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STUDIA UBB. CHEMIA, LV, 4, 2010 EXPRESSION OF PHENYLALANINE AMMONIA-LYASES IN ESCHERICHIA COLI STRAINS KLAUDIA KOVÁCS a , b , ANDRÁS HOLCZINGER c , BEÁTA VÉRTESSY b,c , LÁSZLÓ POPPE a * ABSTRACT. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the non-oxidative conversion of L-phenylalanine into (E)-cinnamate. PAL can be applied in organic synthesis, and can be considered also for enzyme supplementation cure for genetic disorder phenylketonuria. The aim of this study was to find optimal expression parameters of several previously cloned PAL’s (bacterial, plant and a chimera) in pBAD vectors for further functional characterization. Investigation of the expression level of PAL's in E. coli hosts with SDS PAGE analysis as well as activity assay of the recombinant PAL enzymes was performed. Keywords: phenylalanine ammonia-lyase, heterologous protein expression, biocatalysis, phenylketonuria INTRODUCTION Phenylalanine ammonia-lyase (PAL) catalyzes the non-oxidative conversion of L-phenylalanine into (E)-cinnamate (Figure 1) [1]. Figure 1. Non-oxidative deamination of phenylalanine PAL is an important enzyme in both plant development and pathogen defense. In all plants PAL is encoded by a multi-gene family, ranging in copy number from four in Arabidopsis to a dozen or more copies in some higher plants [2]. The PAL participates in five metabolic pathways: tyrosine, phenylalanine and nitrogen metabolism, phenylpropanoid and alkaloid biosynthesis. Because of its key role between the primary and secondary metabolism in plants, PAL is a potential target for herbicides. a Budapest University of Technology and Economics, Department of Organic Chemistry and Technology, and Research Group for Alkaloid Chemistry of HAS, Műegyetem rkp 3, H-1111, Budapest, Hungary, poppe@mail.bme.hu b Institute of Enzymology of the Hungarian Academy of Sciences, Karolina út 29, H-1113, Budapest, Hungary c Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Műegyetem rkp 3, H-1111, Budapest, Hungary
KLAUDIA KOVÁCS, ANDRÁS HOLCZINGER, BEÁTA VÉRTESSY, LÁSZLÓ POPPE By reversing the natural direction of the PAL reaction in the presence of high concentration of ammonia, optically pure L-phenylalanine, which is the precursor molecule of the artificial sweetener aspartame (L-phenylalanyl-Laspartyl methyl ester) can be produced. Similarly, starting from various (hetero)arylacrylates, further enantiopure L-phenylalanine analogues, such as L-piridil/pirimidil-alanines can be also prepared. Since neither cofactor recycling nor other additives are needed in these asymmetric syntheses, they are potentially interesting as industrial processes as well [3a-e]. In addition to its application in synthetic chemistry, PAL can be applied also in human medicine as treatment to avoid the effects (mental retardation, neurotoxic effects) of the most common congenital metabolic disease, phenylketonuria (PKU). Daily oral administration of micro-encapsulated PAL to PKU rats decreased the systemic toxic phenylalanine level by 75 ± 8% in 7 days (P < 0.001) [4]. Because of its many scopes, PAL is an extensively studied enzyme. Although PAL is an ubiquitous higher-plant enzyme, it has only been encountered in a few bacteria, where cinnamic acid is involved in biosynthesis of several specific bacterial products, such as oligosaccharide antibiotics [5a-c]. The major difference between eukaryotic and prokaryotic PAL’s is a ca. 150-residue long C-terminal extension of the eukaryotic PAL’s (per monomeric unit) which is not present in the prokaryotic PAL’s (Figure 2) [6]. In plant and 276 Figure 2. Tetrameric structures of PALs (A) Homology model of P. crispum PAL [7]; (B) Homology model of P. luminescens PAL [6]; (C) Difference between eukaryotic and prokaryotic PAL monomers
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CHEMIA 4/2010
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L.M. PĂCUREANU, A. BORA, L. CRIŞA
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Studia Universitatis Babes-Bolyai C
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MIRCEA V. DIUDEA theorem in multi-s
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MIRCEA V. DIUDEA 4. M.V. Diudea, Cs
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STUDIA UBB. CHEMIA, LV, 4, 2010 DIA
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DIAMOND D 5 , A NOVEL ALLOTROPE OF
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MONICA L. POP, MIRCEA V. DIUDEA AND
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STUDIA UBB. CHEMIA, LV, 4, 2010 EVA
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EVALUATION OF THE ANTIOXIDANT CAPAC
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STUDIA UBB. CHEMIA, LV, 4, 2010 TO
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TO WHAT EXTENT THE NMR “MOBILE PR
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LORENTZ JÄNTSCHI, SORANA D. BOLBOA
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STUDIA UBB. CHEMIA, LV, 4, 2010 DIA
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DIAGNOSTIC OF A QSPR MODEL: AQUEOUS
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STUDIA UBB. CHEMIA, LV, 4, 2010 MOD
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MODELING THE BIOLOGICAL ACTIVITY OF
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MODELING THE BIOLOGICAL ACTIVITY OF
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LILIANA M. PĂCUREANU, ALINA BORA,
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A. R. ASHRAFI, P. NIKZAD, A. BEHMAR
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GHOLAM HOSSEIN FATH-TABAR, ALI REZA
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GHOLAM HOSSEIN FATH-TABAR, ALI REZA
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MODJTABA GHORBANI symmetric but it
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MODJTABA GHORBANI group can be comp
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MODJTABA GHORBANI 10. P.V. Khadikar
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HOSSEIN SHABANI, ALI REZA ASHRAFI,
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MIRCEA V. DIUDEA, CSABA L. NAGY, PE
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STUDIA UBB. CHEMIA, LV, 4, 2010 WIE
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WIENER INDEX OF MICELLE-LIKE CHIRAL
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WIENER INDEX OF MICELLE-LIKE CHIRAL
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STUDIA UBB. CHEMIA, LV, 4, 2010 TUT
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TUTTE POLYNOMIAL OF AN INFINITE CLA
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TUTTE POLYNOMIAL OF AN INFINITE CLA
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ALI REZA ASHRAFI, HOSSEIN SHABANI,
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MOHAMMAD A. IRANMANESH, A. ADAMZADE
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MOHAMMAD A. IRANMANESH, A. ADAMZADE
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RALUCA O. POP, MIHAI MEDELEANU, MIR
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MELINDA E. FÜSTÖS, ERIKA TASNÁDI
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STUDIA UBB. CHEMIA, LV, 4, 2010 APP
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APPLICATION OF NUMERICAL METHODS IN
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APPLICATION OF NUMERICAL METHODS IN
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VLADIMIR R. ROSENFELD, DOUGLAS J. K
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MAHSA GHAZI, MODJTABA GHORBANI, KAT
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MAHSA GHAZI, MODJTABA GHORBANI, KAT
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M. GHORBANI, M. A. HOSSEINZADEH, M.
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MONICA STEFU, VIRGINIA BUCILA, M. V
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MAHBOUBEH SAHELI, RALUCA O. POP, MO
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MAHBOUBEH SAHELI, RALUCA O. POP, MO
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FARZANEH GHOLAMI-NEZHAAD, MIRCEA V.
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FARZANEH GHOLAMI-NEZHAAD, MIRCEA V.
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