Inhibition of hyperthermostable xylanases by superbase ionic liquids

Abstract

The use of enzymes in aqueous solutions of ionic liquids (ILs) could be useful for the enzymatic treatment of lignocellulose. Hydrophilic ILs that dissolve lignocellulose are harmful to enzymes. The toleration limits and enzyme-friendly superbase IL combinations were investigated for the hyperthermophilic Thermopolyspora flexuosa GH10 xylanase (4-β-D-xylan xylanohydrolase EC 3.2.1.8) TfXYN10A and Dictyoglomus thermophilum GH11 xylanase DtXYN11B. TfXYN10A was more tolerant than DtXYN11B to acetate or propionate-based ILs. However, when the anion of the ILs was bigger (guaiacolate), GH11 xylanase showed higher tolerance to ILs. 1-Ethyl-3-methylimidazolium acetate ([EMIM]OAc), followed by 1,1,3,3-tetramethylguanidine acetate ([TMGH]OAc), were the most enzyme-friendly ILs for TfXYN10A and [TMGH] + -based ILs were tolerated best by DtXYN11B. Double-ring cations and a large size anion were associated with the strongest enzyme inhibition. Competitive inhibition appears to be a general factor in the reduction of enzyme activity. However, with guaiacolate ILs, the denaturation of proteins may also contribute to the reduction in enzyme activity. Molecular docking with IL cations and anions indicated that the binding mode and shape of the active site affect competitive inhibition, and the co-binding of cations and anions to separate active site positions caused the strongest enzyme inhibition.

Activity and stability of hyperthermostable cellulases and xylanases in ionic liquids

“…Activities and stabilities of hyperthermostable cellulases and xylanases in ILs are summarized in Table 1. Highly IL-tolerant hyperthermostable glycoside hydrolases have been found to belong to GH3 glucosidases (Gladden et al 2014), GH5 and GH12 endoglucanases (Datta et al 2010;Klose et al 2012;Gladden et al 2014;Rahikainen et al 2018), and GH10 xylanases (Chawachart et al 2014;Anbarasan et al 2017;Rahikainen et al 2018;Hebal et al 2020). These results suggest that higher tolerance to ILs is inherent to common structural motifs in these proteins (Pottk€ amper et al 2009).…”

“…Extremophilic enzymes have been found to be highly tolerant to the presence of ILs (Datta et al 2010;Ferdjani et al 2011;Zhang et al 2011;Klose et al 2012;Ilmberger et al 2012;Gladden et al 2014;Yu et al 2016;Anbarasan et al 2017;Rahikainen et al 2018;Hebal et al 2020). In a comparison between activities of glycosidases in ILs that differ in their thermostability, a close correlation was found between the enzyme thermostability and the activity and stability in ILs (Ferdjani et al 2011).…”

“…These studies suggest that thermostability is not the sole factor in IL tolerance and other factors like halotolerance and surface charges contribute to resistance against the ILs (Pottk€ amper et al 2009;Ilmberger et al 2012;Nordwald and Kaar 2013;Trivedi et al 2013). Furthermore, among the hyperthermostable enzymes, higher thermostability is not a guarantee for high activity and stability in ILs (Li et al 2013;Summers et al 2017;Rahikainen et al 2018;Hebal et al 2020). Resistance to competitive inhibition by the IL was found to play a crucial role in the tolerance of hyperthermostable endoglucanases and xylanases to ILs (Yu et al 2016;Anbarasan et al 2017;Johnson and Snow 2017;Summers et al 2017;Hebal et al 2020).…”

“…Furthermore, among the hyperthermostable enzymes, higher thermostability is not a guarantee for high activity and stability in ILs (Li et al 2013;Summers et al 2017;Rahikainen et al 2018;Hebal et al 2020). Resistance to competitive inhibition by the IL was found to play a crucial role in the tolerance of hyperthermostable endoglucanases and xylanases to ILs (Yu et al 2016;Anbarasan et al 2017;Johnson and Snow 2017;Summers et al 2017;Hebal et al 2020).…”

“…Therefore, the use of these solvents in the processing of biomass requires the development of IL-tolerant enzymes or enzyme-compatible ILs. The use of hyperthermostable glycoside hydrolases is one strategy to obtain tolerance of enzymes to ILs (Datta et al 2010;Rahikainen et al 2018;Hebal et al 2020). However, several studies have shown that hyperthermostability is not the sole factor in tolerance to ILs, and especially, the resistance to competitive inhibition by IL ions plays a pivotal role (Li et al 2013;Chawachart et al 2014;Anbarasan et al 2017;Hebal et al 2020).…”