Enzymatic Synthesis of Aliphatic Acyloins Catalyzed by Thermostable Transketolase

Abstract

The thermostable transketolase from Geobacillus stearothermophilus (TK gst) was successfully engineered for the synthesis of aliphatic acyloins with varying carbon backbone lengths (C 5 À C 10) based on protein structure-guided studies. Efficient TK gst variants were identified with enhanced activities for substrate combinations of aliphatic aldehydes as acceptors together with aliphatic pyruvate homologues as donors. The TK gst single variant L382F was able to catalyze efficiently the transfer of the ketol group from hydroxypyruvate on all targeted aliphatic aldehydes (C 3 À C 8) to give the corresponding 1,3-dihydroxy ketones with good yields and excellent enantioselectivity. The combination of the H102L/H474S mutation previously designed for the improved utilization of aliphatic pyruvate homologues together with a F435I exchange gave the new variant H102L/H474S/F435I, which is able to transfer the acyl goup of 2-oxobutyrate and 2-oxovalerate to aliphatic aldehydes, giving mono hydroxylated ketones.

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

“…Likewise, TK gst had a very similar profile activity towards these substrates and its activity towards heptanal and octanal was very low. 9 More interestingly, our results showed that TK tmar is active towards uronic acids with relative activities towards D-galacturonic acid (GalAc) and D-glucoronic acid of 52% and 53% respectively. This TK-catalysed reaction would lead to the formation of the respective 7-keto-octuronic acids.…”

“…The TK pH-based high-throughput assay proved to be effective for initial substrate scope screening for a series of aldehydes substrates using LiHPA as ketol donor. 9,38 In this work several aldehyde donors were tested including monosaccharides: C 5 (L-rhamnose, L-arabinose, D-ribose, 2-deoxy-D-ribose and Table 1 TK tmar apparent kinetic parameters towards LiHPA and glycoaldehyde (GA), were calculated following the pH-based high-throughput assay with pure TK tmar (0.25 mg mL −1 ), GA (final concentration 0.5 to 50 mM -LiHPA constant at 50 mM) or LiHPA (final concentration 5 to 100 mM -GA constant at 50 mM), phenol red (28 mM), ThDP (2.4 mM), MgCl 2 (9 mM). All reaction components were prepared in TEA 2 mM buffer pH 7 and reactions carried out 65°C.…”

“…This forms a new asymmetric α-hydroxyketone with an (S)-configuration, which is otherwise difficult to prepare chemically. 6,7 WT and engineered TKs from different microbial sources have been used in the synthesis of phosphorylated sugars, 8 non-phosphorylated aliphatic and aromatic acyloins, [9][10][11][12][13] and rare sugars such as L-glucoheptulose, 14,15 as well as in coupled cascade reactions. [15][16][17][18] For synthetic applications, biocatalysis using enzymes such as TK allows a sustainable, one-step stereoselective way for preparing chiral building blocks and fine chemicals.…”

“…26 TK gst has been subjected to multiple protein engineering studies in order to expand its substrate scope. 9,[27][28][29] In this work, we report the biochemical characterisation of a hyperthermophilic TK from T. maritima DSM3109, with extreme thermostability, as well as investigations into substrates accepted including C 5 and C 6 monosaccharides, sugar derivatives (e.g. glucosamine and furfural) and aliphatic aldehydes (C 3 -C 9 ) using LiHPA as the ketol donor.…”

“…This forms a new asymmetric α-hydroxyketone with an ( S )-configuration, which is otherwise difficult to prepare chemically. 6,7 WT and engineered TKs from different microbial sources have been used in the synthesis of phosphorylated sugars, 8 non-phosphorylated aliphatic and aromatic acyloins, 9–13 and rare sugars such as l -glucoheptulose, 14,15 as well as in coupled cascade reactions. 15–18 …”