Characterisation of a hyperthermophilic transketolase fromThermotoga maritimaDSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

Abstract

Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon-carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK fromThermotoga maritimaDSM3109 (TKtmar). TKtmarhas a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmarhas a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/299 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmarcatalysing the efficient conversion ofd-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8uronic acid, in high yields (98%, 49 mM).