Directed laccase evolution for improved ionic liquid resistance

Abstract

In nature, the biodegradation of lignin is a challenging process since lignin is highly cross-linked and poorly water-soluble. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) play a key role in the enzymatic degradation of lignin and ionic liquids (ILs) have been used successfully to dissolve lignin. One limitation in lignin degradation using laccases is their low activity/resistance in the presence of ILs.In order to improve the resistance of laccase in IL, a directed evolution protocol based on the ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))-screening assay in 96-well microtiter plate format was developed. 1-Ethyl-3-methylimidazolium ethylsulfate ([EMIM] [EtSO 4 ]) can dissolve lignin efficiently and its anion does not inhibit laccase. The stability of the ABTS radical cation was not affected in the presence of [EMIM] [EtSO 4 ]. Therefore, ([EMIM] [EtSO 4 ]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2 and CVLG1) from T. versicolor (Trametes versicolor) were expressed in Saccharomyces cerevisiae and finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO 4 ]. After two rounds of directed evolution, the lcc2 variant M3 (Phe265Ser/Ala318Val) displayed about 4.5-fold higher activity than the lcc2 wild type (WT) in the presence of 15% (v/v) [EMIM] [EtSO 4 ] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC 50 value of [EMIM] [EtSO 4 ] towards M3 increases from 392 mM (lcc2 WT) to 497 mM. † Electronic supplementary information (ESI) available. See

Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A

“…[47] Considering BSLA secondary structure elements, more than 85 % of the amino acid positions beneficial for IL resistance were found in the helices and loops of BSLA. As reported for other enzymes, [30][31][32]35] beneficial substitutions were found both on the surface and in buried regions. Analysis of the amino acid substitution patterns of surface-exposed and buried amino acids revealed differences.…”

“…Enzyme classes such as oxidoreductases (e.g., laccase, [32] formate dehydrogenase) [33] and hydrolases (e.g., celullase) [34][35][36] have been successfully improved by protein engineering for increased IL resistance/tolerance. However, at present, there are only about six reports on improving IL resistance of enzymes by protein engineering.…”

“…However, at present, there are only about six reports on improving IL resistance of enzymes by protein engineering. [32][33][34][35][36][37] All focused on identifying substitutions that affect IL resistance. Despite early success on improving IL resistance, general design principles to guide protein engineering campaigns have still to be established.…”

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

“…Overall, more than 24 reports have been published in which (co-)solvent resistance has been improved (≥ 15 reports by directed evolution; ≥ 9 reports by semi-rational design). Till today, improved organic (co-)solvent resistance has been reported for at least four enzyme classes including monooxygenases, hydrolases, oxidoreductases, and transferases (Wong et al 2004;Ogino 2009, 2010;Koudelakova et al 2013;Liu et al 2009Liu et al , 2013Martinez and Arnold 1991). The reported beneficial amino acid substitutions provided the insight that how enzymes can be stabilizes organic (co-)solvents.…”

Using Molecular Simulation to Study Biocatalysis in Ionic Liquids

“…Directed evolution techniques have also recently been applied to enzyme/IL systems as a means of optimizing enzymes with respect to desired properties Liu et al, 2013;Tee et al, 2008). For example, Carter and coworkers mutated the formate dehydrogenase from Candida boidinii with error-prone PCR to improve the enzyme's thermostability and tolerance to ILs (Carter, Bekhouche, Noiriel, Blum, & Doumèche, 2014).…”