Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis

Abstract

We discuss the hydrolysis of cellulose using a pure cellulase: endo-1,4-β-D-glucanase (EG) from the fungus, Aspergillus niger, in buffer, the pure ionic liquid (IL), tris-(2-hydroxyethyl)-methylammonium methylsulfate (HEMA), and various mixtures of the two at different temperatures. Steady-state fluorescence and absorbance studies were performed to monitor the stability and activity of EG using cellulose azure as the substrate. EG attains its highest activity at 45°C in buffer and denatures at ∼55°C. On the other hand, HEMA imparts substantial stability to the enzyme, permitting the activity to peak at 75°C. The relative roles of temperature, viscosity, pH, polarity, and the constituent ions of the ILs on the hydrolysis reaction are examined. It is demonstrated that pretreatment of cellulose with ILs such as BMIM Cl, MIM Cl, and HEMA results in more rapid conversion to glucose than hydrolysis with cellulose that is not pretreated. The percent conversion to glucose from pretreated cellulose is increased when the temperature is increased from 45 to 60°C. Two different ILs are used to increase the efficiency of cellulose conversion to glucose. Cellulose is pretreated with BMIM Cl. Subsequent hydrolysis of the pretreated cellulose in 10-20% solutions of HEMA in buffer provides higher yields of glucose at 60°C. Finally, to our knowledge, this is the first study dealing with a pure endoglucanase from commercial A. niger. This enzyme not only shows higher tolerance to ILs, such as HEMA, but also has enhanced thermostability in the presence of the IL.

Endeavour to simplify the frustrated concept of protein-ammonium family ionic liquid interactions

“…Further, from the activity measurement studies it was concluded that both imidazolium and ammonium cations with similar anions showed opposite effect on the activity of cellulase, ammonium family ILs being stabilizer while imidazolium-based IL being destabilizer. 132 Our conclusion of ammonium-based ILs to be more biocompatible is consistent with the results obtained by Lau et al 95 Thus, it can be concluded that protein-IL interactions vary from protein to protein and IL to IL. It is unreasonable to conclude from a particular result that if a particular IL is destabilizing/stabilizing a particular protein it will destabilize/stabilize another protein also.…”

“…Hence, this study again proved the superiority of ammonium family ILs over imidazolium-based ILs in providing stability to the enzymes. In continuation with their work on the activity and stability of cellulase, Bose and co-workers 132 in 2012, showed that again HEMA bestowed significant stability to the cellulase when compared to imidazolium-based ILs. Since in buffer, the T m was ~55 0 C which increased to ~75 0 C in HEMA.…”

Molecular dynamics simulations of cellulase homologs in aqueous 1-ethyl-3-methylimidazolium chloride

“…Simulations performed at low temperature (300 K) thus provided a useful model for studying protein-solvent interactions while simulations performed at high temperature (450 K) provided an understanding of how ILs might influence protein denaturation.The concentration of ions in solution would be expected to have a large influence on solvent-protein interactions. Previous reports have suggested high concentrations ofILs can dramatically increase viscosity(Bose, Barnes, & Petrich, 2012) and decrease protein fluctuations(Burney & Pfaendtner, 2013;Jaeger & Pfaendtner, 2013). However, we observed no clear correlation between IL concentration and protein RMSF in our simulations(Figure 3f).…”

Novel cellulose pretreatment solvent: phosphonium-based amino acid ionic liquid/cosolvent for enhanced enzymatic hydrolysis

“…The efficiency of cellulase is correlated with the residual amount of ILs associated with regenerated cellulose. Imidazolium-and halogen-based ILs were reported to decrease the cellulase activity (Bose et al 2012;Li et al 2013). Even trace amounts of these ILs may worsen significantly the cellulase activity (Zhao et al 2009) and this makes an extensive removal necessary for the residual IL before enzymatic conversion (Datta et al 2010;Shi et al 2013).…”