A loop engineering strategy improves laccase lcc2 activity in ionic liquid and aqueous solution

  Photo of Anne Wallraf Copyright: Bio VI

A. M. Wallraf, H. Liu, L. Zhu, G. Khalfallaha, C. Simons, H. Alibiglou, M. D. Davari and U. Schwaneberg, Green Chemistry, 2018, DOI: 10.1039/C7GC03776G


Importance of a domain-connecting loop in laccase for increased activity in ionic liquid (IL) was identified.

Laccases are involved in lignin degradation. EMIM- and BMIM-based ionic liquids (IL) show excellent solubilization of wooden biomass but impede laccase activity. Protein engineering to improve the activity and resistance of laccases in ILs is promising for lignin valorization for the sustainable production of fuels and bulk high-value chemicals . We report for the first time an efficient semi-rational design with focus on a domain-connecting loop of a laccase lcc2 (loop L1) from Trametes versicolor.

The loop engineering strategy is based on a KnowVolution campaign and can be divided into three steps. Prediction of seven resistance increasing positions out of 37 amino acids in L1 (residues 284-320) was performed by in silico SSM analysis with FoldX. These seven positions were saturated by SSM and four beneficial positions were subjected to simultaneous SSM using OmniChange. The OmniChange variants OM1 (A285P/A310R/A312E/A318G) and OM3 (A310D/A312P/A318R) showed a 3.9-fold (535.8 ± 36.9 U/mg) and 1.6-fold (216.8 ± 5.3 U/mg) increased specific activity in aqueous solution (lcc2 WT, 138.9 ± 6.5 U/mg), respectively, and up to 8.4-fold increased activity in (35% (v/v)) EMIM EtSO4 and aqueous solution when compared to lcc2 WT. Hydrogen bond pattern analysis revealed that both variants harbor an increased number of hydrogen bonds within the loop and between domains two and three which resulted in increased IL resistance.

  Flow scheme of the loop engineering experiment Copyright: Royal Society of Chemistry

Entropy analysis indicated that the substitution of alanine at each selected amino acid position A285, A310, A312, and A318 reduced the flexibility of the loop L1. Conservational analysis with ConSurf server showed that the long domain-connecting loop L1 is a conserved feature in fungal laccases and suggests loop engineering as a useful strategy for increasing laccase activity in ILs and aqueous solutions. This work was funded by Deutschen Forschungsgemeinschaft DFG in the frame of the research cluster “ Tailor-Made Fuels from Biomass ” TMFB.

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