Enzymatic synthesis of cyclic imino acids

Optically active cyclic imino acids are widely used as important pharmaceutical intermediates and it is necessary to develop cost effective synthetic method for their production. We have already established one-pot synthesis of L-cyclic imino acids from diamino acids by using N-methyl-L-amino acid dehydrogenase (NMAADH) from Pseudomonas putida. In order to make this process more efficient, we established a recombinant Escherichia coli which expresses NMAADH, lysine racemase from P. putida and D-lysine dehydrogenase from Selenomonas ruminantium in a single cell. The recombinant E. coli makes it possible to recycle NADPH by combination of reductive reaction using NMAADH and oxidative reaction using D-lysine dehydrogenase (Fig.1). Please click Additional Files below to see the full abstract

Overcoming Work-Up Limitations of Biphasic Biocatalytic Reaction Mixtures Through Liquid-Liquid Segmented Flow Processes

Adebar N, Choi J-E, Schober L, et al. Overcoming Work-Up Limitations of Biphasic Biocatalytic Reaction Mixtures Through Liquid-Liquid Segmented Flow Processes. ChemCatChem. 2019;11(23):5788-5793.Biphasic biocatalytic reactions have gained much attention in the field of enzyme-catalysed synthesis. As most components being of relevance for the pharmaceutical industry are hydrophobic, often biphasic reaction media turned out to be the solvent system of choice. However, in spite of successful reaction courses practical difficulties in the downstream-processing, in particular extremely difficult phase separations due to emulsification and precipitation, represent a challenge to overcome in process development. In this work, we report our studies on the benefits of a simple flow set-up being capable to minimise such work-up limitations. In detail, a segmented flow system based on a biphasic MTBE/buffer mixture was successfully applied for two types of enzymatic reductions of a hydrophobic ketone in the presence of an alcohol dehydrogenase (ADH) as an enzyme class being known for their excellent enantioselectivity and successful utilization in the synthesis of a range of active pharmaceutical ingredients. The applicability of this flow system was demonstrated with two different enzymes as well as different substrates. Besides an ADH from Lactobacillus brevis, an ADH from Ogatea minuta was utilized for the reduction of acetophenone and 2,2,2-trifluoroacetophenone, respectively

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “receptor down-regulation” process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes