Mechanism of acetaldehyde-induced deactivation of microbial lipases

Benjamin Franken,Karl E Jaeger,Martina Pohl,Thorsten Eggert

doi:10.1186/1471-2091-12-10

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.

Highlights

Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis
We demonstrate that the protonation state of lysine ε-amino groups in microbial lipases has a significant impact on their sensitivity against acetaldehyde
The protonation state determines the sensitivity of lipases toward acetaldehyde Schiff-base formation depends on the protonation state of primary amino groups, as only a deprotonated amino group (-NH2) can nucleophilically attack the acetaldehyde carbonyl group

Summary

Introduction

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. The stoichiometrically generated acetaldehyde can be used in a high-throughput assay to detect transesterification activities of lipases and esterases directly in the organic phase [12]. Despite of these advantages, aldehydes are generally known to act as alkylating reagents by forming Schiff-bases in a Maillard-type reaction, in particular with enzyme lysine ε-amino groups. Activity has been improved by “pH-tuning” of lipases prior to their use in organic solvents, meaning that the enzyme was lyophilized from a buffer with optimal pH for biocatalysis [23]

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Conclusion