Whole-Cell Biocatalytic Processes with Ionic Liquids - ACS Publications

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Whole-cell biocatalytic processes with ionic liquids Pei Xu, Gao-Wei Zheng, Peng-Xuan Du, Min-Hua Zong, and Wen-Yong Lou ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b00965 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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ACS Sustainable Chemistry & Engineering

Whole-cell biocatalytic processes with ionic liquids Pei Xu a,c, Gao-Wei Zheng b, Peng-Xuan Du c, Min-Hua Zong c, Wen-Yong Lou a,c*

a

State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

Guangzhou 510640, China

b

State Key Laboratory of Bioreactor Engineering, East China University of Science and

Technology, Shanghai 200237, China c

Laboratory of Applied Biocatalysis, School of Light Industry and Food Sciences, South China

University of Technology, Guangzhou 510640, China

1

ACS Paragon Plus Environment


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Abstract: Whole-cell based biocatalysis in ionic liquids (ILs)-containing systems has attracted increasing interests in recent years. Compared to bioreactions catalyzed by isolated enzymes, the major advantages of using whole cell in biocatalytic processes are that cells provide a natural intracellular environment for the enzymes to function with cofactors regeneration in situ. An increasing number of renewable ILs are now accessible as a result of the ongoing progress in designing strategy of ILs and the sustainable environment requirement. The toxicity of ILs to microbial cells and the biodegradability are two of the crucial factors which allow the biocatalysis in ILs being applied in practice rather than in bench-scale. Applications of whole-cell biocatalysis in IL-containing system have, to date, been focused on the production of valuable compounds, mainly through reduction, oxidation and hydrolytic reactions. The mechanism research of ILs affecting the whole-cell biocatalysis offer the possibility to effectively integrate ILs with biotransformation. Thus, a comprehensive understanding of the whole cell-based biocatalytic process with ILs will contribute to the discovery of novel solvent for enzymatic reaction and the synthesis of more valuable compounds.

Keywords: Whole-cell, ionic liquid, biocatalysis, biocompatibility, biodegradability

2


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INTRODUCTION Ionic liquids (ILs) are organic salts that exist as liquids at room temperature. The unique physicochemical properties of ILs, such as negligible volatility, thermal stability and relatively high polarity, make them interesting solvents for biocatalytic process.1 ILs are generally considered as green solvents that are good substitutes for traditional organic solvents.2 Also, ILs have also been described as “designer solvents”, since the physicochemical properties of ILs can be tuned by a suitable combination of cation (Table 1) and anion (Table 2).3 Applications of ILs in biotransformations have been extensively studied during the past decade, with a strong focus on catalysis using isolated enzymes, which remain active in ILs and exert good performance.4-6 The beneficial effects of ILs on enzymatic activity and selectivity are partly attributed to the high polarity of these solvents, with ability to increase the solubility of polar substrates while the biocatalyst exhibits high activity.7-9 And the influence of ILs on the structure, activity, stability and selectivity of isolated enzymes, as well as the applications of enzymes in such media have been extensively reviewed.1, 10-12 As “green solvents”,13 fluorous solvents and supercritical fluids (SCFs) have also been broadly studied and generally afforded good results in biocatalytic transformations.14-17 Nevertheless, the relative expensiveness and debatable greenness of production process hinder the application of fluorous solvents in biotransformation. Additionally, as for the strategy of SCFs, certain temperature and pressure are necessary for the reaction process, resulting in the high cost of equipment. 3



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Much attention has been paid to the whole-cell based biocatalytic process in ILs in recent years. However, compared to enzymatic catalysis, studies of whole-cell biocatalysis in the presence of ILs has been less well explored. To date, the use of ILs in whole-cell processes has been mainly limited to “extraction fermentation” or “biphasic transformation” processes,18-20 in which water-immiscible ILs act as a substrate reservoir and in situ extractant for the removal of toxic products to avoid inhibition to the cells.21 In such applications the IL phase can generally be recycled without loss of productivity of the process,22,23 thus making the use of an ionic liquid economically feasible. What’s more, another one of the major advantages of using whole cells rather than isolated enzymes biocatalysts is that they are able to efficiently regenerate expensive cofactors in redox reactions. Such applications also show how whole cells can act as “enzyme reactors” on a nanoliter scale, which are also applicable to multi-step biotransformations. Additionally, cells provide a natural environment for the enzymes, preventing denaturation and conformational changes in the protein structure that may lead to loss of activity in non-conventional reaction media such as ILs. Furthermore, there is no need for costly enzyme purification in whole-cell processes. In this perspective, we highlight the toxicity of ILs to microbial cells, the biodegradability of ILs, and recent advances in whole-cell based biocatalytic processes in ILs-containing system. Applications to date have included oxidation, reduction and hydrolytic reactions, many of which show excellent activity, selectivity, and IL and biocatalyst re-use properties. In addition, the 4


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mechanism of ILs affecting the whole-cell biocatalysis is also discussed and future trends in this expanding area of biocatalysis are suggested.

TOXICITY OF IONIC LIQUIDS TO MICROBIAL CELL

Although ILs are generally regarded as “green” solvents, their toxicity to microorganisms has been recognized and widely studied recently.24-26 Herein, we provide a systematic review of the available data on the toxicity of ILs based on the types of cations. The microorganisms commonly used in such studies are Escherichia coli, Lactobacillus kefir, Saccharomyces cerevisiae etc. Imidazolium-based ionic liquids. The imidazolium-based ILs are the most commonly investigated group, mostly due to their commercial availability and the fact that they are one of the most widely used types of IL on the industrial scale.27 Romero et al.28 investigated the toxicity of several imidazolium liquids with two alkyl substituents in position 3 and 1 of the cation to Photobacterium phosphoreum. It was demonstrated that the toxic effect became stronger with the elongation of the side chain. Similar results have been found in a number of other studies.29-31 In some cases, IC50 (the concentration of chemical at which cell growth, cell viability or metabolism is inhibited at 50%) was measured in order to quantify the toxicity of ILs. As shown in Table 3, some contrasting results have been reported from different studies. For instance, Lee 5



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et al.32 reported that [BMIm][PF6] was toxic towards the growth of E. coli at concentration as low as 0.5% (v/v), while no toxic effects were found by Pfruender et al.20 at a concentration of 20% (v/v) for the same species of microorganism. One important factor that may differ between these studies and may at least in part explain the contrasting results is that the concentration of biomass differs, and toxic effects may be more obvious in a low biomass concentration than in a higher one. Unfortunately, the biomass concentration is not always stated in reports currently in the literatures.24, 33,34 Wood et al.35 studied the toxicity of over ninety ILs from diverse structural classes towards E. coli by high throughput methods, including agar diffusion tests and growth inhibition tests in liquid media. A wide range of ILs containing imidazolium, pyridinium, quaternary ammonium, alkanolammonium and quaternary phosphonium cations, combined with a diverse range of anions, were screened. For the imidazolium-based ionic liquids, [EMIm]+ and [BMIm]+ chlorides and bromides did not produce inhibition zones in the agar diffusion test, but inhibition zones were produced with increasing radii when the alkyl chain on the cation was increased to hexyl or octyl. Similar relationships between increasing length of the alkyl chain/lipophilicity of the imidazolium cation and the increasing toxicity of the ionic liquid have been widely reported elsewhere.36,37 Apart from the alkyl chain length of the cation in ILs, the anion was also found to be crucial for the toxicity against different microorganisms, such as Listeria monocytogenes and 6


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Escherichia coli.38 Santos et al.39 investigated the toxicity of various ILs to nine microorganisms (Bacillus subtilis, Lactobacillus delbrueckii subs. delbrueckii, Pseudomonas aeruginosa, Streptomyces drozdowiczii, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, Aspergillus brasiliensis and Rhizopus oryzae) interesting to the food industry. Interestingly, choline cation proved to be less toxic for the tested microorganisms, while the [NTf2] anion showed the serious toxicity. Besides, the filamentous fungi revealed the best tolerance to the ILs followed by the yeast Yarrowia lipolytica. It was found that increasing concentration of [Bmim][BF4] and [Hmim][BF4] (>1%, w/v) could seriously inhibit the growth of P. freudenreichii,31 suggesting that the toxic effect of imidazolium-based ILs on microbial cell was partly dependent on the IL's concentration, which was also observed by other researches.30,40,41 With regard to the issue of toxicity of imidazolium-based ILs, a complementary effect (either positive or negative) may be held by the additive accompanied with ILs other than those as described above.30 Latala et al.42 observed that salinity could affect the toxicity of ILs towards blue-green algae. The toxicity of ILs such as [BMIm]Cl and [HMIm]Cl was reduced when salinity increased. It was suggested that ion-pairing between the alkylimidazolium cations and elevated concentrations of chlorides competed with and inhibited interaction of the alkylimidazolium cations with negatively charged moieties on the cell walls of eukaryotic algae and cyanobacteria. Also, the elevated concentrations of positively charged inorganic counterions 7



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in the salt may compete with the alkylimidazolium cations for binding sites on the cell wall structures. Pyridinium-based ionic liquids. ILs containing a pyridinium head group have been also well-studied over the past years. Madaan and Tyagi47 reviewed the properties and application of various structures of pyridinium-based ILs, which are often used as surfactants and antimicrobial agents, indicating that they are toxic towards microbes. As was found for imidazolium ILs, the toxicity of pyridinium ILs toward E. coli increased with the elongation of alkoxymethyl substituent in agar diffusion tests and liquid inhibition assays.35 Incorporating different substituents in position R3 in the pyridine ring strongly influenced toxicity. For instance, hydroxyl at this position led to slight toxicity but bromide gave highest toxicity.44 The effect of the anion becomes more pronounced when combined with less toxic cations. It is difficult to make more detailed conclusions on the effect of anions on IL toxicity because no study to date has systematically explored the toxic effects of a wide range of anions. The ILs with pyridinium and imidazolium cations carrying the same-length of alkyl side chains do not necessarily exert equal toxicity to different organisms. For example, compared to [BMIm]Br, [BMPyr]Br was observed to be more toxic towards the bacterium Vibrio fischeri,48 while less toxic to the freshwater snail Physa acuta.49 indicating that the mode of interaction of the cations with the cells and organisms differs among different species. One model of the toxicity of IL cations predicts that pyridinium ions should lead to less toxic ILs than imidazolium 8


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ions with similar alkyl chain lengths because of the fewer number of electronegative ions in the cation ring. Clearly, the above examples indicate that the situation is more complex than this and depends to some extent on the target organism50 and highlight the need for more data to formulate better models to predict the toxicity of ILs to specific cell types.

Quaternary ammonium and quaternary phosphonium ionic liquids. Quaternary ammonium salts are widely used in numerous applications, such as disinfectants, surfactants, antistatic agents and catalysts. Their properties depend on the alkyl chain length, the presence of other functional groups in the cation and the nature of the anion.51,52 Generally, the longer alkoxymethyl chain that a quaternary ammonium IL cation has, the higher toxicity it exerts, and this is consistent with the results found in imidazolium and pyridinium ILs. For instance, the toxicity of an IL with the quaternary ammomium cation [N1888 ]+ toward microbial cells is much greater than that of the [N1124 ]+ cation, which has shorter alkyl chain lengths (see Table 1 for nomenclature of quaternary ammonium ions).35 The substantial influence of the anions associated with quaternary ammonium cations and also the difference in sensitivity of different types of organism is exemplified by [N1124]Cl, which exhibited lower toxicity to bacteria,53 but was more toxic toward duckweed when compared with [N1124][NTf2].54 In recent years, there has been a strong movement to discover new ammonium ILs with higher biocompatibility. Some of the most interesting groups of IL are those containing the 2-hydroxyethyltrimethylammonium (cholinium) cation. Cholinium is a naturally occurring 9



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cation; indeed cholinium chloride (also known as choline) is one of the B-complex vitamins and is considered to have low toxicity, which is a positive indication for the biocompatibility of cholinium ILs. The rational design of low-toxicity ILs by combining cholinium and benign anions may be considered a major breakthrough in development of ILs for biotransformation.55-57 Nockemann et al.58 successfully synthesized two hydrophilic ILs, choline saccharinate and choline acesulfamate, from readily available starting materials (choline chloride and non-nutritive sweeteners). These ILs are much less toxic in aqueous solution when compared with other types of hydrophilic ILs. Subsequently, Hou et al.59 successfully synthesized a series of cholinium amino acids ILs based on their renewable nature and evaluated their application in the pretreatment of biomass. The amino acid-based ILs exerted low toxicity to the tested strain. The good biocompatibility of the cholinium amino acids ILs was also proved by another group, whose research demonstrated that these ILs had an extremely low toxicity, ten times less than imidazolium and pyridinium ILs, to Artemia salina.60 Deep eutectic solvents (DESs), as a new generation of ILs analogues composed of a quaternary ammonium salt and a metal salt or hydrogen bond donor (HBD), has been considered as an alternative to the traditional reaction medium.61-63 Hayyan et al.64,65 found that the tested four choline chloride (ChCl) based DESs exerted extremely low toxicity to all the studied bacteria including B. subtilis, S. aureus, E. coli and P. aeruginosa and showed less cytotoxicity as compared to ILs. However, it was noted that the cytotoxicity of the DESs was higher than their 10


