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Ionic liquid facilitates biocatalytic conversion of hardly water soluble ketones
Abstract
Ionic liquids represent a promising alternative to conventional cosolvents as biocompatible solubilisers for biocatalysis. This was shown using water miscible ionic liquids to facilitate the stereoselective reduction of hardly water soluble, aliphatic ketones catalysed by the alcohol dehydrogenase from Lactobacillus brevis. Ten ionic liquids were screened for activity and solubility. Improved storage stabilities besides improved enzyme activities, as well as reduced substrate surplus and product inhibitions were found, while applying the most promising AMMOENG™ 101 in more detailed investigations. Batch reactions with cofactor regeneration via a glucose dehydrogenase showed increased reaction rates; thus underlining the positive influence of AMMOENG™ 101. For (R)-3-octanol, (R)-2-nonanol, (R)-2-decanol, and (R)-2-octanol space time yields between 250 and 350mmolL−1d−1 were achieved.
... Of the other hydrophilic ionic liquid systems that have been assayed, [C 2 C 1 Im][Et 2 PO 4 ] is strikingly detrimental to the enzymatic activity of L. brevis ADH with only 5% relative activity left at 10% (v/v) for the reduction of 2-octanone. This contrasts with [C 1 C 1 Im][Me 2 PO 4 ], with 85% relative activity, and is comparable to the activity of YADH found for [C 1 C 1 Im][Cl] (above), and [C 2 C 1 Im][MeSO 3 ] with 105% relative activity (Kohlmann et al., 2011). ...
... c) Water-miscible, amphiphilic ionic liquids Hydroxyl-functionalised ionic liquids, including the AmmoEng TM series of task-specific ionic liquids (Figure 2), have been shown to enhance the activity of ADHs even at very high concentrations of up to 90% (v/v) ionic liquid content (De Gonzalo et al., 2007), with the optimal concentration dependant on the experimental set-up. Up to 90% (v/v) is tolerated when whole cell-biocatalysts or lysates thereof are applied (De Gonzalo et al., 2007), whilst for purified enzyme, solution concentrations of 10% (v/v) AmmoEng TM 101 were shown to enhance the relative activity of L. brevis ADH by 180% (Kohlmann et al., 2011). A major advantage of the use of these amphiphilic ionic liquids is the much higher than usual solubility of difficult substrates, as has been demonstrated for, for example, 4'-Br-2,2,2-trifluoroacetophenone and 6-bromoβ-tetralone (Hussain et al., 2008). ...
... Both ADH and GDH 103 enzymes showed 50% (w/w) residual activity in the water-immiscible ionic liquid [C 4 C 1 Pyr][NTf 2 ] (Hussain et al., 2008). The amphiphilic oligoether-based ionic liquids AmmoEng TM 100 and 101, with a 14-carbon coconut oil-derived group, were found to either impart the most activity or increase conversion by 150 and 180%, respectively (De Gonzalo et al., 2007;Kohlmann et al., 2011), with the main difference being the anion [MeSO 4 (100) and Cl (101)]. AmmoEng TM 102, which has an 18-carbon tallow-derived group, a 5-fold longer oligio(ether-) chain than AmmoEng TM 101 and a greater ability to absorb water due to the higher ether content (Ribot et al., 2012), was found to increase activity of L. brevis ADH by 110%. ...
Biopolymer processing and handling is greatly facilitated by the use of ionic liquids, given the increased solubility, and in some cases, structural stability imparted to these molecules. Focussing on proteins, we highlight here not just the key drivers behind protein-ionic liquid interactions that facilitate these functionalities, but address relevant current and potential applications of protein-ionic liquid interactions, including areas of future interest.
... For example, the presence of some hydrophilic ILs as additives can facilitate the biocatalytic processes with Lactobacillus brevis, Rhodotorula sp. AS2.2241 and Trigonopsis variabilis AS2.1611 and provide significant increases in product yield and reaction efficiency compared with conventional solvents (Kohlmann et al., 2011;Lou et al., 2009aLou et al., , 2009b. Moreover, IL was found to have a stabilizing influence on nicotinamide cofactors in the bioreduction. ...
... Additionally, from a practical viewpoint, a hydrophobic IL-containing biphasic system is not good enough for the bioreduction on a large scale, since the biphasic system may cause pronounced emulsification and consequently will give rise to negative effects on the biocatalytic process. On the other hand, relatively few water-miscible ILs have been tested for use in whole-cell biocatalytic reductions of prochiral ketones (Kohlmann et al., 2011;Lou et al., 2009aLou et al., , 2009b. Therefore, we initially evaluated a wide range of hydrophilic ILs as the cosolvents for the bioreduction with Acetobacter sp. ...
... For NO 3 À and Cl À -based ILs, when the cation in ILs changed from C 2 MIM þ to C 2 OHMIM þ (Table 2, entries 2, 3, 7, 13), both the yield and the initial reaction rate were markedly enhanced, demonstrating that the hydroxyl-functionalized C 2 OHMIM þ cation promotes the bioreduction. This finding also confirms the similar previous observations that hydroxylfunctionalized ILs manifested excellent biocompatibility with the biocatalysts and afforded good experimental results (de Gonzalo et al., 2007;Kohlmann et al., 2011;Lou et al., 2009b). For C n MIM Á NO 3 (n¼2, 4) and C n MIM Á Br (n¼2, 4-7), the initial reaction rate and the maximum yield clearly decreased with the elongation of the alkyl chain attached to the cation (i.e., increasing n value) ( Table 2, entries 7, 8, 15-19). ...
The utilization of hydrophilic ionic liquids to improve the synthesis of enantiopure alcohols was successfully performed, via the anti-Prelog asymmetric reduction of ketones with whole cells of Acetobacter sp. CCTCC M209061 newly isolated from Chinese kefir. The best results were obtained with C2OHMIM·NO3, which showed good biocompatibility and also increased moderately cell membrane permeability, thus improving the reaction efficiency. Additionally, the optimal C2OHMIM·NO3 content, buffer pH, reaction temperature and substrate concentration for 4-(trimethylsilyl)-3-butyn-2-one reduction to (R)-4-(trimethylsilyl)-3-butyn-2-ol were 10.0% (v/v), 5.0, 30 °C and 12 mM, respectively. Under the optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 14.0 μmol/min·gcell, 91%, and >99%, respectively, which were much better than the results reported previously. The efficient whole-cell biocatalytic process proved to be feasible on a 400-mL preparative scale, and the immobilized cells still retained above 88.0% of their original activity after successive re-use for 10 batches, showing the good operational stability in the presence of C2OHMIM·NO3. Furthermore, the established biocatalytic system with Acetobacter sp. CCTCC M209061 and C2OHMIM·NO3 was shown to be highly effective for the anti-Prelog asymmetric reduction of other aryl ketones to the corresponding (R)-alcohols.
... Hydrophilic dialkylimidazolium-based ILs, which are similar in structure to cationic surfactants, may be able to increase the permeability of microbial cell membrane and then not only lower the product concentration within microbial cells but also reduce the inhibition and toxic effects of the product [14][15][16]. Furthermore, in some cases, hydrophilic ILs can not only improve the enzyme stability but also act as enzyme activators, and result in an enhanced productivity [17][18][19][20][21]. Therefore, it seems that hydrophilic ILs are promising and attractive co-solvents for use in the cell-based biocatalytic processes. ...
... 3.1 Effect of IL content. Although use of hydrophilic IL could improve enzyme activity and stability, as well as reduced substrate surplus and product inhibitions, the content of hydrophilic IL in the co-solvent system should also be carefully controlled to avoid inactivation of enzyme at high IL content [18,22,34]. As shown in Figure 3, the content of C 2 OHMIM?NO 3 had great influence on the bioreduction of TMSB with immobilized C. parapsilosis cells. ...
Hydrophilic ionic liquids (ILs) were employed as green solvents to construct an IL-containing co-solvent system for improving the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by immobilized Candida parapsilosis cells. Among 14 hydrophilic ILs examined, 1-(2'-hydroxyl)ethyl-3-methylimidazolium nitrate (C(2)OHMIM·NO(3)) was considered as the most suitable IL for the bioreduction with the fastest initial reaction rate, the highest yield and the highest product e.e., which may be due to the good biocompatibility with the cells. For a better understanding of the bioreduction performed in the C(2)OHMIM·NO(3)-containing co-solvent system, the effects of several crucial variables were systematically investigated. The optimal C(2)OHMIM·NO(3) content, substrate concentration, buffer pH, co-substrate concentration and temperature were 10% (v/v), 3.0 mmol/L, 5.0, 98.1 mmol/L and 30°C, respectively. Under the optimal conditions, the initial reaction rate, the maximum yield and the product e.e. were 17.3 µmol/h g(cell), 95.2% and >99.9%, respectively, which are much better than the corresponding results previously reported. Moreover, the immobilized cells remained more than 83% of their initial activity even after being used repeatedly for 10 batches in the C(2)OHMIM·NO(3)-containing system, exhibiting excellent operational stability.
... Hydrophilic dialkylimidazolium-based ILs, which are similar in structure to cationic surfactants, may be able to increase the permeability of microbial cell membrane and then not only lower the product concentration within microbial cells but also reduce the inhibition and toxic effects of the product [14][15][16]. Furthermore, in some cases, hydrophilic ILs can not only improve the enzyme stability but also act as enzyme activators, and result in an enhanced productivity [17][18][19][20][21]. Therefore, it seems that hydrophilic ILs are promising and attractive co-solvents for use in the cell-based biocatalytic processes. ...
... 3.1 Effect of IL content. Although use of hydrophilic IL could improve enzyme activity and stability, as well as reduced substrate surplus and product inhibitions, the content of hydrophilic IL in the co-solvent system should also be carefully controlled to avoid inactivation of enzyme at high IL content [18,22,34]. As shown in Figure 3, the content of C 2 OHMIM?NO 3 had great influence on the bioreduction of TMSB with immobilized C. parapsilosis cells. ...
Hydrophilic ionic liquids (ILs) were employed as green solvents to construct an IL-containing co-solvent system for improving the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by immobilized Candida parapsilosis cells. Among 14 hydrophilic ILs examined, 1-(2'-hydroxyl)ethyl-3-methylimidazolium nitrate (C(2)OHMIM·NO(3)) was considered as the most suitable IL for the bioreduction with the fastest initial reaction rate, the highest yield and the highest product e.e., which may be due to the good biocompatibility with the cells. For a better understanding of the bioreduction performed in the C(2)OHMIM·NO(3)-containing co-solvent system, the effects of several crucial variables were systematically investigated. The optimal C(2)OHMIM·NO(3) content, substrate concentration, buffer pH, co-substrate concentration and temperature were 10% (v/v), 3.0 mmol/L, 5.0, 98.1 mmol/L and 30°C, respectively. Under the optimal conditions, the initial reaction rate, the maximum yield and the product e.e. were 17.3 µmol/h g(cell), 95.2% and >99.9%, respectively, which are much better than the corresponding results previously reported. Moreover, the immobilized cells remained more than 83% of their initial activity even after being used repeatedly for 10 batches in the C(2)OHMIM·NO(3)-containing system, exhibiting excellent operational stability.
... Due to the diverse nature of proteins and ILs, not all proteins are stable in ILs [8,19,39,40,42]. Depending on the IL and its concentration, the structural integrity of proteins may be affected with regard to unfolding or protein aggregation [43][44][45][46]. Some ILs consisting of large organic cations and organic or inorganic anions (Box 1) are able to interact strongly with the amino acids that comprise proteins, consequently affecting the integrity of the proteins. ...
Ionic liquids (ILs) are salts with low melting points that can be used as solvents for mild extraction and selective fractionation of biomolecules (e.g., proteins, carbohydrates, lipids, and pigments), enabling the valorisation of microalgal biomass in a multiproduct biorefinery concept, while maintaining the biomolecules’ structural integrity and activity. Aqueous biphasic systems and emulsions stabilised by core-shell particles have been used to fractionate disrupted microalgal biomass into hydrophobic (lipids and pigments) and hydrophilic (proteins and carbohydrates) components. From nondisrupted biomass, the hydrophobic components can be directly extracted using ILs from intact cells, while the most fragile hydrophilic components can be obtained upon further mechanical cell disruption. These multiproduct biorefinery concepts will be discussed in an outlook on future separations using IL-based systems.
... Some studies suggested that the IL solvents have adverse impacts on the lipase activity by affecting the hydrogen bonding of the protein [23,24]. Others, found out that the ionic liquid solvents increase the stability of cellulases [25]; plus they can refine both the stability and activity of alcohol dehydrogenase [26] and peroxidase [27]. Apart from ionic liquid solvents, the stability and activity of enzymes have been examined in organic solvents by several authors [20,28,29]. ...
Herein, we performed meticulous experimental and computational studies to explore the stability, activity, and dynamics of laccase in the presence of various organic and inorganic solvents. It is well known that laccases are eco-friendly enzymes, which can quickly eliminate recalcitrant chemicals from contaminated media. We determined the Asp96 (COO⁻)---Arg43 (N-H2), Asp131 (COO⁻)---Arg197(N-H1), Asp138(COO⁻)---Arg195 (N-H2), Asp140(COO⁻)---Arg199(N-H2), Asp214(COO⁻)---Arg260(N-H2), Asp224(COO⁻)---Arg423(N-H2), Asp424(COO⁻)---Arg243(N-H1), Asp424(COO⁻)---Arg243(N-H2), Glu288(COO⁻)---Arg176(N-H1) and Glu288(COO⁻)---Arg176(N-H2) salt bridges as the crucial ones in maintaining the structural integrity of laccase. Furthermore, the fluorescence, circular dichroism (CD) spectroscopies, and molecular dynamics simulation outcomes highlighted that the secondary structure elements and tertiary structure of the enzyme did not change significantly in the presence of both selected organic (ethanol and hexane) and inorganic (1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) co-solvents. The obtained experimental results demonstrated that the enzyme was more stable in the hexane and aqueous ionic liquid solutions than in the ethanol solution. Additionally, laccase exhibited more activity in the presence of the hexane and aqueous ionic liquid solutions. Probably due to more accessibility of the substrate to the active site of laccase. Interestingly, the activity of laccase in the presence of the co-solvents was in decreasing order of hexane<[BMIM][PF6] < ethanol and the same order was observed for the number of water molecules in the enzyme's hydration shell. The results also indicated that the biocatalyst may keep a balance between the interactions with both water molecules and co-solvents to maintain its native conformation. The results also showed that there were some co-solvent molecules in the first hydration shell of the enzyme, but they were not enough to make a considerable change in the enzyme structure.
... Sometimes, chemically modified cations of ILs-DMP improve the activity and stability of enzymes, for example, [108]. ILs-DMP have also served as biocompatible solubilizers for hardly water-soluble substances, for example, in the stereoselective reduction of ketones using the alcohol dehydrogenase from Lactobacillus brevis [109]. Interesting and promising are investigations in electroenzymatic syntheses, efforts to combine oxidoreductase-catalyzed reactions with the electrochemical reactant supply [110]. ...
... A recent work presented the effect of 10 different ILs (with either imidazolium or ammonium cations) on the enzyme stability. Improved storage stabilities and improved enzyme activities were found in the most promising, ammonium-based, AMMOENG TM 101 IL [32]. Later, the same group [33] proved the feasibility of continuous production using the previously recommended IL, combined with product separation using a membrane bioreactor (the so-called process integration). ...
... A recent work presented the effect of 10 different ILs (with either imidazolium or ammonium cations) on the enzyme stability. Improved storage stabilities and improved enzyme activities were found in the most promising, ammonium-based, AMMOENG TM 101 IL [32]. Later, the same group [33] proved the feasibility of continuous production using the previously recommended IL, combined with product separation using a membrane bioreactor (the so-called process integration). ...
... It has been demonstrated that the amount of IL in a biphasic system affects significantly on the activity, enantioselectivity and stability of enzymes and microbial cells [39,40]. Therefore, it is necessary to investigate the effect of [C 4 MIM][PF 6 ] content in the biphasic system. ...
Background
Enantiopure (S)-1-(4-methoxyphenyl) ethanol {(S)-MOPE} can be employed as an important synthon for the synthesis of cycloalkyl [b] indoles with the treatment function for general allergic response. To date, the biocatalytic resolution of racemic MOPE through asymmetric oxidation in the biphasic system has remained largely unexplored. Additionally, deep eutectic solvents (DESs), as a new class of promising green solvents, have recently gained increasing attention in biocatalysis for their excellent properties and many successful examples in biocatalytic processes. In this study, the biocatalytic asymmetric oxidation of MOPE to get (S)-MOPE using Acetobacter sp. CCTCC M209061 cells was investigated in different two-phase systems, and adding DES in a biphasic system was also explored to further improve the reaction efficiency of the biocatalytic oxidation.
Results
Of all the examined water-immiscible organic solvents and ionic liquids (ILs), 1-butyl-3-methylimidazolium hexafluorophoshpate ([C4MIM][PF6]) afforded the best results, and consequently was selected as the second phase of a two-phase system for the asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061 cells. For the reaction performed in the [C4MIM][PF6]/buffer biphasic system, under the optimized conditions, the initial reaction rate, the maximum conversion and the residual substrate e.e. recorded 97.8 μmol/min, 50.5 and >99.9 % after 10 h reaction. Furthermore, adding the DES [ChCl][Gly] (10 %, v/v) to the aqueous phase, the efficiency of the biocatalytic oxidation was rose markedly. The optimal substrate concentration and the initial reaction rate were significantly increased to 80 mmol/L and 124.0 μmol/min, respectively, and the reaction time was shortened to 7 h with 51.3 % conversion. The immobilized cell still retained over 72 % of its initial activity after 9 batches of successive reuse in the [C4MIM][PF6]/[ChCl][Gly]-containing buffer system. Additionally, the efficient biocatalytic process was feasible up to a 500-mL preparative scale.
Conclusion
The biocatalytic asymmetric oxidation of MOPE with Acetobacter sp. CCTCC M209061 cells was successfully conducted in the [C4MIM][PF6]-containing biphasic system with high conversion and enantioselectivity, and the reaction efficiency was further enhanced by adding [ChCl][Gly] to the reaction system. The efficient biocatalytic process was promising for the preparation of enantiopure (S)-MOPE.
... It has been demonstrated that the amount of IL in a biphasic system affects significantly on the activity, enantioselectivity and stability of enzymes and microbial cells [39,40]. Therefore, it is necessary to investigate the effect of [C 4 MIM][PF 6 ] content in the biphasic system. ...
... This could be either due to interactions with the enzyme or substrate availability. [88] In agreement with the literature, BAL stability was enhanced using DMSO as the co-solvent, and only 10% loss of activity was observed within 250 h of incubation. Even though DMSO offers excellent stability enhancement, DMSO is not preferred in bio-or chemical-catalysis due to problems in downstream processing. ...
... Additionally, the polarity, hydrophobicity and solvent miscibility behaviour of ILs can be finely tuned through appropriate modification of the cation and anion [8,9] so as to meet the requirements of applications. In recent years, ILs have been widely used in analytical chemistry [10], biodiesel and biomass [11][12][13][14], and biotechnology, particularly in biocatalytic reactions which can be performed by using isolated enzymes or whole cells [15,16]. According to studies, there are several role models in biocatalysis process with ILs. ...
Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs' interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed.
... For instance, the presence of certain hydrophilic ILs as additives in reaction systems can facilitate the biocatalytic reduction reactions with alcohol dehydrogenases from Lactobacillus brevis and Rhodococcus rubber and with whole cells of Rhodotorula sp. AS2.2241, Trigonopsis variabilis AS2.1611 and Candida parapsilosis CCTCC M203011, and provide significant increases in reaction rate, yield, and product e.e. (Kohlmann et al., 2011;de Gonzalo et al., 2007;Zhang et al., 2012b;Lou et al., 2009a,b). In our previous report (Lou et al., 2004), the addition of small amount of hydrophilic [C 4 MIM][BF 4 ] could improve the activity, enantioselectivity and stability of papain during asymmetric hydrolysis of d,l-p-hydroxyphenylglycine methyl ester. ...
A comparative study was made of Mung bean epoxide hydrolases-catalyzed asymmetric hydrolysis of styrene oxide to (R)-1-phenyl-1,2-ethanediol in an n-hexane/buffer biphasic system containing various hydrophilic ionic liquids (ILs). Compared to the n-hexane/buffer biphasic system alone, addition of a small amount of hydrophilic ILs reduced the amount of non-enzymatic hydrolysis, and improved the reaction rate by up to 22%. The ILs with cation containing an alkanol group, namely [C(2)OHMIM][BF(4)] and [C(2)OHMIM][TfO], and the choline amino acid ILs [Ch][Arg] and [Ch][Pro] were found to be the most suitable co-solvents for the reaction, owing to their good biocompatibility with the enzyme, which led to high initial rates (0.99-1.25μmol/min) and high product e.e.s (95%). When substrate concentration was around 30mM, where optimal performance was observed with the IL-containing systems, the product e.e. was improved from 90% without ILs to ≥95% in the presence of ILs.
... Also, they may be explosive and are usually environmentally harmful. Ionic liquids (ILs), have recently emerged as novel green solvents for a great variety of biocatalytic transformations, and are becoming more and more attractive in such applications as a promising alternative to the conventional organic solvents [13][14][15][16]. Many kinds of ILs have proven to be biocompatible with diverse microbial cells, and present many advantages for the biotransformations such as high conversion rates, high enantioselectivity, excellent operational stability and recyclability [17]. ...
Background
Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs), to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS) to (R)-1-trimethylsilylethanol {(R)-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst.
Results
It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim·PF6)/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v), 6.0, 30°C and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1 μmol/min gcwm, 98.6% and >99%, respectively. The efficient whole-cell biocatalytic process was shown to be feasible on a 450-mL scale. Moreover, the immobilized cells remained around 87% of their initial activity even after being used repeatedly for 8 batches in the C4mim·PF6/buffer biphasic system, exhibiting excellent operational stability.
Conclusions
For the first time, we have successfully utilized immobilized Candida parapsilosis CCTCC M203011 cells, for efficiently catalyzing anti-Prelog enantioselective reduction of ATMS to enantiopure (R)-1-TMSE in the C4mim·PF6/buffer biphasic system. The substantially improved biocatalytic process appears to be effective and competitive on a preparative scale.
... Addition of AMMOENG™ 101 as a co-solvent could facilitate stereoselective reduction of aliphatic ketones catalyzed by LbADH. Improved storage stabilities besides improved enzyme activities, as well as reduced substrate surplus and product inhibitions were found, leading to an increase in reaction rates (Kohlmanna et al., 2011). In another type of application, water-immiscible ILs can form biphasic systems. ...
Chiral secondary alcohols play an important role in pharmaceutical, agrochemical, and chemical industries. In recent years, impressive steps forward have been achieved towards biocatalytic ketone reduction as a green and useful access to enantiopure alcohols. An increasing number of novel and robust enzymes are now accessible as a result of the ongoing progress in genomics, screening and evolution technologies, while process engineering provides further success in areas of biocatalytic reduction in meeting synthetic challenges. The versatile platform of these techniques and strategies offers the possibility to apply high substrate loading and thus to overcome the limitation of low volumetric productivity of usual enzymatic processes which is the bottleneck for their practical application. In addition, the integration of bioreduction with other enzymatic or chemical steps allows the efficient synthesis of more complex chiral products.
