A novel phenylalanine ammonia-lyase from Pseudozyma antarctica for stereoselective biotransformations of unnatural amino acids

Varga, Attila; Csuka, Pál; Sonesouphap, Orlavanah; Bánóczi, Gergely; Toşa, Monica Ioana; Katona, Gabriel; Molnár, Zsófia; Bencze, László Csaba; Poppe, László; Paizs, Csaba

doi:10.1016/j.cattod.2020.04.002

Cited by 16 publications

(20 citation statements)

References 54 publications

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“…Finally, we evaluated the performance of Ls PAL3 in the ammonia addition reaction of trans -cinnamic acid derivatives, which is expected to deliver enantiopure phenylalanine derivatives at a maximal theoretical yield of 100%. Although PALs are typically of excellent enantioselectivity in amino addition, they differ significantly in terms of activity and substrate adaptability. ,,, Considering the excellent catalytic activity of Ls PAL3 in ammonia elimination, we increased the substrate loading of the ammonia addition reactions from commonly-used 1–5 ,, to 25 mM.…”

Section: Resultsmentioning

confidence: 99%

“…The ammonia elimination of rac-1j has been investigated previously with wild-type PcPAL and mutants. 16,23,36 After 16 h incubation at a substrate concentration of 2 mM, the wild-type was reported to afford a conversion of 7.9%, while the best mutant I460V afforded 49.8%. 23,36 Here, 1j was accepted by all 5 LsPALs.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…15 They can be applied to produce enantiopure Denantiomers by consuming the L-enantiomers from their racemates (kinetic resolution), or catalyze the ammonia addition reactions of trans-cinnamic acids under an ammonia rich buffer to create L-α-amino acids with high enantioselectivity. 16 PAL-based multi-enzymatic cascade reactions have also been reported to synthesize more diverse structures. 20 exhibit a wide substrate spectrum toward various phenylalanine and cinnamic acid derivatives.…”

Section: ■ Introductionmentioning

confidence: 99%

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Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids

Zhu

Liu

Pei

et al. 2023

J. Agric. Food Chem.

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Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of L-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward L-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55−60 °C and pH 9.0−9.5. However, four of them, LsPAL1−LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids

Zhu

Liu

Pei

et al. 2023

J. Agric. Food Chem.

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“…The exact mechanism of the increase in phenylalanine levels, which can damage the brain, is still unclear. The same can be said about direct damage and a decrease in tyrosine levels, as well as a lack of other large neutral amino acids that interfere with neurotransmitter formation and neuronal development [ 16 , 17 , 18 ].…”

Section: Discussionmentioning

confidence: 99%

Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems

et al. 2021

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The kinetics of L-phenylalanine ammonia-lyase (PAL) penetration into the monolayer of liver cells after its release from capsules was studied. The studies showed the absence of the effect of the capsule shell based on plant hydrocolloids on the absorption of L-phenylalanine ammonia-lyase in systems simulating the liver surface. After 120 min of incubation, in all variants of the experiment, from 87.0 to 96.8% of the enzyme penetrates the monolayer of liver cells. The combined analysis of the results concludes that the developed encapsulated form of L-phenylalanine ammonia-lyase is characterized by high efficiency in correcting the disturbed catabolism of phenylalanine in phenylketonuria, which is confirmed by the results of experiments carried out on in vitro model systems. PAL is approved for the treatment of adult patients with phenylketonuria. The encapsulated L-phenylalanine ammonia-lyase form can find therapeutic application in the phenylketonuria treatment after additional in vitro and in vivo studies, in particular, the study of preparation safety indicators. Furthermore, it demonstrated high efficacy in tumor regression and the treatment of tyrosine-related metabolic disorders such as tyrosinemia. Several therapeutically valuable metabolites biosynthesized by PAL via its catalytic action are included in food supplements, antimicrobial peptides, drugs, amino acids, and their derivatives. PAL, with improved pharmacodynamic and pharmacokinetic properties, is a highly effective medical drug.

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“…Nonetheless, the application of aromatic ammonia-lyases within the synthetically attractive reverse ammonia addition reaction, for the large scale synthesis of various specific target molecules is still challenging, especially due to occurrence of substrate/product inhibition or enzyme stability issues caused by the high (4–6 M) ammonia concentrations employed within the reaction 18 , 26 , 27 . For developing P(T)ALs and P(T)AMs with high operational stability and broad substrate scope novel MIO-enzymes from bacterial (PALs from Nostoc punctiforme 13 , Kangiella koreensis 28 and Planctomyces brasiliensis 29 , PAL/TAL and PAM from Pseudomonas fluorescens 30 ), and/or extremophile sources (PALs from Rubrobacter xylanophilus 31 and Pseudozyma antarctica 32 ) have been explored, some of them with increased thermal- and ionic strength-stability. However, their substrate scope exploration still remains a tedious and laborious task.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Moisă

Amariei

Nagy

et al. 2020

Sci Rep

Self Cite

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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 reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

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A novel phenylalanine ammonia-lyase from Pseudozyma antarctica for stereoselective biotransformations of unnatural amino acids

Cited by 16 publications

References 54 publications

Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids

Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids

Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

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