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

ABSTRACT: BACKGROUND:Pretreating 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. ?-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. RESULTS:We 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 ?-glycosidic substrates (248 µmol/min/mg on pNPGlc and 262 µmol/min/mg on cellobiose) and tolerant to Glc (apparent K i = 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. CONCLUSIONS:H0HC94 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.

SUBMITTER: Goswami S

PROVIDER: S-EPMC4802596 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Goswami Shubhasish S Gupta Neha N Datta Supratim S

Biotechnology for biofuels 20160322

<h4>Background</h4>Pretreating 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 (Gl ...[more]

PMID: 27006691

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Project description:The isomerization of estragole to trans-anethole is an important reaction and is industrially performed using an excess of NaOH or KOH in ethanol at high temperatures with very low selectivity. Simple Ru-based transition-metal complexes, under homogeneous, ionic liquid (IL)-supported (biphasic) and "solventless" conditions, can be used for this reaction. The selectivity of this reaction is more sensitive to the solvent/support used than the ligands associated with the metal catalyst. Thus, under the optimized reaction conditions, 100% conversion can be achieved in the estragole isomerization, using as little as 4 × 10-3 mol % (40 ppm) of [RuHCl(CO)(PPh3)3] in toluene, reflecting a total turnover number (TON) of 25 000 and turnover frequencies (TOFs) of up to 500 min-1 at 80 °C. Using a dimeric Ru precursor, [RuCl(μ-Cl)(η3:η3-C10H16)]2, in ethanol associated with P(OEt)3, a TON of 10 000 and a TOF of 125 min-1 are obtained with 100% conversion and 99% selectivity. These two Ru catalytic systems can be transposed to biphasic IL systems by using ionic-tagged P-ligands such as 1-(3-(diphenylphosphanyl)propyl)-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide immobilized in 1-(3-hydroxypropyl)-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl) imide with up to 99% selectivity and almost complete estragole conversion. However, the reaction is much slower than that performed under solventless or homogeneous conditions. The use of ionic-tagged ligands significantly reduces the Ru leaching to the organic phase, compared to that in reactions performed under homogeneous conditions, where the catalytic system loses catalytic performance after the second recycling. Detailed kinetic investigations of the reaction catalyzed by [RuHCl(CO)(PPh3)3] indicate that a simplified kinetic model (a monomolecular reversible first-order reaction) is adequate for fitting the homogeneous reaction at 80 °C and under biphasic conditions. However, the kinetics of the reaction are better described if all of the elementary steps are taken into consideration, especially at 40 °C.

Dataset Information

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

Publications

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

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