We described an efficient in situ generation of hydroxypyruvate from d-serine catalyzed by a damino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l-serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbonÀ carbon bond. The enzymatic cascade reaction was performed in one-pot in the presence of d-serine and appropriate aldehydes for the synthesis of valuable (3S)-hydroxyketones, which were obtained with high enantio-and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d-serine and aldehydes. Scheme 1. In situ generation of HPA from TA or dAAOcatalyzed reaction coupled with TK-catalyzed reaction.Scheme 2. Reaction of DAAO Rg coupled with l-lactate dehydrogenase (LDH) in the presence of NADH.

Thiamine diphosphate‐dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon‐carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high‐throughput solid‐phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α‐ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.

Here, we present an immobilized enzyme cascade in a basket-type reactor allowing a one-pot, two-step enzymatic synthesis of L-erythrulose from D-serine and glycolaldehyde. Three enzymes, D-amino acid oxidase from Rhodotorula gracilis (DAAORg), catalase from bovine liver (CAT), and transketolase from Geobacillus stearothermophilus (TKgst) were covalently immobilized on silica monolithic pellets, characterized by an open structure of interconnected macropores and a specific surface area of up to 300 m2/g. Three strategies were considered: (i) separate immobilization of enzymes on silica supports ([DAAO][CAT][TK]), (ii) co-immobilization of two of the three enzymes followed by the third ([DAAO+CAT][TK]), and (iii) co-immobilization of all three enzymes ([DAAO+CAT+TK]). The highest L-erythrulose concentrations were observed for the co-immobilization protocols (ii) and (iii) (30.7 mM and 29.1 mM, respectively). The reusability study showed that the best combination was [DAAO + CAT][TK], which led to the same level of L-erythrulose formation after two reuse cycles. The described process paves the way for the effective synthesis of a wide range of α-hydroxyketones from D-serine and suitable aldehydes.