Table 2 Summary of protein engineering strategies for improving tolerance of laccase to ILs or other enzymes to DES

Trametes versicolor laccase (Lcc2)

Directed evolution

CAA

4.5-fold higher activity in 15% (v/v) of [EMIM] [EtSO4] (M3 mutant)

(Liu et al. 2013)

KnowVolution

CAA, computational modeling and evolutionary conservation analysis

Improved activity in ILs; Loop 1 was important for improving laccase resistance with ILs

(Wallraf et al. 2018)

Pleurotus ostreatus

laccase

Directed evolution

CAA, and molecular modeling

Higher thermostability of laccase in acidic and alkaline pH and aqueous betaine-based NADESs

(Piscitelli et al. 2011; Varriale et al. 2022)

Melanocarpus albomyces laccase

KnowVolution

CAA with various substrates: ABTS, 2,6-dimethylphenol and syringaldazine; molecular docking and simulations

Improved stability and activity at alkaline pH; Key residues that located in close proximity of the T1Cu site were identified to increase alkaline tolerance

(Novoa et al. 2019)

Myceliophthora thermophila laccase

Combination of computational-assisted rational design and site-directed mutagenesis

MD simulations

The inhibition mechanism of [C₂C₁Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface

(Stevens & Shi 2022)

Clostridium cellulovorans cellulase

Directed evolution

CAA and MD simulations

Improved tolerance to ILs and DESs; Residue Arg300 was the key for the ionic strength activation through a salt bridge with the neighboring Asp287

(Lehmann et al. 2014)

Penicillium verruculosum

cellobiohydrolase I

Combination of computational-assisted rational design and site-directed mutagenesis

CAA and MD simulations

Improved tolerance to DES; the formation of salt bridges and π–π interaction in variants stabilized surface exposed flexible α‐helix and highly flexible loop in the multi‐domain β‐jelly roll fold structure

(Pramanik et al. 2021)