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 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 SWCNTNH2 -SS-PcPAL 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 PcPAL L134A/S614C and PcPAL 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.

Simultaneous enrichment of grape pomace with γ-linolenic acid and carotenoids by solid-state fermentation with Zygomycetes fungi and antioxidant potential of the bioprocessed substrates.

Two filamentous fungi (Actinomucor elegans and Umbelopsis isabellina), were tested for their ability to enrich white grape pomace simultaneously with both γ-linolenic acid (GLA) and carotenoids through solid-state fermentation (SSF) processes. U. isabellina presented higher ability to produce GLA-rich lipids (composed mainly of neutral fractions) than A. elegans (the 6-th day of SSF: 378.85 mg/100 g of pomace -U. isabellina and 193.36 mg/100 g of pomace- A. elegans). The amounts of ß-carotene and lutein for both SSFs gradually increased until the end of the fermentation processes. The effect of fermentation time on the phenolic content and antioxidant activity of grape pomace was also studied. The SSF with A. elegans increased significantly total phenolic and flavonoid contents and DPPH scavenging activity of grape popmace. These bioprocessed grape pomaces with significant amounts of carotenoids and GLA-rich lipids (>94% nutritionally-valuable polyunsaturated fatty acids at the sn-2 position) could be very attractive for food industry.

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%).

Modern diversification of the amino acid repertoire driven by oxygen.

All extant life employs the same 20 amino acids for protein biosynthesis. Studies on the number of amino acids necessary to produce a foldable and catalytically active polypeptide have shown that a basis set of 7-13 amino acids is sufficient to build major structural elements of modern proteins. Hence, the reasons for the evolutionary selection of the current 20 amino acids out of a much larger available pool have remained elusive. Here, we have analyzed the quantum chemistry of all proteinogenic and various prebiotic amino acids. We find that the energetic HOMO-LUMO gap, a correlate of chemical reactivity, becomes incrementally closer in modern amino acids, reaching the level of specialized redox cofactors in the late amino acids tryptophan and selenocysteine. We show that the arising prediction of a higher reactivity of the more recently added amino acids is correct as regards various free radicals, particularly oxygen-derived peroxyl radicals. Moreover, we demonstrate an immediate survival benefit conferred by the enhanced redox reactivity of the modern amino acids tyrosine and tryptophan in oxidatively stressed cells. Our data indicate that in demanding building blocks with more versatile redox chemistry, biospheric molecular oxygen triggered the selective fixation of the last amino acids in the genetic code. Thus, functional rather than structural amino acid properties were decisive during the finalization of the universal genetic code.

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 kcat/KM 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. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

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 (83.4% conversion after 4h at 35°C, retaining >90% of original activity after 10 cycles).

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.

Total phenolic contents, antioxidant activities, and lipid fractions from berry pomaces obtained by solid-state fermentation of two Sambucus species with Aspergillus niger.

The aim of this study was to investigate the effect of solid-state fermentation (SSF) by Aspergillus niger on phenolic contents and antioxidant activity in Sambucus nigra L. and Sambucus ebulus L. berry pomaces. The effect of fermentation time on the total fats and major lipid classes (neutral and polar) was also investigated. During the SSF, the extractable phenolics increased with 18.82% for S. ebulus L. and 11.11% for S. nigra L. The levels of antioxidant activity of methanolic extracts were also significantly enhanced. The HPLC-MS analysis indicated that the cyanidin 3-sambubioside-5-glucoside is the major phenolic compound in both fermented Sambucus fruit residues. In the early stages of fungal growth, the extracted oils (with TAGs as major lipid fraction) increased with 12% for S. nigra L. and 10.50% for S. ebulus L. The GC-MS analysis showed that the SSF resulted in a slight increase of the linoleic and oleic acids level.

Inhibition of histidine ammonia lyase by heteroaryl-alanines and acrylates.

Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the beta-position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with K(i) values between 18 and 139 microM. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L-histidine can be dissected into 4-methyl-1H-imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a K(i) value of 1.7+/-0.09 mM which is to be compared with the K(m) value of 15.6 mM for the normal reaction. We conclude that 5-methyl-1H-imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation.