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In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient "smart peak parking" algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

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The separation, concentration and transport of the amino acids through membranes have been continuously developed due to the multitude of interest amino acids of interest and the sources from which they must be recovered. At the same time, the types of membranes used in the sepa-ration of the amino acids are the most diverse: liquids, ion exchangers, inorganic, polymeric or composites. This paper addresses the recuperative separation of three amino acids (alanine, phe-nylalanine, and methionine) using membranes from cellulosic derivatives in polypropylene ma-trix. The microfiltration membranes (polypropylene hollow fibers) were impregnated with solu-tions of some cellulosic derivatives: cellulose acetate, 2-hydroxyethyl-cellulose, methyl 2-hydroxyethyl-celluloseand sodium carboxymethyl-cellulose. The obtained membranes were characterized in terms of the separation performance of the amino acids considered (retention, flux, and selectivity) and from a morphological and structural point of view: scanning electron microscopy (SEM), high resolution SEM (HR-SEM), Fourier transform infrared spectroscopy (FT-IR), energy dispersive spectroscopy (EDS) and thermal gravimetric analyzer (TGA). The re-sults obtained show that phenylalanine has the highest fluxes through all four types of mem-branes, followed by methionine and alanine. Of the four kinds of membrane, the most suitable for recuperative separation of the considered amino acids are those based on cellulose acetate and methyl 2-hydroxyethyl-cellulose.

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During manufacturing processes and in the storage period of tea, amino acids may undergo enantiomeric isomerization, converting their l- to d-forms. To examine the hypothesis, a method was developed for the analysis of the enantiomers in tea leaves. After enriched by ion-exchange solid-phase extraction, the enantiomeric pairs were separated by a chiral high performance liquid chromatography (HPLC) and subsequently detected and identified by using a high resolution quadrupole time-of-flight mass spectrometry (QTOF MS). Only l-forms of amino acids were found in fresh tea leaves. A total of 11 d-amino acids were found in 19 tea samples, ranging from trace amount to 43 µg/g. The results indicated that the enantioisomerization of amino acids occurred in post-harvest tea leaves, and affected by process conditions and storage time.

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Several amino acid chiral ionic liquids were introduced as functional monomers to prepare molecularly imprinted polymers for specific recognition of L-phenylalanine. Among them, the imprinted polymers L-Phe@MIPs based on [ViImC3][L-Pro] showed the best selective recognition ability for L-phenylalanine. A series of experiments such as dynamic adsorption, static adsorption, and competitive adsorption were conducted to investigate the specific recognition ability and adsorption capacity of the L-Phe@MIPs. It is found that the adsorption efficiency to L-phenylalanine on L-Phe@MIPs was 3.11 times higher than that to D-phenylalanine. All the results demonstrated that the L-Phe@MIPs possessed good recognition and relatively high adsorption efficiency for L-phenylalanine. Besides, the recovery of L-phenylalanine was above 98%, and the L-Phe@MIPs exhibited good reusability.

RESUMEN

The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein extraction including the potentials of enzyme addition in the form of proteases and/or carbohydrolases. Strategic biomass selection, combined with the appropriate process conditions can increase protein yields after extraction. Enzyme addition, especially of proteases, can be useful when alkaline protein extraction yields are low. These additions can also be used to enable processing at a pH closer to 7 to avoid the otherwise severe conditions that denature proteins. Finally, a protein biorefinery concept is presented that aims to upcycle residual biomass by separating essential amino acids to be used for food and feed, and non-essential amino acids for production of bulk chemicals.

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