Mechanism of acetaldehyde-induced deactivation of microbial lipases

Abstract

BackgroundMicrobial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing.ResultsBased on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules.ConclusionsDifferences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of the enzymes from buffer at pH 6.0 can provide an easy and effective way to stabilize lipases toward inactivation by acetaldehyde.

Immobilization of lipase in cage-type mesoporous organosilicas via covalent bonding and crosslinking

“…Another reason for the reduction in activity might be the generation of acetaldehyde from the tautomerization of vinylalcohol when using vinyl ester as acyl donor and/or the presence of 1-butanol. It has been reported that this leads to a deactivation of various lipases [58][59][60]. This deactivation is more severe under continuous flow conditions than under batch condition, which might due to the higher reactants concentration as well as generation of a larger amount of acetaldehyde in total during the fixed-bed reaction.…”

A lipase‐catalyzed process for green synthesis of temsirolimus

“…There were two possible reasons for the improvement. First, it was reported that the acetaldehyde produced from enol donors was responsible for lipases deactivation . This might be one of the advantages of using our newly designed substrates.…”

Lipase-Catalyzed Transesterification Synthesis of Geranyl Acetate in Organic Solvents and Its Kinetics

“…(Berger andFaber, 1991, Franken et al, 2011). For example, C. cylindracea lipase forms a Schiff base with lysine residue, thus limiting its use (Franken et al, 2011). The poor activity of these enzymes could be accounted for.…”