Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties

Abstract

Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst ) retained 100% activity for one week at 50 8C and for 3 days at 65 8C, and has an optimum temperature range around 60-70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTK gst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTK gst from 1 L of culture. The reaction rate of mTK gst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTK gst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors -glycolaldehyde, d-glyceraldehyde and butyraldehyde -from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTK gst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.

nanoDSF as screening tool for enzyme libraries and biotechnology development

“…Transketolase is a dimeric enzyme from a thermophilic bacterium with a rather high T m of 76 °C , but it shows the highest cosolvent sensitivity in our investigation. Its high sensitivity could originate in the tertiary structure where the two active sites are composed of amino acids from both subunits at the dimer interface, where also the organic thiamine cofactor is bound, thereby rendering the enzyme less stable . Because the TK subunit interface contains several Trp residues, the single peak indicates that subunit dissociation occurs concomitant with unfolding.…”

A thermostable transketolase evolved for aliphatic aldehyde acceptors

“…3 In vitro, TK has been shown to also utilize non-phosphorylated sugars and (2R)-hydroxyaldehyde acceptors with generation of a (3S)-configurated stereocenter. Transketolase (EC 2.2.1.1, TK) is a ThDP-dependent enzyme that performs the transfer of a two-carbon ketol unit among various phosphorylated sugars in the pentose phosphate pathway.…”

“…These residues bind the acceptor substrate and position its electrophilic carbonyl group towards a nucleophilic attack. 3 by amino acid residues with other larger side chains were expected to narrow the acceptor access tunnel and improve the binding of aliphatic aldehyde acceptors such as propanal (Fig. Particularly, Asp470 appears to control the enantioselective binding of 2-hydroxyaldehydes by specific hydrogen bonding to the OH group.…”

Convergent in situ Generation of Both Transketolase Substrates via Transaminase and Aldolase Reactions for Sequential One‐Pot, Three‐Step Cascade Synthesis of Ketoses

“…For biocatalytic applications, mesophilic TKs from Saccharomyces cerevisiae and from Escherichia coli have been largely used and optimized by mutagenesis . We have identified the first thermostable TK from Geobacillus stearothermophilus (TK gst ) that offers significantly improved stability at high temperature, more robustness towards non‐usual reaction conditions and a broadened substrate spectrum obtained by in vitro evolution towards (2 S )‐hydroxylated, aliphatic and aromatic aldehydes and also towards HPA analogs as new donor substrates …”

“…The purpose of this study was to optimize the synthesis of highly valuable natural (3 S ,4 S )‐ketoses from (2 S )‐hydroxyaldehydes. While the synthesis of such compounds is inaccessible using mesophilic TKs, we could recently show that TK gst catalysis enabled best yields upon reaction at 60 °C in a reasonable time frame (8–24 h) . To make this TK gst ‐catalyzed process greener and more sustainable, a major challenge was to generate the donor HPA as well as the acceptor substrates in situ in a one‐pot procedure, not only to avoid the costly purchase or chemical synthesis of these compounds, but also to counteract their limited stability at 60 °C (Scheme ).…”