An Activity, Stability and Selectivity Comparison of Propioin Synthesis by Thiamine Diphosphate‐Dependent Enzymes in a Solid/Gas Bioreactor

Abstract

Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…This extremely low affinity for aliphatic or aromatic aldehyde substrates is probably a specific property of BFD. This behavior was also previously observed for wildtype BFD and propanal as a substrate (Mikolajek et al, 2007(Mikolajek et al, , 2009. indicates that the release of benzoin could be rate-limiting for BAL-catalyzed benzoin synthesis (Chakraborty et al, 2008).…”

Engineering stereoselectivity of ThDP-dependent enzymes

“…With the exception of propioin, which was formed with up to 53% ee for the S‐ enantiomer, both enzymes catalysed the formation of the R‐ enantiomers in excess, yielding highest enantioselectivity for the carboligation of isovaleraldehyde [89% ee ( R )]. Besides, application of both enzymes in a gas‐phase reactor yielded propioin with only very low ee [19% ( S )] . However, studies of Jordan and coworkers demonstrated that acetoin and acetolactate can be accessed in an enantiocomplementary fashion with a variant of Sc PDC‐E477Q yielding ( R )‐acetoin and a variant of the E1 subunit of the pyruvate dehydrogenase complex (PDHc‐E1‐E636A/Q) yielding the S ‐product (Table ).…”

“…Here, the potential to catalyse the formation of mixed carboligation products from a donor substrate and an acceptor substrate in a highly chemoselective and stereoselective manner is of special interest. Among the enzymes that have been most intensively studied are transketolase (TK) from Escherichia coli and Saccharomyces cerevisiae , acetohydroxy acid synthase (AHAS) isoenzymes I–III from E. coli , benzoylformate decarboxylase (BFD) from Pseudomonas putida , benzaldehyde lyase (BAL) from Pseudomonas fluorescens , pyruvate decarboxylase (PDC) from S. cerevisiae , Zymomonas mobilis and Acetobacter pasteurianus , branched chain ketoacid decarboxylase from Lactococcus lactis , cyclohexane‐1,2‐dione hydrolase from Azoarcus sp. and 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate (SEPHCHC) synthase (MenD) from E. coli .…”

Biotechnology of flavours—the next generation

“…It appears a particularly attractive idea to perform the production of volatiles using functional (hydrated) enzymes in the gas phase (Mikolajek et al 2007). The concept was applied to reverse hydrolyses, but also to carboligation.…”