<p>Abstract</p> <p>Background</p> <p>Microbial 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.</p> <p>Results</p> <p>Based on five microbial lipases from <it>Candida rugosa</it>, <it>Rhizopus oryzae</it>, <it>Pseudomonas fluorescens </it>and <it>Bacillus subtilis </it>we demonstrate that the protonation state of lysine <it>ε</it>-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of <it>Bacillus subtilis </it>lipases in the presence of acetaldehyde revealed several stable products such as <it>α,β</it>-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.</p> <p>Conclusions</p> <p>Differences 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 <it>α,β</it>-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.</p
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