Development of an integrated chromatographic system for ω-transaminase-IMER characterization useful for flow-chemistry applications.

An integrated chromatographic system was developed to rapidly investigate the biocatalytic properties of ω-transaminases useful for the synthesis of chiral amines. ATA-117, an (R)-selective ω-transaminase was selected as a proof of concept. The enzyme was purified and covalently immobilized on an epoxy monolithic silica support to create an immobilized enzyme reactor (IMER). Reactor efficiency was evaluated in the conversion of a model substrate. The IMER was coupled through a switching valve to an achiral analytical column for separation and quantitation of the transamination products. The best conditions of the transaminase-catalyzed bioconversion were optimized by a design of experiments (DoE) approach. The production of (R)-1-(4-methoxyphenyl)propan-2-amine and (R)-1-methyl-3-phenylpropylamine, intermediates for the synthesis of the bronchodilator formoterol and the antihypertensive dilevalol respectively, was achieved in the presence of different amino donors. The enantiomeric excess (ee) was determined off-line by developing a derivatization procedure using Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide reagent. The most satisfactory conversion yields were 60% for (R)-1-(4-methoxyphenyl)propan-2-amine and 29% for (R)-1-methyl-3-phenylpropylamine, using isopropylamine as amino donor. The enantiomeric excess of the reactions were 84%R and 99%R, respectively.

Immobilized enzyme-based analytical tools in the -omics era: Recent advances.

Protein analysis is a field under rapid development mainly thanks to technological advances which have granted miniaturization of analytical devices, automation and higher detection sensitivity. The interest in the field has paralleled the expansion of the -omics era, laying down the bases for the current applications in proteomics and glycomics. Advances in protein sample transformation prior to analysis have led to reduction of sample consumption and contamination, enhancing throughput. Within this context, and thanks to the availability of new high performing materials and technologies, increasingly more efficient and miniaturized enzyme-based analytical tools have been proposed to overcome shortcomings encountered in the in-solution enzymatic reactions (protein digestion and protein deglycosylation, for proteomics and glycomics, respectively). In this context, immobilized enzyme reactors (IMERs) and IMER-based platforms have been developed as promising approaches toward automation and higher analysis throughput. The scenario is in continuous development as underlined by thirty-four papers published in the last five years. This review encompasses recent advances in the design and operational set-ups of IMERs purposely developed for the analysis of proteins and glycoproteins. Recently developed dual IMERs, which integrate more than one processing step into a single IMER, and analytical platforms exploiting tandem IMERs are also reviewed and commented.

Immobilized enzyme reactors in HPLC and its application in inhibitor screening: A review.

This paper sets out to summarize the literatures based on immobilized enzyme bio-chromatography and its application in inhibitors screening in the last decade. In order to screen enzyme inhibitors from a mass of compounds in preliminary screening, multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes, and then the immobilized enzyme reactor applied as the immobilized enzyme stationary phase in HPLC. Therefore, a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs. Here, we briefly summarize the selective methods of supports, immobilization techniques, co-immobilized enzymes system and the screening model.