Using the β-glucosidase catalyzed reaction product glucose to improve the ionic liquid tolerance of β-glucosidases

Abstract

BackgroundPretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose is typically recovered through precipitation with an anti-solvent. An industrially feasible pretreatment and hydrolysis process requires robust cellulases that are stable and active in the presence of either small amounts of ILs co-precipitated with recovered cellulose or for saccharifications in the presence of IL. β-glucosidase (BG) hydrolyzes cellobiose into two molecules of glucose (Glc) and is the last step of biomass hydrolysis. These enzymes are prone not only to product inhibition by glucose but also to inactivation by ILs. With increasing interest in IL-based pretreatment methods, there is increasing focus toward a search for Glc-tolerant and IL-tolerant BG.ResultsWe identified a BG belonging to the GH1 family, H0HC94, encoded in Agrobacterium tumefaciens 5A, and cloned and overexpressed the protein in Escherichia coli. H0HC94 exhibited high enzymatic activity with β-glycosidic substrates (248 µmol/min/mg on pNPGlc and 262 µmol/min/mg on cellobiose) and tolerant to Glc (apparent Ki = 686 mM). Further evidence of Glc-based stabilization came from the increase in melting temperature of H0HC94, with increasing Glc concentrations. The half-life of H0HC94 also increased between 2- and 20-fold in the presence of increasing concentrations of Glc. In the presence of 0.9 M of different [C2mim]-based ionic liquids, the specific activity of H0HC94 decreased by around 20–30 %. However, the addition of 100 mM glucose to the IL-enzyme mix resulted in a more stable enzyme as evidenced by the slight recovery of H0HC94 melting temperature and up to tenfold increase in half-life. This higher stability came at a cost of 2–10 % decrease in specific activity. The steady-state kinetic analyses for a subset of the ionic liquids tested indicate that the enzyme undergoes uncompetitive inhibition by glucose and ionic liquid, indicating the possibility of binding of the ionic liquid and glucose to the enzyme–substrate complex.ConclusionsH0HC94 is a Glc-stabilized BG that is also tolerant up to 0.9 M concentrations of different IL’s and indicates the possibilities of using an IL–Glc-based cellulose solvent that displays enzyme-compatibility.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0484-3) contains supplementary material, which is available to authorized users.

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

“…The double and triple mutant containing V169C mutation was highly active, together the V169C/E173L/I246A had the highest increase in residual specific activity, almost 1.9-folder higher at 1 M glucose, compared to wild-type (Supplementary file, Table S3) and (Fig. 5b) These observations are in line with our previous reports when we showed that the β-glucosidase reaction product glucose improved the half-life and the kinetic stability of β-glucosidase, H0HC94 [22] and O08324 [15]. B8CYA8 and all its mutants show elevated melting temperature (Supplementary file, Table S4) with increasing concentrations of glucose, as reported previously [15, 22].…”

“…5b) These observations are in line with our previous reports when we showed that the β-glucosidase reaction product glucose improved the half-life and the kinetic stability of β-glucosidase, H0HC94 [22] and O08324 [15]. B8CYA8 and all its mutants show elevated melting temperature (Supplementary file, Table S4) with increasing concentrations of glucose, as reported previously [15, 22]. The increase was about 4-5 °C in the presence of the 0.5 M glucose, and 6-7 °C in 1 M glucose (Supplementary file, Table S4), and indicated the benefits of glucose accumulation during large-scale high biomass loading saccharification reactions.…”

“…The Phe may prevent the accumulation of glucose near the active site from enhancing the glucose tolerance of W122F. Similar mutations across H0HC94 and O08324 studied in our laboratory, seems to support the role of hydrophobic residues inside the active site pocket [12, 22]. Amino acid residues in the aglycone-binding site have been proposed to be responsible for glucose tolerance [13, 43].…”

“…a: Multiple sequence alignment of B8CYA8 against previously identified glucose tolerant GH1 β-glucosidase by Clustal Omega [50]. B8CYA8 from Halothermothrix orenii [18] was aligned with O93784 from Humicola grisea [51], Q8T0W7 from Neotermes koshunensis [52], H0HC94 from Agrobacterium tumefaciens [22], Td2F2 from compost microbial metagenomics library [52], D5KX75 from a metagenome [53], O08324 from Thermococcus sp. [15] and A0A0F7KKB7 from metagenome [9].…”

“…Based on the initial rate measured, the amount of enzyme to be used, and the assay time was optimized. The specific activity of the mutants was assayed at the T opt and pH opt of each enzyme as per previously published protocol, using saturating concentrations of substrates, p NPGlc, and cellobiose (Clb) [22]. Clb hydrolysis produces two molecules of glucose and the calibration curve used was based on the glucose produced.…”