Cellulases in Ionic Liquids—The Long Term Stability of

It is a well known fact that some ionic liquids (ILs) have the potential to dissolve crystalline, water-insoluble cellulose that could be used as a source of glucose and subsequently derived molecules. Nevertheless, in the presence of high IL concentrations, cellulase activity and, even more so, cellulase stability are still challenging difficulties that need to be overcome. Therefore four fungal cellulase preparations were assayed in the presence of 30% (v/v) of five different ILs. Thereby the cellulase from Aspergillus sp. (Sigma-Aldrich) was not only remarkably active (up to 28 U/mg in 30% (v/v) IL ([BMMIM]Cl)), but furthermore stayed active during several weeks in the presence of 60% (v/v) IL (up to 50% relative activity after 9 weeks).


Introduction
The enzymatic conversion of cellulose into glucose, and subsequently biofuels, could provide a renewable and sustainable energy source.A major difficulty that needs to be mastered in this degradation process is the insolubility of crystalline cellulose in water.Several ionic liquids (ILs) have been shown to dissolve water-insoluble cellulose and/or wood.Among these is 1-butyl-3methylimidazolium chloride ([BMIM]Cl) [1,2].Hence, ILs could be used as a reaction medium for the enzymatic hydrolysis of cellulose, if cellulases were shown to be active and stable in the presence of high IL concentrations.To date only a limited number of cellulases have been described as being

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active in the presence of ILs.The majority of cellulases, including a preparation from Trichoderma reesei, appear to be inactive in the presence of IL concentrations above 20% (v/v) [3,4].In contrast, some thermostable cellulases have shown high activity in the presence of about 30% (v/v) IL [5,6].Nevertheless, the stability of cellulolytic activity in the presence of high IL concentrations during relevant time periods remains to be demonstrated.Fungal enzymes are often more stable due to glycosylation, and have been shown to be persistent in the presence of proteases, high temperatures, and surfactants [7][8][9].

Results and Discussion
The cellulases derived from Trichoderma reesei and T. viride were almost inactive in the presence of 30% (v/v) of all tested ILs (Figure 1), whereby 30% (v/v) IL was chosen, as it turned out to be a critical concentration in previous studies [3].The Aspergillus niger preparation was only noticeably active in the presence of [BMIM]Cl, but the cellulase derived from Aspergillus sp.showed high activities in the presence of 30% (v/v) of all tested ILs, and was altogether the most active and IL-tolerant enzyme preparation (Figure 1).For this reason, this extract was chosen for the evaluation of its long term stability.Correspondingly, the extract was co-incubated with the respective ILs (60% (v/v)) at room temperature, and the cellulolytic activity was measured after up to 63 days, in the presence of 30% (v/v) IL at 37 °C.During this time the enzyme showed stable activities in the presence of

and [EMIM]
[OTF], after a decrease to about 50% relative activity in the first days.For example, 17 U/mg (50% relative activity) were observed after 63 days in the presence of [EMIM] [OTF] (Figure 2) and 12 U/mg (29% relative activity) in the presence of [BMIM]Cl, an IL that has been shown to dissolve crystalline cellulose.To our knowledge, in comparison with bacterial as well as fungal cellulases, the observed high activities over a long time period represent by far the highest and longest stability of cellulolytic activity in the presence of high IL concentrations that have been described.Reasons for this high activity and stability might be that the cellulase extract of Aspergillus sp. is a mixture of different cellulases that work concertedly.Furthermore it has been shown that IL tolerance can be correlated to thermostability and halotolerance [5].Interestingly, different Aspergillus species have been shown to be halotolerant [10].

Conclusions
The observed high activity and stability of Aspergillus sp.cellulase in the presence of high IL concentrations raises hope that an industrial process employing cellulose for the generation of glucose and subsequent molecules via enzymatic hydrolysis in ionic liquids is possible.Future plans for achieving this goal would be tests on crystalline cellulose and IL treated natural cellulosic substrates like switchgrass or wood.The increase of IL content in the reactions would also be a future prospect.

Figure 1 .
Figure 1.Specific activity of cellulase extracts of A. niger, A. sp., T. reesei and T. viride in the presence and absence of 30% (v/v) IL at 37 °C after 30 min.

Figure 2 .
Figure 2. Stability of specific activity of cellulase extracts of A. niger, A. sp., T. reesei and T. viride after incubation in the presence and absence of 60% (v/v) ILs at room temperature; activity was measured in 30% IL (v/v) at 37 °C after 30 min.