The applications of ionic liquids in biotransformation have attracted much attention during the last decade due to their unique properties, such as negligible vapor pressure, nonflammability, high thermal and excellent chemical stability, and ability to dissolve a variety of materials. Moreover, their properties can be finely tuned by selecting the combinations of appropriate ion identities, allowing ionic liquids to be customized for specific processes. They have been used as solvents, cosolvents, or additives in many enzyme or whole cell processes, which often led to improved process performance. In this chapter, a variety of enzymes and whole cell catalysts as well as their catalyzed reactions in ionic liquids are discussed. In addition, the effects of ionic liquids' properties on the enzymes and the methods to improve the enzyme activity and stability in ionic liquids are also described.
There are numerous applications of ionic liquids (ILs) particularly in biology since, some of the ILs stabilizes the protein and augment enzyme activity while some of them have opposite effect. We have explored the influence of imidazolium-based ILs, 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) and 1-butyl-3-methylimidazolium iodide ([Bmim][I]) on the stability of stem bromelain (BM) using UV–visible spectroscopy, steady-state and thermal fluorescence, circular dichroism spectroscopy and dynamic light scattering (DLS) measurements. We attempt to understand the effect of imidazolium-based ILs on the stability of BM based on the variation of anion of the IL. All of these ILs acted as destabilizer for the native state of BM except at 0.01 M. The destabilizing behaviour of these ILs increased with increase in the concentration of ILs in the order of Cl⁻ > Br⁻ > I⁻. On the other hand, anions are found to follow the well accepted Hofmeister series where the destabilization behaviour increased with increase in the chaotropicity or decrease in the kosmotropicity of the anion of the IL.
Over the past years since the discovery of ionic liquids (ILs), there is an increased demand to consider ILs as novel biocompatible co-solvents for proteins. Due to their tunable physical properties ILs can adjust themselves in any required experimental conditions starting from protein extraction to enzyme catalysis at elevated temperature. In recent years, large numbers of ILs have been synthesized and their effect on protein stability has been illustrated. With the rapid growth in various kinds of ILs, our understanding of protein stability in ILs has substantially increased. It is not necessary that a particular IL that is biocompatible to a protein will behave same for the other. Therefore, it is extremely essential to collect the literature dealing with the direct involvement of ILs in protein folding/unfolding studies under the same roof. This review focuses the tremendous accomplishments achieved in recent years in the field of protein stability in ILs. We hope that this would also help to set a stage where we can identify, explore and compare the mechanistic behavior of protein folding/unfolding in ILs. This review will surely bring a new boost in protein folding studies from the chemical biology perspective.
The application of biocatalytic methods for synthetic purposes plays an important role in synthetic chemistry since the discovery that the action of enzymes goes beyond performing hydrolytic reactions, and that biocatalysts can act with excellent levels of activity and selectivity in organic solvents as well as in neoteric systems. Enzyme-catalyzed processes can provide benefits to existing methods for accessing bulk and fine chemicals in a selective and nonselective fashion under mild reaction conditions. This chapter provides an update of relevant transformations carried out in ionic liquids (ILs) for the synthesis of valuable products; this chapter is divided according to the classes of enzymes used for biotransformations developed in ILs. First, an introduction regarding the most common ILs used in combination with enzymes, and the main advantages of using ILs with synthetic applicability is discussed. Then, selected reactions using a variety of enzymes is presented. The use of mainly hydrolases and oxidoreductases is also presented, paying attention to the lesser developed lyase or isomerase-catalyzed reactions in ILs. Nonsolvent applications of these neoteric solvents are also included, to provide a better understanding about the potential of ILs in organic chemistry. Finally, the state of the art regarding the use of enzymes in deep eutectic solvents is explained, which is a very promising emerging type of media that can fill the gap between the application of these solvents not only in academia but also hopefully in industrial biotechnology in the near future.
Ionic liquids have drawn much attention as reaction media for enzymatic reactions due to their better activity, thermal stability, stereoselectivity, enantiomers selective and recyclability compared with conventional organic solvent and water. The recent developments of enzyme catalysis using lipase, oxidoreductase and protease in ionic liquids were reviewed in this paper.
We investigated the asymmetric bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-[3,5-bis(trifluoromethyl) phenyl] ethanol ((R)-BTPE) in a hydrophilic quaternary ammonium-based ionic liquid (IL)-containing system to improve the efficiency of bioreduction catalyzed by recombinant Escherichia coli cells overexpressing carbonyl reductase. Based on the low toxicity to microbial cells and moderately increased cell membrane permeability, tetramethylammonium cysteine ([N1,1,1,1][Cys]) was selected and employed as co-solvent. Some key reaction parameters involved in the bioreduction were also investigated in the [N1,1,1,1][Cys]-containing system. The optimum conditions for the process were found to be: 3.5% (w/v) [N1,1,1,1][Cys], 20% (v/v) isopropanol, 1 M BTAP, 12.7 g/L of recombinant E. coli cells, pH 6.8, reaction for 12 h at 30 °C. A 98.7% yield (with >99 % of enantiomeric excess (ee)) was obtained under the optimum conditions. The biocatalytic process was scaled up to a 5 L fermentor afforded high reaction yield in IL-containing system. The results demonstrated that the IL [N1,1,1,1][Cys] is a useful co-solvent to improve bioreduction process and may has potential applications in various biocatalytic reactions.
Biocatalytic reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] with Acetobacter sp. CCTCC M209061 cells was successfully conducted in ionic liquid (IL)-based biphasic systems. Several water-immiscible ILs were used to construct biphasic systems. The best IL investigated was 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim·PF6), which also had good biocompatibility. Several influential variables were examined. The optimum parameters were as follows: volume ratio of buffer to C4mim·PF6, 1/2 (v/v); substrate concentration, 55 mmol/L; buffer pH, 5.5; cosubstrate concentration, 80 mmol/L; reaction temperature, 35 °C; and shaking speed, 220 rpm. Under these optimal conditions, the initial reaction rate, the yield, and the product e.e. were 0.39 mmol/(L min), 90.8%, and >99%, respectively, which were much better than results reported previously. This efficient whole-cell biocatalytic process was feasible on a 450-mL preparative scale, and the immobilized cells showed excellent operational stability and could be reused for at least 10 batches.
Previously, it could be demonstrated, that the monophasic, enzymatic reduction of aliphatic 2-ketones into the corresponding (R)-2-alcohols is an adequate and viable method as carried out in a cascade of two enzyme–membrane reactors (Leuchs, S.; Na’amnieh, S. N.; Greiner, L. Green Chemistry 2013, 15, 167–176.). In the present work, the process metrics of the ketone reduction were calculated. A cost analysis revealed that the enzyme costs are negligible, but the cost for nicotinamide cofactor NADP+ is dominating the overall cost of the chemical raw material followed by the ionic liquid (TEGO IL K5) used as solubiliser and the buffer. The overall cost of chemicals was €148/kgproduct. To assess the environmental impact of the process, the E-factor (kgwaste/kgproduct) 132 and the process mass intensity 133 (PMI, kgsubstrate/kgproduct) were calculated. A process model based on initial rate experiments was elaborated and used to improve the process under cost and environmental aspects. Applying several measures to enhance the cofactor utilisation, the cost base could be reduced by 65% and the E-factor (PMI) to 17 (18).
The screening and use of benign chemicals for enhanced oil recovery (EOR) applications is important because of their properties and relationship to the embedded fluids. We investigated a special type of ionic liquids (ILs) called “Ammoeng” for potential use in surfactant EOR to replace the currently used surfactants that have many disadvantages. The interfacial tension (IFT) between a representative oil sample from Saudi reservoirs and solutions of Ammoeng™ ILs at different concentrations in 10 wt% NaCl aqueous solutions were measured as a function of temperature. It was found that the IFT values decreased with the increase of IL concentration. However, the effect of temperature on the IFT depended on the type of IL. Ammoeng™ 102 gave the lowest IFT values among the screened ILs. The comparison of the results to those resulting from TritonX100, a commercially used surfactant, showed that the IFT values using Ammoeng™ 102 were smaller than the corresponding values when TritonX100 was used at the same conditions. The possibility of having a synergetic effect when using a mixture of Ammoeng™ 102 and Triton X 100 was also investigated. The results showed that the IFT values depended on the total concentration, the surfactant to IL mass ratio, and the temperature.
Imidazolium-based ionic liquids (ILs) were designed and synthesised for their use as non-conventional media in alcohol dehydrogenase (ADH)-catalysed reactions. Screenings with several ADHs and various ketone or alcohol substrates for their selective reductions or oxidations, respectively, showed that when containing up to 50 % of the IL the overall conversion of the reactions could be improved in some cases, while the stereoselectivity of the enzyme remained unaltered. Attempts at using these ILs as co-substrates for the recycling of the nicotinamide cofactor led to promising results, opening a new possibility for a substrate-coupled recycling system.
Graphical Abstract
The chemoenzymatic epoxidation of a terpene alcohol, citronellol, is reported. Some experimental conditions, such as the use of lipases from different sources, oxidizing agents (H2O2 or urea–hydrogen peroxide, UHP), reaction time, acyl donor type (C6–C16), temperature (15–40 °C) and the influence of organic media, were evaluated. In most cases, citronellol oxide 2 or the ester citronellol oxide 3 were obtained. Depending on the reaction conditions, high yields of products 2 or 3 were obtained (>99%). CAL-B was the most effective catalyst in this reaction. For epoxide 2, the highest yields of 80% and 77% were obtained at 20 °C and 25 °C, respectively, using UHP as an oxidizing agent and octanoic acid as an acyl donor. The organic medium appears to be one of the most important parameters in the reaction. Using chloroform or dichloromethane, product 2 was obtained at a >99% yield after 24 h. When different mixtures consisting of varied organic solvents and an imidazolium-based ionic liquid (IL) were used, the results were dependent on both the solvent and IL counter-ion (18–75%).
Over the past decade, disaccharide phosphorylases have received increasing attention as promising biocatalysts for glycoside synthesis. Unfortunately, these enzymes typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of cosolvents to increase the dissolved concentration of these acceptors. However, commonly applied solvents such as methanol and dimethyl sulfoxide (DMSO) are not compatible with many intended applications of carbohydrate-derived products. In this work, the solubility of a wide range of relevant acceptors was assessed in the presence of ionic liquids (ILs) as alternative and ‘green’ solvents. The IL AMMOENG 101 was found to be the most effective cosolvent for compounds as diverse as medium- and long-chain alcohols, flavonoids, alkaloids, phenolics and terpenes. Moreover, this IL was shown to be less deleterious to the stability and activity of sucrose phosphorylase than the commonly used dimethyl sulfoxide. To demonstrate the usefulness of this solvent system, a process for the resveratrol glycosylation was established in a buffer containing 20% AMMOENG 101, 1 M sucrose and saturated amounts of the acceptor. A single regioisomer 3-O-α-D-glucopyranosyl-(E)-resveratrol was obtained as proven by NMR spectroscopy.
Low solubility of starting materials and products in water and low enzyme utilisation are the two main obstacles for the production of enantiopure long-chain alcohols with alcohol dehydrogenases. A combination of techniques was used to overcome these limitations. A previously identified ionic liquid was used as a detergent to increase the low solubility for the starting materials as well as for the products. The low enzyme utilisation was increased by using ultrafiltration in an enzyme membrane reactor. Based on kinetic characterisation and stability data of the alcohol dehydrogenase from Lactobacillus brevis and glucose dehydrogenase from Bacillus sp. used for ketone reduction and cofactor regeneration, respectively, a continuous process was realised. In a configuration of a cascade of two enzyme membrane reactors the process could be demonstrated for more than 1000 hours with turnover numbers of more than 106 and space time yields of up to 34 g L−1 d−1 with 99.9% enantioselectivity. Furthermore, downstream processing via adsorption of the alcohols was included, allowing 90% recycle of the aqueous buffer. Thus, the E-factor (amount of waste stream per product) was reduced to 13.
