Directed laccase evolution for improved ionic liquid resistance

• Published in: Green chemistry : an international journal and green chemistry resource : GC 2013-01, Vol.15 (5), p.1348-1355
• Main Authors: HAIFENG LIU, LEILEI ZHU, BOCOLA, Marco, CHEN, Nora, SPIESS, Antje C, SCHWANEBERG, Ulrich
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Oxidoreductases; Saccharomyces
• ISSN: 1463-9262; 1463-9270
• DOI: 10.1039/c3gc36899h

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 sub(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 sub(4)]. Therefore, ([EMIM] [EtSO sub(4)]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2and CVLG1) from T. versicolor(Trametes versicolor) were expressed in Saccharomyces cerevisiaeand finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO sub(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 sub(4)] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC sub(50) value of [EMIM] [EtSO sub(4)] towards M3 increases from 392 mM (lcc2 WT) to 497 mM.