Fine tuning enzyme activity assays for monitoring the enzymatic hydrolysis of PET.

Efficient monitoring of the enzymatic PET-hydrolysis is crucial for developing novel plastic-degrading biocatalysts. Herein, we aimed to upgrade in terms of accuracy the analytical methods useful for monitoring enzymatic PET-degradation. For the HPLC-based assessment, the incorporation of an internal standard within the analytic procedure enabled a more accurate quantification of the overall TPA content and the assessment of molar distributions and relative content of each aromatic degradation product.

Ancestral l-amino acid oxidase: From substrate scope exploration to phenylalanine ammonia-lyase assay.

In this study we assessed the applicability of the recently reported ancestral l-amino acid oxidase (AncLAAO), for the development of an enzyme-coupled phenylalanine ammonia-lyase (PAL) activity assay. Firstly, the expression and isolation of the AncLAAO-N1 was optimized, followed by activity tests of the obtained octameric N-terminal His-tagged enzyme towards various phenylalanine analogues to assess the compatibility of its substrate scope with that of the well-characterized PALs. AncLAAO-N1 showed high catalytic efficiency towards phenylalanines mono-, di-, or multiple-substituted in the meta- or para-positions, with ortho- substituted substrates being poorly transformed, these results highlighting the significant overlap between its substrate scope and those of PALs.

Phenylalanine ammonia-lyases: combining protein engineering and natural diversity.

In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) provided tailored PALs active towards challenging, highly valuable di-substituted substrates, such as the L-DOPA precursor 3,4-dimethoxy-L-phenylalanine or the 3-bromo-4-methoxy-phenylalanine. The rational design approach and saturation mutagenesis strategy unveiled identical PcPAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of PcPAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V PcPAL variants, we imprinted these beneficial mutations to PALs of different origins.

Engineered, Scalable Production of Optically Pure l-Phenylalanines Using Phenylalanine Ammonia-Lyase from .

An efficient preparative-scale synthetic procedure of l-phenylalanine derivatives has been developed using mutant variants of phenylalanine ammonia-lyase from (PAL). After rigorous reaction engineering, the PAL-catalyzed hydroamination reaction of cinnamic acids provided several unnatural amino acids of high synthetic value, such as ()-- and ()--methoxyphenylalanine; ()-- and ()--methylphenylalanine; and ()-- and ()--bromophenylalanine at preparative scale, significantly surpassing the catalytic efficiency in terms of conversions and yields of the previously reported PAL-based biotransformations. The PAL variants tolerated high substrate and product concentrations, representing an important extension of the PAL-toolbox, while the engineered biocatalytic procedures of improved E-factor and space-time yields fulfill the requirements of sustainable and green chemistry, providing facile access to valuable amino acid building blocks.

Towards a general approach for tailoring the hydrophobic binding site of phenylalanine ammonia-lyases.

Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL).

Toolbox for the structure-guided evolution of ferulic acid decarboxylase (FDC).

The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC).

Reporting system of suspected adverse effects from electronic cigarettes and refill liquids in Europe.

In line with Article 20 (9) of the Tobacco Products Directive (TPD) 2014/40/EU, a guidance document outlining a reporting system of suspected adverse effects from electronic cigarettes and their refill liquids was produced by Work Package 4 entitled 'Integration into national policies and sustainability' of the Joint Action on Tobacco Control (JATC). The JATC was a European project that lasted three years (2017-2020), with the general objective of providing support for the implementation of the TPD throughout the 27 European Union Member States (EU MS). The reporting system described in the guidance document includes a short reporting template for the reporting of adverse effects on human health from electronic cigarettes and refill liquids, both by Economic Operators (EO) and by National Competent Authorities (NCA).

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

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to -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.

Countrywide population movement monitoring using mobile devices generated (big) data during the COVID-19 crisis.

Mobile phones have been used to monitor mobility changes during the COVID-19 pandemic but surprisingly few studies addressed in detail the implementation of practical applications involving whole populations. We report a method of generating a "mobility-index" and a "stay-at-home/resting-index" based on aggregated anonymous Call Detail Records of almost all subscribers in Hungary, which tracks all phones, examining their strengths and weaknesses, comparing it with Community Mobility Reports from Google, limited to smartphone data. The impact of policy changes, such as school closures, could be identified with sufficient granularity to capture a rush to shops prior to imposition of restrictions.

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases.

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.

Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties.

Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PAL compared with the other possible mutant variants.

Efficient Biodiesel Production Catalyzed by Nanobioconjugate of Lipase from .

The Amano lipase from (L-AK) was covalently immobilized on various carbon nanomaterials (functionalized single-walled carbon nanotubes and graphene oxide) and tested for biodiesel production. Using the most active lipase preparation (covalently immobilized L-AK on SwCNT derivatized with glycerol diglycidyl ether) under optimal conditions, quasi-complete conversion (>99%) of sunflower oil was obtained after only 4 h reaction time. Moreover, the biocatalyst maintained more than 99% of its initial activity in the batch system after multiple recycling experiments.

Efficient and Stable Magnetic Chitosan-Lipase B from Bioconjugates in the Enzymatic Kinetic Resolution of Racemic Heteroarylethanols.