When challenged by a difficult reduction reaction, a chemist should always also consider biocatalysis in synthesis planning. The inherent selectivity of enzymes has been known for many decades now and the practical applicability of biocatalysis has undergone dramatic improvements rendering it a true alternative to established chemocatalysis. In this contribution recent developments in the field of enzymatic reduction using whole cells and isolated enzymes are reviewed.
BACKGROUND: Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs), to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS) to (R)-1-trimethylsilylethanol {(R)-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst. RESULTS: It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim * PF6)/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v), 6.0, 30 degreesC and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1 mumol/min gcwm, 98.6 % and >99 %, respectively. The efficient whole-cell biocatalytic process was shown to be feasible on a 450-mL scale. Moreover, the immobilized cells remained around 87 % of their initial activity even after being used repeatedly for 8 batches in the C4mim * PF6/buffer biphasic system, exhibiting excellent operational stability. CONCLUSIONS: For the first time, we have successfully utilized immobilized Candida parapsilosis CCTCC M203011 cells, for efficiently catalyzing anti-Prelog enantioselective reduction of ATMS to enantiopure (R)-1-TMSE in the C4mim * PF6/buffer biphasic system. The substantially improved biocatalytic process appears to be effective and competitive on a preparative scale.
The alcohol dehydrogenase from Lactobacillus brevis (LbADH) is a versatile cata-lyst for enantioselective reduction of ketones. Its substrate scope is wide with high regio-and enantioselectivity. In this critical review, we have gathered the information available on the substrate scope as well as the applications reported. Quantitative information such as productivity per catalyst, space-time yield (STY), cofactor utilisation, and stability are derived to allow comparison and assessment of practical value.
The continuous biocatalytic synthesis of (R)-2-octanol carried out in an enzymemembrane reactor showed an excellent process stability of the biocatalysts, resulting in a turnover number of 15 million for the applied alcohol dehydrogenase. Utilisation of the ionic liquid AMMOENG™ 101 as a feasible cosolvent increased substrate concentration, and improved space time yields and turnover numbers of the cofactor by factors of 3 and 6, respectively. Moreover, 80% less waste was generated, while producing the same amount of product. Integrated product separation was realized via solid phase extraction. Extraction of the applied solid phase with supercritical carbon dioxide allowed more than 30 reuses of the solid phase.
Major advances dealing with reductases and oxidases are summarized and organized according to important concepts in asymmetric biosynthesis. It is found that compartmentalization is necessary to avoid reversible reactions through consumption of both cofactor forms by the same enzyme. The reaction catalyzed by Old Yellow Enzyme (OYE) is stereoselective, and variability is observed throughout the whole family and has allowed the development of enzyme- and substrate-based stereocontrols. A wide range of possible substrates with bulky hydrophobic side chains are synthesized and converted by leucine dehydrogenase (LeuDH) and phenylalanine dehydrogenase (PheDH).Toluene dioxygenases (TOD) catalyze the oxidation of aromatic compounds to furnish the corresponding cis-cyclohexadiene diols and present great synthetic potential as there is currently no synthetic equivalent.
This review describes the recent developments of enzymatic catalysis in ionic liquids, reporting the use of different biocatalysts in organic synthesis. Several ionic liquids appear as an alternative to conventional organic solvents, providing comparable or higher rates and, in some cases, improved enantioselectivity.
Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space-time yields, as well as stability as far as they can be extracted from the literature.
Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid [BMIM][(CF(3)SO(2))(2)N] compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether.
Ionic liquids as novel solvents for enzymatic reactions can improve selectivity and yield. The enantioselectivity of a lipase catalysed kinetic resolution can be increased at higher temperatures. For a galactosidase catalysed synthesis of a disaccharide the secondary hydrolysis is suppressed doubling the yield.
Over the last decade ionic liquids have achieved much attention and are not any longer just a class of esoteric compounds, but are proving to be valuable and useful in a multitude of different applications. So far, ionic liquids have mainly been considered to be an alternative to conventional solvents in reaction and separation processes. Hence, it is not surprising that most of the recent publications report on the use of ionic liquids as a solvent for chemical and biochemical syntheses. This may be rationalized by the possibility of carrying out biphasic reactions for the separation and recovery of otherwise homogenous precious metal catalysts or enzymes, as ionic liquids form two phases with many organic product mixtures. In contrast, the use of ionic liquids as performance additives or as reactive component in the preparation of oligomers or pre-polymers has gained far less attention. The authors believe that due to their unique properties ionic liquids have a great potential to be used as performance additives in many materials and applications. The application of ionic liquids is in accordance with the chemical industry's guidelines and principles concerning the initiatives “sustainable development” and “responsible care”. The authors demonstrate this by provision of an example taken from Degussa's ongoing ionic liquids research program: The use of ionic liquids as secondary dispersant in universal pigment pastes, i.e. in the white base paint to be tinted.
The effect of hydrophilic ionic liquids (ILs) on the activity of chloroperoxidase (CPO) was checked through kinetic and stereochemical studies. The possibility to employ this enzyme in synthesis has been demonstrated investigating the chemo- and stereoselectivity of oxidation of phenyl methylsulfide in several citrate buffer–IL mixtures.
Ionic liquids such as [BMIM][PF6] and [BMIM][NTF] are already known as good alternatives to organic solvents in biphasic biotransformation. Herein, we report about a systematic procedure based on physical properties to identify more commercially available ionic liquids exhibiting the potential to improve the efficiency of whole cell biocatalyses. This approach resulted in the identification of seven other water immiscible ionic liquids. These ionic liquids were rated by their biocompatibility, their substrate- and product-specific distribution coefficients and by for example performed asymmetric reductions of several prochiral ketones. With the use of a recombinant Escherichia coli as biocatalyst, overproducing a Lactobacillus brevis alcohol dehydrogenase and a Mycobacterium vaccae N10 formate dehydrogenase for cofactor regeneration, the great potential of asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems were demonstrated in simple batch processes.
