj Napoca, Romania. *Corresponding authors: poppe@mail.bme.hu; paizs@chem.ubbcluj.ro https://orcid.org/0000-0002-7403-7098 (unauthenticated)

Keywords:

phenylalanine ammonia-lyase, Petroselinum crispum, molecular cloning, expression and expression optimization

Abstract

In the present study the molecular cloning, expression and purification of recombinant PcPAL, with a cleavable N-terminal His-tag is described. The PcPAL gene was cloned into pET-19b vector and transformed to different E.coli host cells. The optimization of expression and purification processes provided recombinant protein with high purity in its native, tetrameric fold with a yield of 7-8 mg protein / 1 L culture. The activity of the recombinant protein was tested towards its natural substrate L-Phe, the K_M, and k_cat values suggesting excellent catalytic properties of the recombinant enzyme.

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