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46776-GB1
Effect of Ionic Liquid Properties on the Enzyme Stabilization Under Microwave Radiation

Hua Zhao, Savannah State University

Ionic liquids (ILs) as neoteric solvents and microwave irradiation as energy source are becoming two important tools for many enzymatic reactions. However, it is not well understood what properties of ILs govern the enzyme stabilization, and whether the microwave irradiation could activate enzymes in ILs. To tackle these two important issues, the synthetic activities of immobilized Candida antarctica lipase B (Novozyme 435) were examined in more than twenty ILs through microwave heating. Under microwave irradiation, enhanced enzyme activities were observed when the enzyme is surrounded by a layer of water molecules. However, such enhancement diminished when the reaction system was dried. To understand the effect of IL properties, the enzyme activities under microwave irradiation were correlated with the viscosity, polarity and hydrophobicity (log P) of ILs respectively. The initial reaction rates bear no straight relationship with the viscosity and polarity (in terms of dielectric constant and ) of ILs, but have a loose correlation (a bell shape) with log P values. The enzyme stabilization by ILs was explained from aspects of hydrogen-bond basicity of anions, dissolution of the enzyme, ionic association strength of anions, and substrate ground-state stabilization by ILs.

We designed a series of ILs that are able to dissolve carbohydrates but do not considerably inactivate the immobilized lipase B from Candida antarctica. These ILs consist of glycol-substituted cations and acetate anions. They could dissolve more than 10% (wt) cellulose and up to 80% (wt) D-glucose. The transesterification activities of the lipase in these ILs are very comparable with those in hydrophobic ILs. The hydrogen-bond forming anions, oxygen-containing cations, and low cation bulkiness promote the carbohydrate dissolution, while the low anion concentration appears essential for the enzyme stabilization. Therefore, an optimization could be achieved through a fine design of IL structures. To demonstrate the potential applications of these ILs, we performed the enzymatic transesterifications of methyl methacrylate with D-glucose and cellulose, respectively, both fully dissolved in ionic media. In the case of D-glucose, conversions up to 80% were obtained; and in the case of cellulose, conversions up to 89% and isolated yields up to 66% were achieved.

The pretreatment of lignocelluloses is known as the key to fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs) were found capable of dissolving more than 10% (wt) cellulose. Preliminary investigations (Biotechnol. Bioeng. 2006, 95, 904; Chinese Sci. Bull. 2006, 51, 2432; Appl. Biochem. Biotechnol. 2007, 137-140, 407) suggest that celluloses regenerated from IL solutions are subjected to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and non-volatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is corrosive, toxic and extremely hygroscopic solid (m.p. ~70 C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several newly developed ILs by our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirmed that all regenerated celluloses are less crystalline (58-75% lower) and more accessible by cellulase (> 2 times) than untreated substrates. As a result, the regenerated Avicel^® cellulose, filter paper and cotton were hydrolyzed 2-10 times faster than their respective untreated celluloses. A complete hydrolysis of Avicel^® cellulose could be achieved in 6 hrs given the Trichoderma reesei cellulase/substrate ratio (wt/wt) of 3:20 at 50 C. In addition, we observed that cellulase is more thermally stable (up to 60 C) in the presence of regenerated cellulose. Furthermore, our systematic studies suggest that the presence of various ILs during the hydrolysis induced different degrees of cellulase inactivation. Therefore, a thorough removal of IL residues after cellulose regeneration is highly recommended, and a systematic investigation on this subject is much needed.

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