This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.
Electroenzymatic syntheses combine oxidoreductase-catalysed reactions with electrochemical reactant supply. The use of ionic liquids as performance additives can contribute to overcoming existing limitations of these syntheses. Here, we report on the influence of different water-miscible ionic liquids on critical parameters such as conductivity, biocatalyst activity and stability or substrate solubility for three typical electroenzymatic syntheses. In these investigations promising ionic liquids were identified and have been used as additives for batch electrolyses on preparative scale for the three electroenzymatic systems. It was possible to improve the space-time-yield for the electrochemical regeneration of NADPH by a factor of three. For an amino acid oxidase catalysed resolution of a methionine racemate with ferrocene-mediated electrochemical regeneration of the enzyme-bound cofactor FAD a 50% increase in space time yield and 140% increase in catalyst utilisation (TTN) were achieved. Furthermore, for the chloroperoxidase-catalysed synthesis of (R)-phenylmethylsulfoxide with electrochemical generation of the required cosubstrate H2O2 the space time yield and the catalyst utilisation were improved by a factor of up to 4.2 depending on the ionic liquids used.
Extraction of electrons from cellobiose is realised by cellobiose dehydrogenase (CDH) in hydrated choline dihydrogen phosphate (dhp). Both inter- and intra-electron transfer of CDH were observed in the hydrated choline dhp suggesting a potential candidate of a non-aqueous solvent for enzymatic treatment of depolymerised biomass.
For the prediction and optimisation of the equilibrium conversion in biphasic catalysed reactions, the equilibrium constant of the desired reaction and the partition coefficients of all reactants have to be known. Within this contribution we have examined the alcohol dehydrogenase-catalysed reduction of several linear and aromatic ketones in biphasic reaction media with respect to equilibrium conversion. In this example, the equilibrium constant can be expressed in terms of differences in oxidation-reduction potentials Delta E-0. However, for a large variety of organic compounds, these data are quite rare in the literature. To overcome this lack of data, we have utilised methods of computational chemistry to calculate data for the Gibbs free energy Delta G(R) leading to the equilibrium constants of a homologous series of linear ketones. To obtain comparable data for the reduction of substituted acetophenone derivatives, the Hammett relation leads to the necessary equilibrium constants. Furthermore, we compare the equilibrium conversions of a set of cofactor regeneration methods for the alcohol dehydrogenase-catalysed reductions. These results lead to a time-saving experimental design for the enantioselective reduction of 2-octanone to (R)-2-octanol on a preparative scale utilising biphasic reaction conditions.
Bi- and monophasic ionic liquid (IL)/buffer systems were successfully employed for the biocatalytic reduction of ketones catalysed by the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber via hydrogen transfer. Two different catalyst preparations were employed, namely recombinant ADH-‘A’ ‘immobilised’ in Escherichia coli and partially purified ADH-‘A’. For biphasic systems conversions were acceptable until 20% vv−1 of IL. In contrast, hydroxy-functionalised ‘Tris-like’-ILs were successfully employed in monophasic systems up to 90%vv−1 IL. The use of these solvents allowed highly stereoselective enzymatic carbonyl reductions at substrate concentrations from 1.2 to 1.5M.
A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L−1 d−1, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.
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. (c) 2005 Elsevier B.V. All rights reserved.
Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.
In recent years researchers have started to explore a particular class of organic solvents called room temperature ionic liquids - or simply ionic liquids - to identify their unique advantages for biocatalysis. Because they lack vapour pressure, ionic liquids hold potential as green solvents. Furthermore, unlike organic solvents of comparable polarity, they often do not inactivate enzymes, which simplifies reactions involving polar substrates such as sugars. Biocatalytic reactions in ionic liquids have also shown higher selectivity, faster rates and greater enzyme stability; however, these solvents present other challenges, among them difficulties in purifying ionic liquids and controlling water activity and pH, higher viscosity and problems with product isolation.
Functionalized, hydrogen-bonding ionic liquids have been successfully evaluated as media for the performance of cofactor-dependent enzyme catalysed oxidations; the effects of incorporating hydroxyl groups into both the cation and anion have been studied and the dependence of activity upon water content has been evaluated.
The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.
Various issues that surround biocatalysis in ionic liquids are reviewed and presented in details. The effects of ionic liquids (ILs) on the structure and activity of enzymes as well as their thermal and operational stability is surveyed. Included also in the review is the effects of ILs on the (enantio)selectivity of biocatalytic transformations in comparison with conventional reaction media and the design of efficient reaction procedures based on its unconventional solvent characteristics. The study showed that there is hardly an IL that is not tolerated by any enzyme, and the impression is generally tolerated to higher concentrations than water-miscible molecular solvents. Ionic liquids have potential as reaction media for biotransformation of highly polar substrates, such as polysaccharides, which cannot be performed in water, owing to equilibrium limitations. It is expected that green and biocompatible ionic liquids will become available to contribute to a greener chemical industry.
The ionic liquid (IL) Ammoeng110 contains cations with oligoethyleneglycol units and was found to be highly effective for the formation of aqueous two-phase systems (ATPS) that can be used for the biocompatible purification of active enzymes. Above critical concentrations of the IL and an inorganic salt in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a salt-enriched lower phase. For the optimization of the composition of IL-based ATPS with regard to the extraction of catalytically active enzymes, the Box-Wilson method of experimental design was successfully applied; IL-based ATPS proved to be suitable for the purification and stabilization of two different alcohol dehydrogenases (from Lactobacillus brevis and a thermophilic bacterium). Both enzymes were enriched in the IL-containing upper phase resulting in an increase of specific activity by a factor of 2 and 4 respectively. Furthermore, the presence of IL within the system provided the opportunity to combine the extraction process with the performance of enzyme-catalyzed reactions. The IL was found to exhibit a stability improving effect on both enzymes and a solubility enhancing effect on hydrophobic substrates. Thus the conversion and volumetric productivity of ADH catalyzed reduction of acetophenone could be increased significantly.