Evaluation of deep eutectic solvents as new media for (original) (raw)
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Process Biochemistry, 2012
This study aimed at analyzing the advantages and limitations of several deep eutectic solvents (DESs) as 'green solvents' for biotransformation using immobilized Candida antarctica lipase B as catalyst. The transesterification of vinyl laurate was chosen as model reaction and the influence of substrate polarity was assessed using alcohols of various chain lengths. Results showed that grinding of immobilized lipase was essential parameters for good lipase activity. Moreover, in our model reaction some hydrogen-bond donor component from the DES can compete with the alcoholysis reaction. Indeed, side reactions were observed with DES based on dicarboxylic acid or ethylene glycol, leading to some limitations of their use. However, the results showed that other DESs such as choline chloride:urea and choline chloride:glycerol could exhibit high activity and selectivity making them promising solvents for lipase-catalyzed reactions. Finally, the best DES's specific activity -and stability up to five days incubation time -were analyzed and compared with conventional organic solvents. Experiments revealed that iCALB is less influenced by the chain length of alcohol in DES than organic solvents and it is preserves its activity with minimally destructive to protein structure.
Shedding Light on Lipase Stability in Natural Deep Eutectic Solvents
Chemical and Biochemical Engineering Quarterly, 2018
This study presents the potential role of natural deep eutectic solvents (NADESs) in a lipase-catalyzed hydrolysis reaction as both a co-solvent in an aqueous solution and as a main solvent. Ammonium salts such as choline chloride (ChCl) were paired with different hydrogen bond donors such as glycerol and malonic acid and sugars (glucose, fructose and sucrose). The hydrolysis of p-nitrophenyl palmitate by six different lipases: lipase from porcine pancreas (PR), lipase from Candida rugosa (CR), Amano lipase PS, from Burkholderia cepacia (AM), lipase from Rhizopus niveus (RN), lipase acrylic resin from Candida antartica (ARC), lipase B Candida antartica immobilized on Immobead 150, recombinant from Aspergillus oryzae (CALB), were tested in five NADESs. The results showed that NADES3 prepared from ChCl/sucrose was the most promising solvent as it enhanced the activities of both CALB and lipase from porcine pancreas to 355 % and 345 %. The kinetics investigation confirmed the higher catalytic efficiency (k cat /K m) of lipases in the 40 % of (NADES3) and compared with the aqueous form. The trend achieved by NADES may be a promising approach for applications and further perspectives as genuinely green industrial solvents.
Green Chemistry, 2013
This work reports the first lipase-catalyzed reactions between substrates of different polarities using deep eutectic solvents as a medium. The model reaction consisted of a lipophilization process based on the alcoholysis of phenolic esters using immobilized Candida antarctica lipase B as a biocatalyst. Results showed that water could dramatically improve the lipase activity and change the reactivity of phenolic substrates. Indeed, very low conversions (<2%) were observed in pure DES, whereas in DES-water binary mixtures, quantitative conversions were achieved. After investigating the role of various parameters, such as the substrate concentration and ratio, pH or thermodynamic activity of water, the effect of the presence of water in pure DES based on urea or glycerol was discussed. In this paper, we propose new perspectives for the enzymatic modification of polar substrates using this novel generation of green, inexpensive and easy-to-handle solvents.
Screening of lipase carriers for reactions in water, biphasic and pure organic solvent systems
Acta biochimica Polonica, 2014
In bioprocesses lipases are typically used in immobilized form, irrespective of type of reaction systems, to ensure an even distribution of catalysts in water restricted media and/or to facilitate separation and reuse. In these studies we report on the selection of appropriate enzyme-carrier preparation for hydrolysis reaction in aqueous and biphasic systems and transesterification in organic solvent. For this Candida rugosa lipase was bound by adsorption or covalent attachment onto various carriers to give 24 preparations. Selection of proper preparation was based on reactivity, thermal stability (4 h at 60°C), possibility of drying and operational stability in 17-23 successive batch processes of 4-nitrophenyl palmitate hydrolysis in water. Activity of preparations varied from 20 to 5100 U∙mL(-1) but the most stable preparations were those of moderate activity: bound by adsorption or covalent attachment to NH₂-Kieselgel or acrylic carrier (retained activity over 90%). Selected prep...
Influence of Betaine- and Choline-based Eutectic Solvents on Lipase Activity
Current Biochemical Engineering
BACKGROUND: Eutectic solvents are a mixture of two compounds which possess a lower melting temperature than compounds by themselves at low temperatures (below 100ºC), using quaternary ammonium salts, as choline and betaine, and organic acids, polyols and amides as hydrogen bond donors. These solvents can be an alternative as non-aqueous media for enzymatic reactions, mainly using lipases. The objective of this work is to evaluate enzymatic activity and stability of commercial lipases), immobilized or at free form (Thermomyces lanuginosus: Lipozyme TL IM, iTL and Lipolase 100 L, fTL; Candida antarctica: Novozym 435, iCALB; Novozym 735, iCALA and Novozym CALB L, fCALB; and a phospholipase (Lecitase Ultra), in the presence of eutectic solvents (choline chloride (ChCl):urea, ChCl:glycerol, betaine hydrochloride (BeHCl):urea and BeHCl:glycerol. METHODS: Initially, lipases were maintained by 2 hours at solutions of choline and betaine-based eutectic solvents (1 to 20% m/m) at 25ºC, compar...
Journal of Molecular Catalysis B: Enzymatic, 2001
Following a simple and quick treatment based on dissolving the crude lipase of Candida rugosa in different percentages Ž. Ž. vrv of several polar organic solvents methanol, ethanol, acetone, 1-propanol, 2-propanol, 1-butanol and 2-butanol , and a further dialysis, different preparations with enhanced activity and thermal stability were obtained. This improved catalytic Ž. behaviour was observed in aqueous media more efficient triglyceride hydrolysis as well as in anhydrous organic solvents Ž. better enantioselectivity andror yield in the esterification of R-and S-ketoprofen. Combination of this treatment with organic solvents with a correct control of water activity in the reaction media allowed a much better catalytic behaviour, dramatically increasing the reaction yield and the enantiodiscrimination ability in the esterification of S-ketoprofen vs. the R counterpart.
Tetrahedron: Asymmetry, 1993
Chiral resolutions of racemic 3-hydroxy esters were performed in organic phases with lipases from PseudomoMs cepacia, Ckomobacterium viscosum and Porcine pancreas. The reaction conditions have been optimized with 3-hydroxy octanoic acid methyl ester. Different organic solvents have been tested showing a tendentious correlation with the hydrophobicity of the solvents expressed as log P. The reaction time was shortened six fold by using irreversible acylating agents. We have found solvent type, lipase type and acylating agent acting as tools for changing the enantioselectivity. Lipase from Pseuahonas cepacia was lyophilized at different pH and the influence of the amount of water added was investigated, resulting in the highest activity at the pH optimum and a denaturation of the lipase above 1 % water (w/wb,&. The water activity was measured on-line with a humidity sensor. Water activities greater than 0.4 led to a decrease in enantioselectivity and reaction rate. In the optimized system the resolutions of other 3-hydroxy esters were tested. Aliphatic compounds reacted with lower enantioselectivity, only the substrates could be isolated in high enantiomeric purity. In contrast, aromatic 3-hydroxy esters were acylated by lipases with high stereoselectivity. A model of the active site of lipase from Pseudomonas sp. explained these experimental observations.
A spectrophotometric transesterification-based assay for lipases in organic solvent
Analytical Biochemistry, 2009
A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30°C or in decane at 50°C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification (R 2 = 0.999) and to results from p-nitrophenyl palmitate (R 2 = 0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 ± 350 U g À1 ) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 ± 0.04 U g À1 ). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.
Covalent immobilization of lipase from Candida rugosa on Eupergit®
Acta periodica technologica, 2005
An approach is presented for the stable covalent immobilization of lipase from Candida rugosa on Eupergit ® with a high retention of hydrolytic activity. It comprises covalent bonding via lipase carbohydrate moiety previously modified by periodate oxidation, allowing a reduction in the involvement of the enzyme functional groups that are probably important in the catalytic mechanism. The hydrolytic activities of the lipase immobilized on Eupergit ® by two conventional methods (via oxirane group and via glutaraldehyde) and with periodate method were compared. Results of lipase assays suggest that periodate method is superior for lipase immobilization on Eupergit ® among methods applied in this study with respect to both, yield of immobilization and hydrolytic activity of the immobilized enzyme.
The role of the reaction medium in lipase-catalyzed esterifications and transesterifications
Chemistry and Physics of Lipids, 1998
The nature of the reaction medium (different organic solvents and supercritical fluids) markedly influenced the microbial lipase activity in esterification and transesterification reactions employing as substrates natural and synthetic compounds in terms of reaction rates and lipase enantioselectivity. The experimental data obtained show that while there are no substantial correlations between enantioselectivity and some physicochemical characteristics of the solvent as hydrophobicity and dielectric constant, the solvent polarity and hydrophobicity is able to modulate greatly lipase activity. The possible effects of the solvent characteristics on the catalytic performance of the enzymes are discussed and a rationale is proposed to explain the results obtained.