cellulase and 1-butyl-3-methylimidazolium-chloride

Thermostability and stability in ionic liquids are essential properties of cellulases that are applied in industrial processes of bioconversion. Engineering of protein surface of endoglucanase II from Penicillium verruculosum was used to improve the enzyme thermostability and stability in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The engineering was based on analysis of the protein surface topography and enhanced by multiple sequence alignment and ΔΔG calculations. In the case of the thermostability, half-life time was improved in 1.3-1.6 times at 70 °C and 1.2-1.4 times at 80 °C. In the case of the stability in [Bmim]Cl, the residual activity after 72 h of incubation in the presence of [Bmim]Cl (50 g/L, 50 °C, pH 4.5) was 1.7-1.9 times greater for the tailored enzyme. The yield of reducing sugars after enzymatic hydrolysis of aspen wood pretreated with [Bmim]Cl was 10-20% higher with the tailored endoglucanase.

Topics: Cellulase; Engineering; Imidazoles; Ionic Liquids; Penicillium

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations.

Topics: Actinobacteria; Cellulase; Enzyme Inhibitors; Enzyme Stability; Imidazoles; Ionic Liquids; Molecular Dynamics Simulation; Structure-Activity Relationship

Acid enhanced ionic liquid (IL) 1-n-butyl-3-methylimidazolium chloride ([C4 mim]Cl) pretreatment has shown great potential for boosting the yield of sugars from biomass cost-effectively and environmental-friendly. Pretreatment with shorter processing time will promote the commercial viability. In this work, pretreatment of reduced Amberlyst catalysis time of 34 min was demonstrated to be the most effective among time-varying pretreatments, evidenced by partial removal of hemicellulose and cellulose crystal transformation of Arundo donax Linn. A higher fermentable sugar concentration of 10.42 g/L (2% substrate) was obtained after 72 h of saccharification than the others. Total processing time to reach 92% glucose yield was cut down to approximately 26 h. Progressive deconstruction of crop cell wall was occurred with increased catalysis time by gradual releasing of H3O(+) of Amberlyst. However, vast lignin re-deposited polymers on fibers could hinder further enzymatic hydrolysis. These discoveries provide new insights into a more economic pretreatment for bioethanol production.

Topics: Acids; Biomass; Carbohydrates; Catalysis; Cellulase; Cellulose; Crystallization; Fermentation; Hydrolysis; Imidazoles; Ionic Liquids; Ions; Lignin; Poaceae; Polysaccharides; Spectroscopy, Fourier Transform Infrared

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved.

Topics: Biotechnology; Cellulase; Enzymes, Immobilized; Graphite; Hydrolysis; Imidazoles; Ionic Liquids; Lignin; Nanostructures; Oryza; Oxides; Plant Shoots; Polyethylene Glycols

The aim of this work was to study an environmentally friendly method for pretreating rice straw by using 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) as an ionic liquid (IL) assisted by surfactants. Different temperatures, reaction times, and surfactant concentrations were studied. Compared with [BMIM]Cl only pretreatment, the addition of 1% sodium dodecyl sulfate (SDS) and 1% cetyl trimethyl ammonium bromide (CTAB) increased lignin removal to 49.48% and 34.76%, respectively. Untreated and pretreated rice straw was thoroughly characterized through FTIR, XRD, and FE-SEM. Cellulose crystallinity and surface morphology of the rice straw were substantially altered after surfactant-assisted IL pretreatment. In conclusion, surfactant-assisted IL pretreatment is an effective method for producing fermentable sugars from lignocellulosic substrates.

Topics: Carbohydrate Metabolism; Cellulase; Cellulose; Crystallization; Hydrolysis; Imidazoles; Ionic Liquids; Oryza; Spectroscopy, Fourier Transform Infrared; Surface-Active Agents; Temperature; Waste Products; X-Ray Diffraction

The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains.

Topics: Catalysis; Cellulase; Cellulose; Hydrolysis; Imidazoles; Ionic Liquids; Lipid Bilayers; Liposomes; Phosphatidylcholines; Phosphatidylglycerols; Trichoderma

In this study, a pretreatment by combining acidified aqueous ionic liquid 1-butyl-3-methylimidazolium chloride (IL [Bmim]Cl) solution with dilute NaOH extraction was employed to pretreat high crystallinity index (CrI) of corn stover before its enzymatic saccharification. After NaOH extraction, [Bmim]Cl-HCl-water (78.8:1.2:20, w/w/w) media was used for further pretreatment at 130 °C for 30 min. After being enzymatically hydrolyzed for 48 h, corn stover pretreated could be biotransformed into reducing sugars in the yield of 95.1%. Furthermore, SEM, XRD and FTIR analyses of untreated and pretreated corn stovers were examined. It was found that the intact structure was disrupted by combination pretreatment and resulted in a porous and amorphous regenerated cellulosic material that greatly improved enzymatic hydrolysis. Finally, the recovered hydrolyzates obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. In conclusion, the combination pretreatment shows high potential application in future.

Topics: Carbohydrate Metabolism; Cellulase; Crystallization; Ethanol; Fermentation; Hydrolysis; Imidazoles; Sodium Hydroxide; Waste Products; Water; Zea mays

Recently, application of ionic liquids (ILs) has received much attention due to their special solvency properties as a promising method of pretreatment for lignocellulosic biomass. Easy recovery of ionic liquids, chemical stability, temperature stability, nonflammability, low vapor pressure, and wide liquidus range are among those unique properties. These solvents are also known as green solvents due to their low vapor pressure. The present study was set to compare the effect of five different ILs, namely, 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]), 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-ethyl-3-methyl imidazolium diethyl phosphate ([EMIM][DEP]), 1-allyl-3-methylimidazolium chloride ([AMIM][Cl]), and 1-ethyl-3-methylimidazolium-hydrogen sulfate ([EMIM][HSO₄]), on corn stover in a bioethanol production process. The performance of ILs was evaluated based on the change observed in chemical structure, crystallinity index, cellulose digestibility, and glucose release. Overall, [EMIM][Ac]-pretreated corn stover led to significantly higher saccharification, with cellulose digestibility reaching 69% after 72 hr, whereas digestibility of untreated barley straw was measured at only 21%.

Topics: Allyl Compounds; Aspergillus niger; beta-Glucosidase; Biotechnology; Cellulase; Cellulose; Glucose; Imidazoles; Ionic Liquids; Organophosphates; Trichoderma; Zea mays

To improve the cellulose digestibility of energy crop Arudo donax Linn. with cost-efficient, a novel pretreatment of protic acid resin Amberlyst 35DRY catalyzed inexpensive ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) was developed in this work. The pretreatment was performed at 160°C with [C4mim]Cl for 1.5h, followed by Amberlyst 35DRY catalyzed at 90°C for 1h. The IL-Amberlyst pretreatment was demonstrated to be effective, evidenced by the reduction in cellulose crystallinity (31.4%) and increased porosity caused by extensive swelling the undissolved biomass and partial depolymerization of the longer cellulose chain of the dissolved biomass by Amberlyst. Consequently, a higher glucose yield (92.8%) was obtained than for the single [C4mim]Cl pretreatment (42.8%) at an enzyme loading of 20 FPU/g substrate. Overall, the enhanced pretreatment was competitive by using inexpensive and recyclable IL-Amberlyst 35DRY pretreated system with shorter processing time and reduced enzyme usage.

Topics: Anion Exchange Resins; Biomass; Cellulase; Crystallization; Hydrolysis; Imidazoles; Poaceae

The present study investigated the impact of regeneration process on the crystalline structure and enzymatic hydrolysis behaviors of microcrystalline cellulose (MCC) regenerated from ionic liquid 1-butyl-3-methylimidazolium chloride. The crystalline structures of these regenerated samples were analyzed by X-ray diffraction. Results suggested that almost amorphous cellulose was obtained by regenerating MCC in acetone (DRC-a), while partial cellulose II structure could be found in these regenerated samples from water and ethanol. Additionally, the enzymatic hydrolysis behaviors of MCC and its regenerated samples were comparatively studied. Results showed that above 90% of cellulose could be converted into glucose within 4h for DRC-a and regenerated cellulose without drying (WRC-w) as compared to that of MCC (9.7%). Therefore, the regeneration process could significantly influence the crystallinity and digestibility of cellulose.

Topics: Aspergillus niger; beta-Glucosidase; Cellulase; Cellulose; Hydrolysis; Imidazoles; Ionic Liquids; Trichoderma; X-Ray Diffraction

The three highest hydrolysis-capacity-value isolates of Bacillus subtilis (A 002, M 015, and F 018) obtained from Thai higher termites, Microcerotermes sp., under different isolation conditions (aerobic, anaerobic, and anaerobic/aerobic) were tested for cellulase activities--FPase, endoglucanase, and β-glucosidase--at 37 °C and pH 7.2 for 24 h. Their tolerance to an ionic liquid, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), was also investigated. The results showed that the isolate M 015 provided the highest endoglucanase activity whereas the highest FPase and β-glucosidase activities were observed for the isolate F 018. The isolate F 018 also showed the highest tolerance to [BMIM]Cl in the range of 0.1-1.0 vol.%. In contrast, the isolate A 002 exhibited growth retardation in the presence of 0.5-1.0 vol.% [BMIM]Cl.

Topics: Adaptation, Physiological; Animals; Bacteria; Bacterial Proteins; Cellulase; Imidazoles; Ionic Liquids; Isoptera; Temperature; Thailand