Isoamyl acetate synthesis in imidazolium-based ionic liquids using packed bed enzyme microreactor

The acylation of isoamyl alcohol with acetic anhydride catalyzed by immobilized Candida antarctica lipase B was studied in ionic liquids (ILs) based on quaternary imidazolium cations with alkyl, alkenyl, alkynyl, benzyl, alkoxyl or N-aminopropyl side chains. Among the tested ILs, the highest enzyme activity together with the highest isoamyl acetate yield were obtained in [C₇mmim][Tf₂N]. No loss of lipase B activity was observed during one-month incubation in this hydrophobic IL without the presence of substrates. Isoamyl acetate synthesis using [C₇mmim][Tf₂N] as solvent was further studied in a continuously operated miniaturized enzymatic packed bed reactor at various flow rates and temperatures. Up to 92% isoamyl acetate yield could be obtained within 15 min by using 0.5 M acetic anhydride and 1.5 M isoamyl alcohol inlet concentrations at 55 °C, corresponding to the volumetric productivity of 61 mmol l^?1 min^?1, which to the best of our knowledge is the highest reported so far for this reaction. No decrease in productivity was experienced during the subsequent runs of continuous microbioreactor operation performed within 14 consecutive days. The benefits of reactor miniaturization along with the green solvent application were therefore successfully exploited for the development of a sustainable flavour ester production.     

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C(12)mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C(2)mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C(12)mim]Trp have more stability than AuNPs synthesized at [C(2)mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Theoretical study on the interactions between methanol and imidazolium-based ionic liquids

To better understand the property of the binary systems composing of imidazolium salt, [emim]⁺Aˉ (A=Clˉ, Brˉ, BF₄ˉ, and PF₆ˉ) and methanol, we have investigated in detail the interactions of methanol molecule with anions Aˉ, cation [emim]⁺, and ion pair [emim]⁺Aˉ of several ionic liquids (ILs) based on 1-ethyl-3-methylimidazolium cation by performing density functional theory calculations. It is found that H-bonds are universally involved in these systems, which may play an important role for the miscibility of methanol with imidazolium-based ILs. The interaction mechanisms of methanol molecule with anion and cation are found to be different in nature: the former mainly involves LP_X-s_{O - H}^* \sigma_{{O - H}}^{*} interaction, while the latter relates with the decisive orbital overlap of the type of LP_O-s_{C - H}^* \sigma_{{C - H}}^{*} . Based on the present calculations, we have provided some reasonable interpretations for properties of the binary mixtures of ILs and alcohol and revealed valuable information for the interaction details between ILs and alcohols, which is expected to be useful for the design of more efficient ILs to form superior solvent system with alcohol.     

Scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters

The scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters have been studied. Herein, we report the effects of modifying the structure of the imidazolium cation and how important the choice of counter ion becomes on model glycosylation reactions of thioglycosides at room temperature in the presence of N-iodosuccinimide (NIS).     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.     

Sugarcane bagasse oxidation using a combination of hypochlorite and peroxide

Reactive oxygen species (ROS) such as singlet oxygen ((1)O(2)), superoxide (O(2)(-)), hydroxyl radicals (OH(*)), or hypochlorite ion (OCl(-)), can remove both hemicellulose and lignin from lignocellulose. Ox-B (US Patent 6,866,870), an ROS producing solution containing sodium hypochlorite and hydrogen peroxide, was investigated for its ability to oxidize sugarcane bagasse. Treatment with equivalent amounts of hypochlorite produced similar results. Ox-B differentiated from hypochlorite when low concentration treatments were used and they were followed by a caustic wash. Cellulases hydrolyzed 80-100% of the cellulose present after Ox-B/caustic treatment compared to 40% or less for NaOCl/caustic treatment. Ox-B treatment was temperature independent and complete within 3h. It was pH dependent, with best results obtained when the pH was controlled at 8. Although highly effective, in order for Ox-B to be industrially feasible for alcohol production, the chemical cost must decrease to justify its use.     

Imidazolium-based ionic liquids for cellulose pretreatment: recent progresses and future perspectives

Applied Microbiology and Biotechnology - As the most abundant biomass in nature, cellulose is considered to be an excellent feedstock to produce renewable fuels and fine chemicals. Due to its...     

Insights into the effect of imidazolium-based ionic liquids on chemical structure and hydrolytic activity of microbial lipase

Bioprocess and Biosystems Engineering - This work studied the effect of the cation alkyl chain length of 1-alkyl-n-methylimidazolium chloride ([Cnmim]Cl)-based ILs on the activity of Aspergillus...     

Mild pretreatment and enzymatic saccharification of cellulose with recycled ionic liquids towards one-batch process

The development of second-generation bioethanol involves minimizing the energy input throughout the processing steps. We report here that efficient ionic liquid pretreatments of cellulose can be achieved with short duration times (20min) at mild temperature (45°C) with [Emim](+)[MeO(H)PO(2)](-) and at room temperature (25°C) with [Emim](+)[CH(3)COO](-). In these conditions, yields of glucose were increased by a factor of 3. In addition, the recycling of these two imidazolium-based ILs can be performed in maintaining their efficiency to pretreat cellulose. The short time and mild temperature of cellulose solubilization allowed a one-batch processing of [Emim](+)[MeO(H)PO(2)](-) IL-pretreatment and saccharification. In the range from 0 to 100% IL in an aqueous enzymatic medium, the glucose yields were improved at IL proportions between 10 and 40%. The maximum yield at 10% IL is very promising to consider one batch process as efficient as two-step process.     

Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids

The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water.     

Study of thermodynamic properties of imidazolium-based ionic liquids and investigation of the alkyl chain length effect by molecular dynamics simulation

In this paper, structural and dynamical properties of five imidazolium-based ionic liquids (ILs) [amim]Br (a = methyl, ethyl, butyl, pentyl, hexyl) were studied by molecular dynamics simulations. United atom force field (UAFF) has been used for the representation of the interaction between ions. Good agreement with experimental data was obtained for the simulated density based on the UAFF. The calculated densities gradually decrease with an increase in the length of alkyl side chain, which is a result of weakening the electrostatic interaction between ions. The simulated heats of vaporisation are higher than that of non-ILs and decrease with an increase in temperature. Radial distribution function (RDF) was employed to analyse the local structure of ILs. Cation–anion RDFs show that the anions are well organised around the cation in two shells (0.41 and 0.6 nm). The velocity autocorrelation functions of the anion and cations show that the relaxation time increased with an increase in the length of the alkyl side chain. The diffusion coefficients of ions were calculated by mean square displacement of the centre of mass of the ions at 400 K. The calculated diffusion coefficients using UAFF agree well with other all atom force fields. Also diffusion coefficients decrease with an increase in the length of the alkyl side chain. The calculated transference numbers show that the cation contributes more than anion in the electrical current. The diffusion coefficients increase with temperature.     

Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid‐liquid extraction

Flurbiprofen is a kind of nonsteroidal anti‐inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S‐flurbiprofen shows good performance on clinic anti‐inflammatory treatment, while R‐enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S‐flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2‐dichloroethane as organic solvent, 1‐butyl‐3‐methylimidazole L‐tryptophan ([Bmim][L‐trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L^?1, and [Bmim][L‐trp] concentration was 0.02 mol L^?1. Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale‐up of the extraction process.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.     

Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse

Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could beused as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulasecomplex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkalinetreatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatmentmethod. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production ofCMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated andpurified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single bandcorresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°Cand pH 5.0-7.5.     

Analysis of enzyme kinetics using electrospray ionization mass spectrometry and multiple reaction monitoring: fucosyltransferase V

An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.     

Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO₂) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD⁺). Oxidative stress, as a resultof which the NAP(P)⁺/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO₂ to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO₂ levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.