Lipase B from immobilized by covalent binding on sebacoyl-activated chitosan-coated magnetic nanoparticles proved to be an efficient biocatalyst (49.2-50% conversion in 3-16 h and >96% enantiomeric excess) for the enzymatic kinetic resolution of some racemic heteroarylethanols through transesterification with vinyl acetate. Under optimal conditions (vinyl acetate, -hexane, 45 °C), the biocatalyst remains active after 10 cycles.

The production of L- and D-phenylalanines using engineered phenylalanine ammonia lyases from Petroselinum crispum.

The biocatalytic synthesis of L- and D-phenylalanine analogues of high synthetic value have been developed using as biocatalysts mutant variants of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL), specifically tailored towards mono-substituted phenylalanine and cinnamic acid substrates. The catalytic performance of the engineered PcPAL variants was optimized within the ammonia elimination and ammonia addition reactions, focusing on the effect of substrate concentration, biocatalyst:substrate ratio, reaction buffer and reaction time, on the conversion and enantiomeric excess values. The optimal conditions provided an efficient preparative scale biocatalytic procedure of valuable phenylalanines, such as (S)-m-methoxyphenylalanine (Y = 40%, ee > 99%), (S)-p-bromophenylalanine (Y = 82%, ee > 99%), (S)-m-(trifluoromethyl)phenylalanine (Y = 26%, ee > 99%), (R)-p-methylphenylalanine, (Y = 49%, ee = 95%) and (R)-m-(trifluoromethyl)phenylalanine (Y = 34%, ee = 93%).

Exploring the substrate scope of ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae.

Ferulic acid decarboxylase from Saccharomyces cerevisiae (ScFDC1) was described to possess a novel, prenylated flavin mononucleotide cofactor (prFMN) providing the first enzymatic 1,3-dipolar cycloaddition mechanism. The high tolerance of the enzyme towards several non-natural substrates, combined with its high quality, atomic resolution structure nominates FDC1 an ideal candidate as flexible biocatalyst for decarboxylation reactions leading to synthetically valuable styrenes. Herein the substrate scope of ScFDC1 is explored on substituted cinnamic acids bearing different functional groups (-OCH, -CF or -Br) at all positions of the phenyl ring (o-, m-, p-) as well as on several biaryl and heteroaryl cinnamic acid analogues or derivatives with extended alkyl chain.

Heterocycles 48. Synthesis, Characterization and Biological Evaluation of Imidazo[2,1-][1,3,4]Thiadiazole Derivatives as Anti-Inflammatory Agents.

Non-steroidal anti-inflammatory drugs (NSAIDs) are an important pharmacological class of drugs used for the treatment of inflammatory diseases. They are also characterized by severe side effects, such as gastrointestinal damage, increased cardiovascular risk and renal function abnormalities. In order to synthesize new anti-inflammatory and analgesic compounds with a safer profile of side effects, a series of 2,6-diaryl-imidazo[2,1-][1,3,4]thiadiazole derivatives ⁻ were synthesized and evaluated in vivo for their anti-inflammatory and analgesic activities in carrageenan-induced rat paw edema.

Tailored Mutants of Phenylalanine Ammonia-Lyase from for the Synthesis of Bulky l- and d-Arylalanines.

Tailored mutants of phenylalanine ammonia-lyase from (PAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding ()-arylacrylates. The wild-type PAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates.

Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids.

A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or β-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well.

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k/K value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site.

Making sense of big data in health research: Towards an EU action plan.

Medicine and healthcare are undergoing profound changes. Whole-genome sequencing and high-resolution imaging technologies are key drivers of this rapid and crucial transformation. Technological innovation combined with automation and miniaturization has triggered an explosion in data production that will soon reach exabyte proportions.

Heterocycles 36. Single-Walled Carbon Nanotubes-Bound N,N-Diethyl Ethanolamine as Mild and Efficient Racemisation Agent in the Enzymatic DKR of 2-Arylthiazol-4-yl-alanines.

In this paper we describe the chemoenzymatic synthesis of enantiopure l-2-arylthiazol-4-yl alanines starting from their racemic N-acetyl derivatives; by combining the lipase-catalysed dynamic kinetic resolution of oxazol-5(4H)-ones with a chemical and an enzymatic enantioselective hydrolytic step affording the desired products in good yields (74%-78%) and high enantiopurities (ee > 99%). The developed procedure exploits the utility of the single-walled carbon nanotubes-bound diethylaminoethanol as mild and efficient racemisation agent for the dynamic kinetic resolution of the corresponding oxazolones.

Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production.

Carboxylated single-walled carbon nanotubes (SWCNTCOOH) were used as support for covalent immobilization of Candida antarctica lipase B (CaL-B) using linkers with different lengths. The obtained nanostructured biocatalysts with low diffusional limitation were tested in batch mode in the ethanolysis of the sunflower oil. SWCNTCOOH-CaL-B proved to be a highly efficient and stable biocatalyst in acetonitrile (83.

Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles.

Phenylalanine ammonia-lyase (PAL), found in many organisms, catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in-line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates.