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Enzymatic esterification of fatty acid esters by tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase
Abstract
Enzymatic production of fatty acid esters from the esterification of oleyl alcohol with various fatty acids was investigated by using two new tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase (ILs-CRL) as biocatalysts in hexane. Both enzyme derivatives were prepared by mixing Candida rugosa lipase with tetraethylammonium l-histidinate (IL1) and tetraethylammonium l-asparaginate (IL2). The ILs-CRL system containing the equivalent protein concentration as in CRL showed higher esterification activity especially on medium chain fatty acids (C 12 –C 16) as compared to non-coated CRL. Hydrophilicity of ILs may play an important role in hydrogen bonding with enzyme surface and consequently stabilize the ILs-CRL.
... Moreover, the enzymes can act on a broad spectrum of substrates, enantioselective as well as performing specific biotransformation (Treichel et al., 2010). Among others, Candida rugosa lipase (CRL) has been prevalently used in oil hydrolysis, transesterification, esterification and interesterification as well as catalyzing long chain of fatty acid esters (Abdul Rahman et al., 2012). However, the free form of CRL is (a) often unstable, (b) exhibiting low activity in organic solvents and (c) prone to deactivation when exposed to prolonged exposure of high temperature and extreme pH (Zhou et al., 2012). ...
... The reaction was initiated by addition of free CRL or CRL-MWCNTs nanoconjugates and the mixture was stirred at 200 rpm in a paraffin oil bath. Ester accumulation was monitored by removing aliquots (1 mL) of the reaction mixture at designated intervals and titrated with NaOH (0.02 M) using phenolphthalein as the indicator (Abdul Rahman et al., 2012). The methyl oleate obtained was expressed in percentage conversion i.e. percent of oleic acid converted versus the total acid in the reaction mixture. ...
Acid functionalization of multi-walled carbon nanotubes (F-MWCNTs) using a mixture of HNO3 and H2SO4 (1:3, v:v) was used as support materials for the adsorption of Candida rugosa lipase (CRL) as nanoconjugates (CRL-MWCNTs) for producing methyl oleate. To evaluate the competency of the CRL-MWCNTs nanoconjugates, parameters viz. reaction time, surfactant as well as thermostability and reusability were investigated. The characterization of CRL-MWCNTs nanoconjugates using Fourier transform infrared spectroscopy, Field Scanning Electron Microscopy and Transmission Electron Microscopy revealed successful attachment of CRL onto the F-MWCNTs. Utilization of CRL-MWCNTs nanoconjugates resulted in a higher acid conversion in the synthesis of methyl oleate (79.85% at 11 h of reaction time) when compared with the free CRL i.e. an approximately 1.5-fold improvement over the free CRL. The highest percentage of esterification (83.62%) was observed following the use of nonionic surfactant when compared with the anionic and cationic ones. The CRL-MWCNTs nanoconjugates could be used up to 5 cycles, retaining 50% of its residual activity. Since the preparation of the CRL-MWCNTs nanoconjugates was facile and cheap while producing reasonable yield, the CRL-MWCNTs nanoconjugates developed here were found as promising biocatalysts for the production of methyl oleate.
... He achived the highest conversion of oleic acid (50%) for ionic liquid [bmim]Ac and sodium di-2-ethylhexysulfosuccinate as the surfactant. In turn, Rahman et al. used as a catalyst lipase immobilized on amino acid ionic liquid (Scheme 13) [92]. ...
... The esterification was carried out in the presence of Candida rugosa lipase in the mixture of [bmim]Ac / [bmim]BF 4 .Esterification of starch with palmitic acid leads to a product having a higher, in comparison to starch, hydrophobicity, but at the expense of reduction heat resistance and lower crystallinity. Aminoacidic ionic liquid (Rahman[92]).Lue et al. investigated the possibility of using various ionic liquids in the synthesis of esters of fatty acids (palmitic or oleic acid) and selected flavonoids (rutin, esculin etc.)[110].Novozyme ® 435 was used as an enzymatic catalyst for this reaction. He concluded, that the best results are obtained for the hydrophobic ionic liquids i.e. [bmim]BF 4 , [bmim]PF 6 , [bmim]TfO and [bmim]NTf 2 . ...
This review is the first attempt to collect papers about possible use of ionic liquids for the chemical processing of oleochemical raw materials. The main focus is put on the processes based on vegetable oils, due to the fact that the majority of published reports in this area were dedicated to the synthesis of fatty acid methyl esters (FAME, biodiesel), which are originated mainly from vegetable oils. A significant amount of reports, including several review papers, were dedicated to the use of ionic liquids in the synthesis of biodiesel, but also other processes such as the synthesis of fatty acid esters or reactions with the use of unsaturated bonds still remain underrated. However, it is important enough to justify their collection in one paper in order to adequately appreciate the potential use of ionic liquids and benefits resulting from the chemistry of vegetable raw materials.
... Another route is to immobilize the enzyme in an IL that is solid at room temperature, forming the so-called IL-coated enzymes. In this case, it is required to melt the IL before enzyme addition and to solidify it afterwards by cooling [47][48][49]. Too thick solid IL coatings were associated to diffusional mass transfer limitations. ...
Poly(ionic liquid)s are interesting materials for CO2 separation due to their affinity towards CO2. Carbonic anhydrase, as an enzyme that catalyzes the hydration reaction of CO2, represents tremendous opportunities for enhanced CO2 capture in aqueous solutions. Yet, when used as a free enzyme solubilized in the liquid medium, it has limited practicability. Here, we report the study on carbonic anhydrase immobilization in poly(ionic liquid)s by entrapment. Due to limited enzyme solubility in the hydrophobic ionic liquid monomer solution, carbonic anhydrase was suspended instead of solubilized prior to bulk polymerization. The immobilization protocol was optimized to maximize the catalytic efficiency of the resulting solid by evaluating the effect of lyophilized suspension hydration and particle size. Immobilized carbonic anhydrase properties were investigated by measurement of kinetic parameters, reusability, thermal and storage stability. Using p-NPA hydrolysis as a model reaction, kinetic parameters were 0.22 s⁻¹ mM⁻¹ (kcat/Km) for free enzyme and 0.06 s⁻¹ mM⁻¹ for the immobilized biocatalyst, indicating a decrease of activity upon immobilization. Immobilized enzyme, however, presented better storage stability than free enzyme as after one month the activity did not change. In the case of dissolved carbonic anhydrase, it decreases by about 30%. The entrapped enzyme could be utilized in 5 consecutive cycles of CO2 hydration while keeping a relative activity superior to 60%.
... According to Rahman et al. (2012) [9], CILCE could be prepared from tetraethylammonium amino acid ILs and Candida rugosa lipase; and later be used to catalyze the esterification of oleyl alcohol with various fatty acids. CILCE is a useful catalyst in the biocatalysis of organic solvents. ...
The utilization of renewable feedstock is paramount for the sustainable production of materials, and not merely for the chemical industry. Ionic liquid (IL) catalytic conversions of mono-, di-, and polysaccharides give 5-hydroxymethylfurfural (HMF) – a versatile intermediate. In this study, local bamboo was the source of biomass. Three different anions of ILs had a noticeable effect on the HMF yield. The chromium chloride catalyst was found to be the most effective in the 1-butyl-3-methylimidazolium chloride system among all tested ILs since it provided a HMF yield of 48%, with more than 95% of glucose
conversion after 3 h at 120°C.
... The high activity, substrate specificity and non-toxicity nature of CRL makes it the popular choice among industrial enzymes, apart from the fact that the enzyme is reasonably priced and easily available (Ferrer et al. 2001;Barriuso et al. 2016;Jafarian et al. 2018). CRL has been used to catalyse a number of sustainable and environmentally-friendly processes involving hydrolysis, esterification as well as inter-and trans-esterification of triglycerides (Abdul Rahman et al. 2012;Barriuso et al. 2016); all of which occurred at the water-oil interface (Grochulski et al. 1993;Zhao et al. 2008) that can be highly similar to the condition of water-immersed latent fingerprints. Moreover, CRL has a wide range of substrate specificity that includes fatty acids (chain lengths of C4, C8, C10 and C12) (Schmitt et al. 2002) and lipids, attributable to its three catalytic triad viz. ...
Objective
Optimisation of the green novel nanobio-based reagent (NBR) for rapid visualisation of groomed fingerprints on wet non-porous substrates using response surface methodology and assessment of its stability and sensitivity were attempted for forensic applications.
Results
Scanning electron microscopy images demonstrated successful attachments of NBR onto the constituents of fingerprints on the substrates. The highest average quality of visualised fingerprints was attained at the optimum condition (100 mg of CRL; 75 mg of acid-functionalised multi-walled carbon nanotubes; 5 h of immobilisation). The NBR produced comparable average quality of fingerprints with the commercially available small particle reagent, even after 4 weeks of storage (without any preservatives) in both chilled and sultry conditions. The NBR was sensitive enough to visualise the increasingly weaker fingerprints, particularly on glass slides.
Conclusion
The optimised novel NBR could be the relatively greener option for visualising latent fingerprints on wet, non-porous substrates for forensic applications.
... The high activity, substrate specificity and non-toxicity nature of CRL makes it the popular choice among industrial enzymes, apart from the fact that the enzyme is reasonably priced and easily available (Ferrer et al. 2001; Barriuso et al. 2016;Jafarian et al. 2018). CRL has been used to catalyse a number of sustainable and environmentally-friendly processes involving hydrolysis, esterification as well as inter-and trans-esterification of triglycerides (Abdul Rahman et al. 2012;Barriuso et al. 2016); all of which occurred at the water-oil interface (Grochulski et al. 1993;Zhao et al. 2008) that can be highly similar to the condition of water-immersed latent fingerprints. Moreover, CRL has a wide range of substrate specificity that includes fatty acids (chain lengths of C4, C8, C10 and C12) (Schmitt et al. 2002) and lipids, attributable to its three catalytic triad viz. ...
Objective
Optimisation of the green novel nanobio-based reagent (NBR) for rapid visualisation of groomed fingerprints on wet non-porous substrates using response surface methodology and assessment of its stability and sensitivity were attempted for forensic applications.
Results
Scanning electron microscopy images demonstrated successful attachments of NBR onto the constituents of fingerprints on the substrates. The highest average quality of visualised fingerprints was attained at the optimum condition (100 mg of CRL; 75 mg of acid-functionalised multi-walled carbon nanotubes; 5 h of immobilisation). The NBR produced comparable average quality of fingerprints with the commercially available small particle reagent, even after 4 weeks of storage (without any preservatives) in both chilled and sultry conditions. The NBR was sensitive enough to visualise the increasingly weaker fingerprints, particularly on glass slides.
Conclusion
The optimised novel NBR could be the relatively greener option for visualising latent fingerprints on wet, non-porous substrates for forensic applications.
... is an ubiquitous enzyme which offers a number of advantages as one of the most sought and cheap industrial biocatalysts [29], attributable to its remarkable properties like versatility, nontoxicity, biodegradability and high reactivity [30,31]. Additionally, the specificity and selectivity of CRL depend on its high affinity towards triglycerides and lipids, which makes it a valuable biocatalyst in hydrolysis, esterification, inter-and trans-esterification of fatty acids [32]. Canopied by a polypeptide lid/flap (in a closed form), its active pocket consist of catalytic triad Ser-209 (nucleophile), His-449 (base) and Glu-341 (acid), which are located along the β-strands of the large β-sheet of CRL [33]. ...
Considering the significant evidential values of fingerprints in underwater criminal investigations and the need to visualise them using a user- and environmentally-friendly reagent, development of a novel, rapid and relatively greener nanobio-based reagent (NBR) deemed beneficial. Lipase from the commercial Candida rugosa immobilised onto acid-functionalised multi-walled carbon nanotubes (NBR) was used as the safer and cheap lipid-sensing reagent to visualise groomed whole/split fingerprints on non-porous objects immersed in stagnant tap water for up to 30 days under laboratory-controlled setting. Attenuated Total Reflectance – Fourier Transform Spectrometry, Field Emission Scanning Electron Microscopy and bioinformatics (molecular docking and molecular dynamics simulations) were employed to characterise and confirm the attachment of NBR onto the lipid constituents of wet fingerprints. Chromatographic results further confirmed the presence of n-hexadecanoic and octadecanoic acids on fingerprints up to 30 days of immersion. Thus, NBR may potentially be useful as the future state-of-the-art fingerprint visualisation technology.
... The reuse of the enzymatic biocatalyst was performed in the optimized condition, i.e., without addition of water. Second to Rahman et al. [39], lipase in non-aqueous medium often undergoes reduction of activity, selectivity, and stability. Trentin et al. [29] also observed for soybean oil transesterification using enzymatic catalyst (Novozym 435) in a solvent-free in the reuse that the activity decreases with a drop in ethyl ester conversion, obtaining yields of 6.2% and 3.6% in the third and fourth cycles, respectively. ...
This work aimed to produce ethyl esters from Chlorella vulgaris microalgae biomass, using an immobilized enzymatic catalyst associated with pressurized fluid (propane) by direct transesterification. In order to optimize the ethyl conversion, different temperatures (46.7–68.1 °C) and pressures (59.5–200.5 bar) were applied a central composite design rotational (CCDR) obtaining the high conversion (74.39%) at 50 °C and 180 bar. The molar ratio also was investigated showing conversions ~ 90% using a molar ratio of 1:24 (oil:ethanol). From the best transesterification conditions, 50 °C, 180 bar, 20% enzymatic concentration, and 1:24 oil:ethanol molar ratio were obtained with success 98.9% conversion in 7 h of reaction. The enzyme reuse maintained its activity for three successive cycles. Thus, this simple process was effective to convert microalgal biomass into ethyl ester by direct transesterification and demonstrate high yields.
... Candida rugosa lipase was used as biocatalysts by enzyme coating with the two ILs to catalyse oleyl alcohol esterification with fatty acids in hexane. The coated lipase disclosed an enhanced activity in catalysis than the lipase without coating [65]. ...
Ionic liquids (ILs) - as environmentally friendly “green” solvents- have been progressively used in various reactions as reagents, solvents and co-solvents including lipase-catalysed reactions. In fact, lipase-catalysed reactions in ILs are considered as a “green”-reaction and are more advantageous than chemical methods owing to the easy recovery of the product and the mild conditions of the reactions. The use of lipase in ILs provides many technological advantages compared to conventionally used solvents, such as selectivity enhancement, enzyme stability improvement, higher conversion rate, and better recyclability and recovery system. Nevertheless, in some cases, especially in hydrophilic ILs, lipase exhibits activity reduction when compared with organic solvents. Currently, various attempts have been made to enhance the performance of lipases in ILs. The main objective of this review is to demonstrate recent developments in the technology of using ILs as reaction media for lipase. It is expected that this review might be an inspiration in ILs assisted lipase-catalysed reactions to produce value-added materials including biofuels as well as biodiesel.
... In addition, as pointed out by Wang et al. (2001), the cost involved in enzyme immobilization together with utilization cycles should be taken into account in the global economic analysis of an industrial biodiesel plant. It has been argued by Rahman et al. (2012) that lipases may lose their activity in non-aqueous medium and selectivity and stability may also be affected. Undoubtedly, the possibility of catalyst reuse is crucial for enzymatic process feasibility and accordingly reaction cycles for immobilized enzyme were performed under ultrasound irradiation. ...
Transesterification of non-edible oils using immobilized lipase is a promising process for biodiesel production. Thus, this study aimed to evaluate the enzymatic transesterification of the non-edible Jatropha curcas oil for FAEE production under ultrasound irradiation in a solvent-free system. The effects of enzyme concentration, water concentration, molar ratio of ethanol to oil and ultrasound power on the FAEE conversion have been evaluated. The results show that enzyme concentration and irradiation power have a positive significant effect on FAEE conversion, where an increase in these variables leads to higher conversions. Conversion above 54% of FAEE was achieved with 1.5 hours of reaction time using ultrasound irradiation, reducing reaction time by at least 3 times, when compared with the same experimental conditions without ultrasound irradiation. Results showed that ultrasound can improve reaction conversion mainly by enhancing the mass transfer between the constituents of the reactions.
... The preparations containing the free CRL or CRL-MWCNTs at 3 mg/mL (unless stated otherwise) were reacted in a working volume of 30 mL, and the mixtures were stirred at 200 rpm in a paraffin oil bath. Aliquots (1 mL) of the reaction mixture were withdrawn each hour for 12 h and analyzed following titration with NaOH (0.02 M) using phenolphthalein as an indicator [25]. The methyl oleate obtained was expressed in terms of percentage conversion, i.e., percent of oleic acid converted versus the total acid in the reaction mixture. ...
The chemical production of methyl oleate using chemically synthesized fatty acid alcohols and other toxic chemicals may lead to significant environmental hazards to mankind. Being a highly valuable fatty acid replacement raw material in oleochemical industry, the mass production of methyl oleate via environmentally favorable processes is of concern. In this context, an alternative technique utilizing Candida rugosa lipase (CRL) physically adsorbed on multi-walled carbon nanotubes (MWCNTs) has been suggested. In this study, the acid-functionalized MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) was used as support for immobilizing CRL onto MWCNTs (CRL-MWCNTs) as biocatalysts. Enzymatic esterification was performed and the efficiency of CRL-MWCNTs was evaluated against the free CRL under varying conditions, viz. temperature, molar ratio of acid/alcohol, solvent log P, and enzyme loading. The CRL-MWCNTs resulted in 30-110 % improvement in the production of methyl oleate over the free CRL. The CRL-MWCNTs attained its highest yield (84.17 %) at 50 °C, molar ratio of acid/alcohol of 1:3, 3 mg/mL of enzyme loading, and iso-octane (log P 4.5) as solvent. Consequently, physical adsorption of CRL onto acid-functionalized MWCNTs has improved the activity and stability of CRL and hence provides an environmentally friendly means for the production of methyl oleate.
... According to Wang et al. [19] the cost involved in enzyme immobilization together with cycle utilization should be taken into account in the global economic analysis of an industrial biodiesel plant. It has been argued by Rahman et al. [20] that lipases may lose its activity in non-aqueous medium and selectivity and stability may also be affected. Repeated cycles of Novozym 435 were adopted by Hajar et al. [21] in the methanolysis of canola oil in solvent-free system and the authors noted that after 5 cycles and 432 h reaction, reaction conversion was kept at 97%, but, unfortunately, enzyme activity was not reported in such research work. ...
The enzymatic reaction is highly respected from an environmentally‐friendly point‐of‐view. Optimization of the reaction media and supporting materials of enzymes must be investigated in parallel with the effort to develop new enzymes. Lipases are frequently used for organic syntheses as synthetic tools even industry because of their acceptance of having a broad range of substrates, stability, and availability. We have investigated the possibility of ILs as both a solvent and activating or stabilization agent of enzymes, in particular, lipase as a model enzyme. ILs allowed recyclable use of a lipase and significant acceleration of transesterification, and also enhanced the stability and reaction activity of a lipase by immobilization through a lyophilization process. We discuss how we enhanced the enzyme capability using the IL engineering focusing on lipase‐catalyzed reactions, i. e., realization of the recyclable use of an enzyme, how ILs mediated the enhanced reaction rate, and improved the stability of the enzyme. Ionic Liquids allowed recyclable use of lipases, improved stability and acceleration of the lipase‐catalyzed reactions, in particular, using the IL‐coated‐enzyme through the immobilization of alkyl‐PEG sulfate IL via lyophilization process.
Twenty new triethanolammonium amino acid salts (TEA AA) have been prepared from triethanolammonium hydroxide and L-amino acids. The physicochemical properties of TEA AA depended on the applied amino acid. Five of the synthesised salts, i.e. mono- and bis-salts of L-glutamic acid, L-aspartic acid, and TEA salt of l-glutamine were solids with melting points between 127.32 °C to 171.51 °C. The other TEA AA exhibited glass transition temperatures from -68.45 °C for TEA Ser to -6.27 °C for TEA Trp and were assigned as amino acid ionic liquids (AAILs). The TEA His was characterised by the highest thermal stability, with an average temperature of 5 % weight loss at 186.4 °C, whereas the lowest stability was determined for TEA Asp (107.5 °C). The developed salts were tested as reaction medium additives for proteolytic enzymes (papain, subtilisin, bromelain). Most AAILs showed an inhibitory effect on tested proteases but with different mechanisms related to the enzyme substrate specificity and structural diversity. The TEA Ser was the most effective competitive inhibitor (Ki = 0.24 10-4 mol/L) for bromelain, while TEA Val uncompetitive inhibitor for papain (Ki = 0.25 10-4 mol/L). The developed TEA AA salts exhibit potential as enzyme-controlling agents for use in industrial processes.
The enzymatic reaction is highly respected from an environmental-friendly aspect. Optimization of reaction media and supporting materials of enzymes must be investigated in parallel with the effort to develop new enzymes. Ionic liquids (ILs) have very good properties as reaction media in chemical reactions and even for biotransformation. ILs allowed significant improvement of the stability of enzymes by immobilization through a lyophilization process. In this chapter, we discuss the possibility of IL engineering focusing on lipase-catalyzed reactions and laccase-catalyzed reactions, that is, ILs mediated the enhanced reaction rate and improved the stability of enzymes.
By using amino acids (AAs) as anions, eleven ionic liquids composed of tetraethylammonium (N2222) as a cation were synthesized, namely, [N2222][AA]s. Their structures were characterized by FT-IR and NMR. The alkalinity of the synthesized AAILs was measured using a pH meter. In the transesterification reaction of soybean oil with methanol, tetraethylammonium arginine ([N2222][Arg]) presented the best catalytic capability. The optimal reaction conditions for the transesterification reaction of soybean oil to achieve 98.4% biodiesel conversion were a methanol/oil of 10:1 mol/mol, reaction temperature of 100 °C, catalyst dosage of 20 wt% (based on the oil weight) and reaction time of 60 min. The kinetics study was evaluated and followed the pseudo-first order reaction mechanism, and the activation energy and pre-exponential factor were 34.53 kJ/mol and 1.21 × 10⁴ min⁻¹, respectively. [N2222][Arg] is a promising, operationally simple, sustainable, biodegradable and possibly renewable catalyst for the conversion of vegetable oil or animal fat into biodiesel.
Enzymes are important contributors to the current industrial development as they activate the reactions through enormous pathways. In recent years, ionic liquids (ILs) have become qualified media for clean extraction, photochemistry, green processing, electro-synthesis, and pharmaceutical applications. Although many enzymes have been studied in ILs media, lipases showed exceptional stability, selectivity and production yields. This chapter briefly outlines some merit as well as the downsides of current ILs applications in lipases reactions including the production of biodiesel, esterification and other established applications. List of abbreviations Cations 1-heptyl-3-methylimidazolium [C 7 MIM] 1-butyl-3-methylimidazolium [BMIM] 1-hexyl-3-methylimidazolium [C 6 MIM] 1-ethyl-3-methylimidazolium [EMIM] 1-dodecyl-3-methylimidazolium [C 12 MIM] 1-penty-3-methylimidazolium [PMIM] 1-ethyl-2,3-dimethylimidazolium [EDMIM] 1-hexadecyl-3-methylimidazolium [C 16 MIM]
The use of ionic liquids to replace organic or aqueous solvents in biocatalysis processes has recently received great attention, and much progress has been made in this area; the lipase-catalyzed reactions are the most successful. Recent developments in the application of ionic liquids as solvents in lipase-catalyzed reactions for organic synthesis are reviewed, focusing on the ionic liquid mediated activation method of lipase-catalyzed reactions.
Three types of triazolium cetyl-PEG10 sulfate ionic liquids were synthesized and their property for activation of Burkholderia cepacia lipase investigated; both the reaction rate and enantioselectivity depended on the cationic part of the coating ILs and 1-butyl-3-methyltriazolium cetyl-PEG10 sufate (Tz1)-coated lipase PS, especially suitable for the transesterification of 1-(pyridin-2-yl)ethanol, 1-(pyridin-3-yl)ethanol, and 1-(pyridin-4-yl)ethanol, among 12 types of tested secondary alcohols. The most important result was obtained when these enzymes were stored in an IL ([N221MEM][Tf2N]) solvent: TZ1-PS showed an amazing stability and it exhibited an excellent activity after 2 years when the enzyme was stored in [N221MEM][Tf2N].
Three types of quaternary ammonium salts, tris(diethylamino)cyclopropenium cetyl-PEG10 sulfate (TAC1), N,N,N,N-tetraethylammonium cetyl-PEG10 sulfate (TEA), and N,N-diethylpiperidinium cetyl-PEG10 sulfate (PP22), have been prepared and evaluated for their activation property of Burkholderia cepacia lipase using them as coating materials of the enzyme. Among the three tested IL-coated enzymes, the TAC1-coated lipase PS (TAC1-PS) was especially suitable for the transesterification of 1-(pyridin-2-yl)ethanol, 1-(pyridin-3-yl)ethanol, 1-(pyridin-4-yl)ethanol, and 4-phenylbut-3-en-2-ol. Although the reaction rate of 1-phenylethanol of the TAC1-PS-catalyzed reaction was inferior to that of IL1-PS, the enzyme was coated with the equivalent amount of L-methionine with TAC1, the resulting enzyme (TAC1-L-Met-PS) exhibited a higher activity compared to that of IL1-PS, and this was mainly contributed to the increasing Kcat value. We further succeeded in demonstrating the recyclable use of TAC1-PS in the [N221MEM][Tf2N] solvent system.
Ionic liquids (ILs) have now been acknowledged as reaction media for biotransformations. The first three examples were reported in this field in 2000, and since then, numerous applications have been reported for biocatalytic reactions using ILs. Two topics using ILs for enzymatic reactions have been reviewed from the standpoint of biocatalyst mediating organic synthesis; the first is “Biocatalysis in Ionic Liquids” which includes various types of biocatalytic reactions in ILs (section 2): (1) recent examples of lipase-mediated reactions using ILs as reaction media for biodiesel oil production and for sugar ester production, (2) oxidase-catalyzed reactions in ILs, (3) laccase-catalyzed reactions, (4) peroxidase-catalyzed reactions, (4) cytochrome-mediated reactions, (5) microbe-mediated hydrations, (6) protease-catalyzed reactions, (8) whole cell mediated asymmetric reduction of ketones, (10) acylase-catalyzed reactions, (11) glycosylation or cellulase-mediated hydrolysis of polysaccharides, (12) hydroxynitrile lyase-catalyzed reaction, (13) fluorinase or haloalkane dehydrogenase-catalyzed reaction, (14) luciferase-catalyzed reactions, and (15) biocatalytic promiscuity of enzymatic reactions for organic synthesis using ILs. The second is “Enzymes Activated by Ionic Liquids for Organic Synthesis”, particularly describing the finding story of activation of lipases by the coating with a PEG-substituted IL (section 3). The author’s opinion toward “Future Perspectives of Using ILs for Enzymatic Reactions” has also been discussed in section 4.
A two-step strategy was attempted to develop the best amino acid-based basic ionic liquids for catalytic production of biodiesel via transesterification. Cholinium with various amino acids as paired anions were first synthesized to screen anionic moiety. Arginine and histidine were selected for further structural evolution by varying the substituents of tetraammonium cation. Tetrabutylammonium arginine ([TBA][Arg]) was found to be the most effective catalyst to obtain 98.0-99.8% yield of biodiesel at 80 °C within 15 min with catalyst loading of 28.84 mmol/100 g high oleic sunflower oil. 13C NMR spectra of reactants and products certified the progress of transesterification structurally. Biodiesel yield of 98.80% was obtained under the optimal conditions: catalyst loading 6% (oil basis, w/w), temperature 90 °C, methanol to oil mole ratio 9:1, and 15 min reaction. The catalytic transesterification by [TBA][Arg] was applicable for different alkyl alcohols but the activity decreased with increasing alkyl chain length. The catalyst did not show specificity and preference to different glycerides and different fatty acids. The strong protonizability of the guanidine moiety in [Arg]- and stability of [TBA]+, [TMA]+ and [Ch]+ in methanol are suggested to be responsible for the high catalytic activity of the ILs. The developed catalyst significantly reduced the reaction time and might be greener and more sustainable due to the properties of the substrates and the preparation in water.
This chapter discusses various factors that influence the lipase activity and stability in ionic liquids, as well as different methods that improve the lipase activity and stability in ionic liquids. This chapter further reviews recent applications of deep eutectic solvents in lipase activation.
White biotechnology is the use of enzymes and microorganisms in industrial production through applied biocatalysis. This allows for milder reaction conditions (pH and temperature) and the use of more environmentally-compatible catalysts and solvents. This, in turn, leads to processes which are shorter, generate less waste, making them both environmentally and economically more attractive than conventional routes.
This book describes the use of white biotechnology within the sustainable chemistry concept, covering waste minimization; the use of alternative solvents (supercritical fluids, pressurized gases, ionic liquids and micellar systems) and energies (microwaves and ultrasound); sustainable approaches for the production of fine and bulk chemicals (aromas, polymers, pharmaceuticals and enzymes); the use of renewable resources and agro-industrial residues; and biocatalysts recycling.
Covering industrial processes and new technologies, this book combines expertise from academia and industry. It is a valuable resource for researchers and industrialists working in biotechnology, green chemistry and sustainability.
The eco-efficient lipase catalyzed synthesis of sugar fatty acid ester surfactants from renewable commodities is a stimulating challenge in biotransformation. The biocatalytic investigations went from solvent-free and minimal ionic liquid use to bulk organic solvents, and binary solvents thereof. Disaccharide maltose was acylated with linoleic acid (C18:2) on the primary O-6,6′ hydroxyl functions. The ionic liquid [emim][MeSO3] and the potentially renewable acetone enabled best conversions. Binary solvents like acetone/DMF and [emim][MeSO3]/[bmpyr][PF6] tended to double conversions. The lipase selection is also crucial and the enzymes Pseudomonas cepacia and immobilized Candida antarctica allowed highest yields in a screening with 10 different lipases. Also other non-solvent parameters such as reaction time and molecular sieve content improved maltose transformation further up to 82%. Analysis by HPLC, ESI-MS, and NMR indicated the formation of mono-6 or 6′-O-linoleyl-α-d-maltose as a mixture of two regioisomers in a 1.4:1 ratio. From an applied point of view, the best solvent is acetone. Unlike ionic liquids and binary mixtures, it is easily removable from reaction mixtures. Moreover, acetone is to some degree a green solvent as it can be produced directly from renewable feedstock.
In this report, molecular dynamics simulations were applied in order to investigate the effect of Room Temperature Ionic Liquid (RTIL) anions toward the structure and dynamic properties of lipases. Two lipases were studied; Candida antarctica lipase B and Candida rugosa lipase were solvated by five RTILs that contained the same cation, with increasing hydration levels. Several properties were investigated: structural deviations and flexibility of the protein conformation, the behaviour of RTILs at the protein surface, and the interactions between RTILs and water molecules in the systems. Both lipases' conformations showed an increased structural stability in RTILs when compared to an aqueous solution. The lowest structural deviation was observed around 15 to 20 percent of water content (w/w protein). The RTIL with chloride anion was shown to be the exception however: inducing the least structural stability at low water percentages. The flexibility of both lipases was clearly affected when transferred from aqueous into RTILs. The flexible regions found for both lipases in water were significantly more rigid in RTILs. Around the protein surface, the behaviour of RTIL anions and the water molecules was similar to other conventional organic solvents. The water retention ability for all RTIL anions was consistent for both lipases accept for the bis(trifluoromethylsulfonyl)imide anion, which showed distinctive behaviour toward different protein surface properties. The effect of water content was more profound compared to the difference between the RTILs anions studied. However, it was found that the structural and dynamic properties of the lipases were affected by the behaviour of anions toward the hydration layer of the enzymes.
The main objective of this short communication is to show the technical feasibility of biodiesel production using a low-cost polyurethane support for Candida antarctica B (CAL-B) immobilization. For this purpose, batch experiments were performed under ultrasound irradiation with enzyme reuse. From the results one can notice conversions to fatty acid ethyl esters up 81% at 132 W output power, molar ratio of soybean fatty acid charge to ethanol of 1:6 and 10 wt% enzyme loads in 180 min reaction time.
The present study deals with the efficient chemo-enzymatic synthesis of enantiopure (R)-1-chloro-3-(piperidin-1-yl) propan-2-ol 3, as an intermediate for (R)-arimoclomol. This intermediate was synthesized from racemic alcohol (RS)-3 using lipases with vinylacetate as the acyl donor in three different ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4], 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF6] ,and 1-ethyl-3-methyl imidazolium tetrafluoroborate [EMIM][BF4]) in combination with an organic solvent (toluene). Various reaction parameters, such as temperature, time, substrate and enzyme concentration, and reaction medium (organic solvent and ionic liquid) were optimized using Pseudomonas aeruginosa MTCC 5113 lipases (PAL). It was observed that 30 mM of (RS)-3 and 30 mg/mL of PAL in 4 mL of solvent (toluene:[BMIM][BF4]::70:30) using vinyl acetate as the acyl donor at 30 °C gave good conversion (C = 50.02%) and enantiomeric excess (eeP = 98.91% and eeS = 99%) in 18 h. The (S)-1-chloro-3-(piperidin-1-yl) propan-2-yl acetate 4 and (R)-3 were separated by flash chromatography. Compound (R)-3 is a key intermediate for the synthesis of enantiopure arimoclomol and bimoclomol.
This paper reports the synthesis of a series of new tetraethylammonium-based amino acid chiral ionic liquids (CILs). Their physico-chemical properties, including melting point, thermal stability, viscosity and ionic conductivity, have been comprehensively studied. The obtained results indicated that the decomposition for these salts proceeds in one step and the temperature of decomposition (T(onset)) is in the range of 168-210 degrees C. Several new CILs prepared in this work showed high ionic conductivity compared to the amino acid ionic liquids (AAILs) found in the literature.
Candida antarctica lipase B, Cal-B, was (i) chemically modified with various epoxides, or (ii) cross-linked with various diepoxides in order to improve enzyme activity and thermal stability. Changed enzyme structure was confirmed by kinetic resolution of p-nitrophenol acetate with methanol and determination of the kinetic parameters. Thermal stability measurements were carried out for each Cal-B derivative, proving that Cal-B modified with 1,2-epoxypropane and 1,2-epoxypentane, and cross-linked with 1,2,7,8-diepoxyoctane and 1,2,9,10-diepoxydecane showed higher stability than soluble enzyme. The influence of the length of the epoxide arm in the modification process, as well as the length of the spacer arm in the cross-linking process, on the final enzyme preparation properties was investigated. The amount of epoxides and diepoxides used for structural rearrangement played an important role in the derivatives' characteristics. The influence of the precipitant used and the presence of additives, such as surfactant or crown-ether, during modification and cross-linking processes was also revealed.
The chemical modification and immobilisation of lipase B from Candida antarctica (CalB) onto three different types of mesoporous silicate (MPS) were undertaken. Soluble CalB was modified by two bifunctional reagents, ethylene glycol bis(succinimidyl succinate) (EGNHS) and glutaraldehyde, and by the monofunctional citraconic anhydride. Both chemically modified and untreated enzyme were then immobilised onto SBA-15-, CNS- and MCM-type MPS by adsorption. Thermal stabilities of chemically modified CalB in solution and of the immobilised preparations were evaluated and compared. Citraconic anhydride dramatically reduced the stability of CalB whereas both bifunctionals yielded an eightfold increase in stability over the native free CalB at 70°C. Following immobilisation of the EGNHS-treated preparation onto CNS-MPS, the stability gain increased to over 60-fold and this combination proved to be the most effective stabilisation strategy. CalB also showed a preference for MPS with larger pores, namely SBA-15. Immobilisation of CalB in alginate beads was also stabilising.
Several citronellyl esters ( acetate, propionate, butyrate, caprate and laurate) were synthesized by immobilized Candida antarctica lipase B ( Novozym) in high yields (> 99%) using equimolar mixtures of citronellol and alkyl vinyl ester as substrates in solvent-free medium. The best results were obtained for citronellyl butyrate synthesis (17.4 mu mol min(-1) mg IME-1) at 70 degrees C, which could be improved up to two-fold by coating the biocatalyst particles with alkyl imidazolium-based ionic liquids, which favoured partitioning of the substrate and product molecules.
Outstanding activation of an enzyme in ionic liquids (ILs) has been demonstrated by covalent modification with comb-shaped poly(ethylene glycol) (PEG) (PM13). Candida rugosa lipase modified with PM13 (PM13–CRL) was readily solubilized in all the ILs tested ([Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]) containing 0.5% (v/v) of water, whereas native lipase did not dissolve in any of the ILs. The results for transesterification of 2-phenyl-1-propanol with vinyl acetate using lipase in ILs revealed that the PM13–CRL conjugate exhibits a high catalytic activity while suspended native lipase shows little activity. The hydrophobicity of ILs somewhat affected the enzyme activity and a more hydrophobic IL such as [Emim][Tf2N] was preferable for the lipase reaction, as was also observed in enzymatic reaction in conventional organic solvents. The enzyme activities in ILs were much higher than those in organic solvents, the excellent activity being associated with unique properties such as the hydrophobicity and the high polarity of ILs. Furthermore, the PM13-–CRL conjugate exhibited a high storage stability in [Emim][Tf2N].
Lipase from Candida rugosa was coated with surfactants and immobilized on silica to catalyze esterification reaction to produce ethyl butyrate, a flavour ester. Various surfactants were tested amongst which non-ionic surfactant performed better than cationic surfactant, whereas inhibitory effect was observed with an anionic surfactant. The optimal temperature of the esterification reaction was 37 °C. Triton X 100 and cetyltrimethyl ammonium bromide (CTAB) were further characterized in order to achieve the optimum catalytic activity of lipase. A low amount of surfactant proved to be most effective.
High performance enzymatic synthesis of oleyl oleate, a liquid wax ester was carried out by lipase-catalysed esterification of oleic acid and oleyl alcohol. Various reaction parameters were optimised to obtain high yield of oleyl oleate. The optimum condition to produce oleyl oleate was reaction time; 5 min, organic solvents of log P ≥ 3.5, temperature; 40-50°C, amount of enzyme; 0.2-0.4 g and molar ratio of oleyl alcohol to oleic acid; 2:1. The operational stability of enzyme was maintained at >90% yield up to 9 cycles. Analysis of the yield of the product showed that at optimum conditions, >95% liquid wax esters were produced.
(R)-Pyrrolidine-substituted imidazolium cetyl-PEG10-sulfate (D-ProMe) derived from D-proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that (R)-isomer of the imidazolium salt worked better than (S)-isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity.
The objective of this study was to develop a suitable method for immobilizing lipase. Although many investigators have studied lipase immobilization, detailed optimization of each step for lipase immobilization using cross-linking methods has been lacking. Therefore, we focused on establishing and optimizing each step of the immobilization procedure to improve procedural efficiencies. Lipase produced from Rhizopus oryzae was immobilized on silica gel using a cross-linking method. The immobilization procedures, such as the pretreatment of silica gel, silanization, crosslinking, glutaraldehyde modification, and elimination of unreated aldehyde groups, were all optimized. Furthermore, the stability of the immobilized lipase and its suitability for reuse were also evaluated.
The dramatic activation of enzymes in nonaqueous media upon co-lyophilization with simple inorganic salts has been investigated as a function of the Jones-Dole B coefficient, a thermodynamic parameter for characterizing the salt's affinity for water and its chaotropic (water-structure breaking) or kosmotropic (water-structure making) character. In general, the water content, active-site content, and transesterification activity of freeze-dried subtilisin Carlsberg preparations containing >96% w/w salt increased with increasing kosmotropicity of the activating salt. Degrees of activation relative to the salt-free enzyme ranged from 33-fold for chaotropic sodium iodide to 2,480-fold for kosmotropic sodium acetate. Exceptions to the general trend can be explained by the mechanical properties and freezing characteristics of the salts undergoing lyophilization. The profound activating effect can thus be attributed in part to the stabilizing (salting-out) effect of kosmotropic salts and the phenomenon of preferential hydration.
A rational design of phosphonium ionic liquid for ionic liquid coated-lipase (IL1-PS)-catalyzed reaction has been investigated, and very rapid transesterification of secondary alcohols accomplished when IL1-PS was used as catalyst in 2-methoxyethoxymethyl(tri-n-butyl)phosphonium bis(trifluoromethanesulfonyl)amide ([P444MEM][NTf2]) as solvent while perfect enantioselectivity was maintaining. Increased Kcat value was suggested to be the most important factor in IL1-PS working the best in [P444MEM][NTf2] solvent.
In this paper, three nucleic acids such as cytosine, cytidine, and thymine and two amino acids, such as D‐tryptophan and N‐carbobenzyloxy‐D‐phenylalanine, were chromatographed using ionic liquid as an additive for the mobile phase in high performance liquid chromatography (HPLC). Ionic liquid, 1‐butyl‐3‐methylimidazolium tetrafuloroborate ([BMIm] [BF4]), was used. The nucleic acid eluent was 5 vol. % of the modifier (methanol:acetonitrile=95:5 (vol. %)), in 10 mM of sodium phosphate monobasic, with addition of 0.5, 2.0, and 4.0 mmol/L of ionic liquid. Separation of nucleic acids was obtained on a commercially available octadecyl silica column (4.6×150 mm i.d., and particle size 5 µm). In the case of D‐amino acids, the mobile phase was 65% methanol in water, with additions of 0.5, 1.0, 2.0, 8.0, 12.0, and 15.0 mmol/L of ionic liquid. The experiments were performed on a stainless steel column, 3.9×300 mm i.d., and particle size 15 µm, packed with octadecyl‐bonded silica at the laboratory. Effects of the concentration of ionic liquid for retention and separation of some nucleic and amino acids were discussed. The results showed the potential application of ionic liquid as a mobile phase additive in liquid chromatography.
For Abstract see ChemInform Abstract in Full Text.
Novel imidazolium salt ionic liquids were prepared derived from polyoxyethylene(10) cetyl sulfate and used as an additive for lipase-catalyzed transesterification in organic solvent; a remarkable enhanced enantioselectivity was obtained when the salt was added in 3 to 10 mol%
vs. substrate in the Pseudomonas cepacia lipase-catalyzed transesterification of 1-phenylethanol using vinyl acetate in a diisopropyl ether or hexane solvent system. There was a remarkable acceleration when the lipase was coated with this novel ionic liquid and used as catalyst for transesterification in i-Pr2O or in hexane.
A homologous series of biosurfactants has been synthesized by a novel sustainable biotransformation technique and compared with three other enzymatic processes. 6-O-Alkanoyl-methyl-a-D-glucopyranosides were obtained by lipase mediated esterification of methyl-a-D-glucopyranoside with capric acid C 10:0 , lauric acid C 12:0 , myristic acid C 14:0 , palmitic acid C 16:0 , and oleic acid C 18:1 . Solvent free transformations were compared with the use of ionic liquids and organic solvents. The lipase from Candida antarctica B, immobilized on macroporous acrylic acid beads (Novozyme 435), was employed either untreated or coated with small amounts of ionic liquids. This resulted in superior efficiencies (80%) with 1-butyl-4-methylpyridine hexafluorophosphate [4bmpy][PF 6 ] and broader substrate tolerance in comparison to solvent free transformation. The results show a positive correlation with increasing polarity of the ionic liquids used as liquid film-coating, which was in opposition to the use of the same ionic liquid as solvent. The analysis of the ionic liquid film coated catalyst carriers was performed by optical and scanning electron microscopy (SEM).
The new ionic liquid-modified polymer sorbent was developed by surface chemical modification of the synthesized polymer particles using ionic liquids. The obtained ionic liquid-modified polymer was successfully used as a special sorbent in a solid-phase extraction (SPE) process to isolate caffeine and theophylline from green tea. A comparison of different SPE cartridges using the blank polymer, C18, and ionic liquid-modified polymer revealed that the highest recovery was obtained using ionic liquid-modified polymer sorbent. Quantitative analysis was carried out by using a C18 column (5 µm, 150 × 4.6 mm) as an analytical column. Good linearity was obtained from 5 × 10−4 to 0.5 mg/mL (r2>0.999) for the two analytes with relative standard deviations <4.5%.
Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.
Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C1–C18). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process.
The chiral pyrrolidine-substituted imidazolium cetyl-PEG10-sulfate (D-ProMe) derived from D-proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that the D-isomer of the imidazolium salt worked better than the L-isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity.
The room temperature solid-phase ionic liquid (RTSPIL) co-lyophilized enzyme exhibited markedly enhanced activity in organic solvent. The enzyme co-lyophilized with a dodecyl-imidazolium salt was 660-fold more active compared to its RTSPIL-free counterpart. The activity enhancement by RTSPILs was mainly attributable to the reduced particle sizes and improved dispersion of enzymes suspended in organic solvent. Also, the RTSPIL co-lyophilized enzyme displayed significantly enhanced enantioselectivity. Its enantioselectivity was 2.5-fold higher than that of its RTSPIL-free counterpart.Graphical abstractIonic liquid co-lyophilized enzyme is described as a useful catalyst for biocatalysis in organic solvent. The some room-temperature-solid-phase ionic liquids were synthesized and used as the lyoprotectant material for the lyophilization of enzyme.Research highlights▶ The room temperature solid-phase ionic liquids (RTSPILs) co-lyophilized enzyme exhibited markedly enhanced reactivity for biocatalysis in organic solvent. ▶ The enantioselectivity of RTSPIL co-lyophilized enzyme presents the enhanced enantioselectivity. ▶ The enhanced activities by RTSPILs are mainly attributable to the size affection of enzyme catalyst after co-lyophilization with RTSPIL.
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Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF. Rapid Science Ltd. 1998
In this study, we invetigated the hydrolysis of olive oil catalyzed by a surfactant-coatedCandida rugosa lipase in a hydrophilic polyacrylonitrile hollow fiber membrane reactor and then compared the results to those using the
native lipase. The organic phase was passed through the hollow inner fibers of the reactor and consisted of either the coated
lipase and olive oil dissolved in isooctane or the coated lipase dissolved in pure olive oil. The aqueous phase was pumped
through the outer space. After 12 h and with conditions of 30°C, 0.12 mg enzyme/mL and 0.62 M olive oil, the substrate conversion
of the coated lipase reached 60%. This was twice the conversion for the same amount of native lipase that was pre-immobilized
on the membrane surface. When using pure olive oil, after 12 h the substrate conversion of the coated lipase was 50%. which
was 1.4 times higher than that of the native lipase.
Propyl-glycoside lactate, a new α-hydroxy acids derivative, was synthesized by esterification between lactic acid and propyl-glycoside, instead of transesterification as reported previously, in a non-aqueous medium using immobilized lipase as biocatalyst. A conversion of 66% was achieved by the optimization of the reaction conditions. Lipase activity was found to be reduced when exposed to high concentration of glycoside. Presence of silica gel in reaction medium improved conversion and initial rate significantly without altering the kinetic mechanism of the enzymatic esterification reaction. Addition of molecular sieves eliminated hydrolysis of the produced ester and allowed glycoside conversion rising to 75%.
The enzymatic synthesis of methyl adipate via green esterification of adipic acid and methanol in hexane has been studied. Lipase from Candida rugosa immobilised onto various layered double hydroxides (LDHs) by a reproducible and simple method of physical adsorption was used as biocatalyst with promising result. Mg/Al–NO3−, Zn/Al–NO3− and Ni/Al–NO3− of LDHs with molar ratio of M2+/M3+ = 4:1 were synthesised by co-precipitation method with continuous agitation. The percentages of protein loading on Mg/Al–NO3−, Zn/Al–NO3− and Ni/Al–NO3− were 71%, 67% and 58%, respectively, due to the larger surface area, porosity and basal spacing of the supports. Parameter studies of reaction time, reaction temperature, water activity, thermostability, storage, leaching and reusability were investigated and optimised. Optimum conditions to produce adipate ester upto 80 % were reaction time; 2.5 h, temperature; 50 °C, and water activity; 0.53, respectively. Increased in optimisation conditions and enhanced stability properties were found after immobilisation compared to the native lipase.
Optically active (−)-menthyl butyrate was synthesized by enantioselective esterification of racemic (±)-menthol and butyric anhydride using lipase from Candida rugosa immobilized onto epoxy-activated supports of Eupergit C and Eupergit C 250 L through physical adsorption method. The effects of various temperature, storage condition, stability in organic solvent and lipase recyclability were investigated for their influence on the enzymatic enantioselective formation of (−)-menthyl butyrate. The immobilized lipases retained high catalytic activity of up to 31% yield and 100% enantiomeric excess of the desired product, and showed better stability compared to the native lipase. They also exhibited about 50% retained activity even after incubation at higher temperatures, storage at room temperature and after long incubation in hexane. Immobilized lipases also showed considerably efficient reusability.
Geotrichum sp. lipase with enhanced activity and operational stability was prepared for use in non-aqueous media. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum sp. lipase. The modified lipase exhibited 18.4-fold enhanced esterification activity towards methyl oleate synthesis, and retained 90% activity following repeated use in 10 cycles. The combined strategy exhibited a significant synergistic effect and was suitable for lipase modification, dramatically enhancing the enzyme activity and operational stability. This approach is applicable to the preparation of other enzyme biocatalysts, since the methods are effective for upgrading crude enzyme to a refined product with high activity and stability for use in non-aqueous media.
Mica was modified either by acid treatment, grafting with aminopropyl-, octyl-, vinyl-, mercapto- and glycidoxy-triethoxysilanes, and activation of pre-treated support with glutaraldehyde (Glu). The derivatives were characterized by X-ray diffraction (XRD), infra-red spectroscopy (FTIR), surface area and porosity analysis, scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) and transmission electron microscopy (TEM) techniques. The modified micas were used for immobilization of lipase from Candida rugosa (CRL). Activity of the lipase was determined by esterification and exhibited the improved activity than the free enzyme following the order; Amino-CRL > Glu-Amino-CRL > Octyl-CRL > Vinyl-CRL > Glycidoxy-CRL > Mercapto-CRL > Mica-CRL. Lipase immobilized mica showed enhanced protein loading (up to 8.22 mg protein/g support) and immobilization (up to 78%) compared to the free lipase and unmodified mica.
The synthesis of butyl propionate in a recirculating bioreactor in room temperature ionic liquid/supercritical carbon dioxide biphasic systems at 50 °C and 80 bar was studied. In these systems, α-alumina microporous membranes with immobilized Candida antarctica lipase B were coated with four different ionic liquids based on 1-n-alkyl-3-imidazolium cations and hexafluorophosphate and bis{(trifluoromethyl)sulfonyl}imide anions. Selectivity increased (reaching >99.5%) when room temperature ionic liquid/supercritical carbon dioxide biphasic systems were used rather than in supercritical carbon dioxide alone. To understand the behaviour of the enzyme and the mass-transfer phenomena in these biphasic systems, the reaction was also carried out in ionic liquids and in ionic liquid/hexane biphasic systems, and the ionic liquid/hexane partition coefficients of the compounds involved in the transesterification reaction were determined. It was observed that the activity in room temperature ionic liquid/supercritical carbon dioxide biphasic systems depends on the effect of the ionic liquid media on the enzyme and the diffusional limitations across the IL-layer around the biocatalyst.
The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH2 reduced the activity to 40%.The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase.
A new way of solubilizing hydrolytic enzymes so that they can be used in organic solvents is to coat them with lipid monolayers. Lipid-coated enzymes have been used successfully for a number of different types of reactions in organic media or organic-aqueous, two-phase systems; for example, enantioselective esterification by lipase, transphosphatidylation of water-insoluble phospholipids by phospholipase D, hydrolysis of a lipophilic substrate by a catalytic antibody and transglycosylation by β-d-galactosidase.
Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min(-1)mg(-1)) as compared to unmodified pH tuned lipase (128 nmol min(-1) mg(-1)). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min(-1) mg(-1), respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content.
This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.
Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent. An ionic liquid, [PPMIM]-[PF(6)] (1, [PPMIM] = 1-(3'-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53 degrees C, was synthesized in two steps from N-methylimidazole. The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53 degrees C and then allowing the mixture to cool. A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity.
Recent studies on biocatalysis in water—organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a
w
) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.
A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.
Four ionic liquid (IL) salts containing the 1-butyl-2,3-dimethylimidazolium (BDMIM) and 1-allyl-2,3-dimethylimidazolium (ADMIM) cations have been prepared; the characterization was based on IR spectroscopy and single-crystal structure determination. The compounds BDMIM[HSO4], BDMIMCl, ADMIMBr, and (BDMIM)4[FeIICl4][FeIIICl4]2 were chosen to incorporate anions significantly differing in hydrogen-bond-acceptor strength in order to elucidate the influence of directional bonding on crystal packing. The cations adopt different arrangements with respect to the counterions. The role of hydrogen bonding in these compounds is discussed with respect to its general significance for lattice energies of IL salts.
Recent developments associated with voltammetric studies in ionic liquid media have been critically reviewed. Initially, advantageous electrochemical properties of ionic liquids are summarised, as are limitations encountered by the presence of impurities and problems related to purification and recovery of ionic liquids. Subsequently, the use of IUPAC recommended ferrocene oxidation and cobalticenium reduction processes as potential reference scales in ionic liquids and the application of voltammetry of adhered solid and microchemical approaches to the measurement of formal potentials and kinetics of coupled first order chemical reactions are discussed. Finally, the possible use of volatile ionic liquids is considered as an alternative to use of the non-volatile ionic liquids media, presently emphasized in most studies.
Lipases co-lyophilized with water-soluble gemini-type amphiphiles were found to have high enzyme activity in nonaqueous media without washing out of the amphiphile with anhydrous organic solvent. In this study, we obtained freeze-dried complexes of Candida rugosa lipase (CRL) with six water-soluble twin glusitol-headed amphiphiles bearing different types of hydrophobic tails, including newly synthesized ones, and their transesterification activity in organic solvent was evaluated. The results indicate that the increased enzyme activity upon CRL modification at 200 molar ratio of amphiphile/CRL, which are restricted to the ester-containing amphiphiles, is probably due to the surface activation by the interaction between ester-carbonyl of the amphiphile and phenyl group of the tyrosine residue situated on the surface of the lid in the CRL.
Ionic liquids are defined today as liquids which solely consist of cations and anions and which by definition must have a melting point of 100 degrees C or below. Originating from electrochemistry in AlCl(3) based liquids an enormous progress was made during the recent 10 years to synthesize ionic liquids that can be handled under ambient conditions, and today about 300 ionic liquids are already commercially available. Whereas the main interest is still focussed on organic and technical chemistry, various aspects of physical chemistry in ionic liquids are discussed now in literature. In this review article we give a short overview on physicochemical aspects of ionic liquids, such as physical properties of ionic liquids, nanoparticles, nanotubes, batteries, spectroscopy, thermodynamics and catalysis of/in ionic liquids. The focus is set on air and water stable ionic liquids as they will presumably dominate various fields of chemistry in future.
Several types of imidazolium salt ionic liquids were prepared derived from poly(oxyethylene)alkyl sulfate and used as an additive or coating material for lipase-catalyzed transesterification in an organic solvent. A remarkably increased enantioselectivity was obtained when the salt was added at 3-10 mol % versus substrate in the Burkholderia cepacia lipase (lipase PS-C)-catalyzed transesterification of 1-phenylethanol by using vinyl acetate in diisopropyl ether or a hexane solvent system. In particular, a remarkable acceleration was accomplished by the ionic liquid coating with lipase PS in an iPr(2)O solvent system while maintaining excellent enantioselectivity; it reached approximately 500- to 1000-fold acceleration for some substrates with excellent enantioselectivity. A similar acceleration was also observed for IL 1-coated Candida rugosa lipase. MALDI-TOF mass spectrometry experiments of the ionic-liquid-coated lipase PS suggest that ionic liquid binds with lipase protein.
Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF(6)]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF(6)]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98-99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58-60 degrees C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF(4)]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF(4)]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.
Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.
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