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individual components using different target organisms, either Artemia salina or ICR mice.. Additionally, the various toxicity of DESs depended on the structure difference of the HBDs and the tested model to some degree. This puts a way to design DESs with less toxicity through combining various “green” compounds according to their different use. Compared with quaternary ammonium ILs, the toxicity of phosphonium ILs has been little investigated. Some phosphonium ILs exhibit similar levels of toxicity to imidazolium ILs carrying the same chain length, such as [P4444]Br and [BMIm]Br.49 It was found that phosphonium-based ILs were relatively more toxic than ammonium-based ILs against Vibrio fischeri, mainly due to the electrostatic potential difference of the central atom, namely P and N.66 The structure of the cation plays a major role in the toxicity of phosphonium ILs, but the effect of anion cannot be neglected. For example, exchange of the halide by other anions, e.g. [NTf2]-, [OTf]-, [NO3]-, [N(CN)2]-, [BF4]-,or [PF6]-, resulted in a loss of antimicrobial activity.67 [P666 14]+ combined with chloride led to high toxicity against E. coli, while toxicity was much lower when chloride was replaced with [NTf2]- as the anion.19 Other phosphonium halides such as [P4444]Br, [P666 14]Br and [P666 14]Cl were also reported to be highly toxic.68,69 Besides, the toxicological behavior of phosphonium-based DESs to microorganisms was found to be similar to that of the ammonium-based DESs.70 Other cation-based ionic liquids. In contrast to ILs containing imidazolium and pyridinium cations, which are widely used in industrial applications, little attention has been paid to the ILs 11



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containing other cation head groups. ILs based on quinolinium have been observed to be extremely toxic, for example [Cnquin]+ was more toxic than [CnMIm]+ (n>4) by at least one order of magnitude.24 Therefore, such quinolinium cations are not well suited for use in biotransformation. The toxicity of pyrrolidinium-, piperidinium- and morpholinium-based ILs has been also comprehensively uninvestigated, but data currently available indicate that they are generally less toxic to the bacterium V. fischeri, when compared with imidazolium and pyridinium ILs.54 Similarly, [BMPyr]+ combined with different anions such as [N(CN)2]- or lactate were less toxic than the corresponding imidazolium ILs, as was the related IL and [BMPIp][O2CMe].71,72 The morpholinium IL cation, in which the cation ring contains both nitrogen and oxygen atoms confer low toxicity; for instance, [C2mmor]Br and [C4emor]Br were apparently nontoxic to the alga Pseudokirchneriella subcapitata.73

BIODEGRADABILITY OF IONIC LIQUIDS

The biodegradability of ILs must be characterized before they can be widely applied in a whole cell-based biocatalytic processes, even when the solvent will be reused many times, because eventually they must be disposed of or may be accidentally released. Reports on ILs biodegradability are numerous, most of which lead to the common conclusion that imidazolium-based ILs are not biodegradable,28,46,74 although some studies suggest that such ILs 12


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could be degraded by bacteria and fungi.75,76 These apparently contradictory results show that further evidence is necessary to establish a better understanding of IL degradability. In general, the biodegradability of ILs was investigated in three ways.77 Firstly biological oxygen demand (BOD5) tests were performed to determine the amount of oxygen required by an activated sludge to metabolize the ILs over five days. The biodegradation was determined as a percentage of oxygen depletion relative to theoretical chemical oxygen demand (COD). The ILs were considered as readily biodegradable if the percentages were higher than 60%. The second approach was based on long-term biodegradability (LTB) tests, where the ILs were added to activated sludge and then maintained in bottles for 30 days. Two ILs, [BMIm][PF6] and [BMIm][NTf2], showed no degradation based on the BOD5 method and the similar result was obtained in the latter test method with no significant decrease of the IL concentration. Another approach, the CO2 headspace test, was also used to investigate the biodegradability of sixteen ILs containing a pyridinium cation in previous study,78 in which the IL with an easily hydrolizable 1-(2-hydroxyethyl) group in the side chain displayed high levels of biodegradation under aerobic condition. However, poor biodegradability was observed in the cases of ILs with methyl or ethyl in the side chain. A more detailed study of the effect of various anions, such as Br−, Cl−, [NTf2]− and [BF4]−, on the biodegradability of imidazolium-based ILs revealed that none of the ILs was biodegradable.74 However, the activated sludge could degrade the akyl chains of long-chain imidazolium-based 13



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cations; for instance, C8 chains were easily degraded. Biodegradation of the alkyl chains ceased when the chain length was reduced to two carbon atoms. Given the relatively good degradation of ILs by activated sludge, nine strains were isolated from activated sewage sludge with the selective pressure of [OMIm][Cl] by Markiewicz et al..75 Higher degradation of the IL was afforded by the isolated consortium as compared to the sewage sludge, mainly due to the difference of the cell density which had an influence on the degradation rate. Impressively, pyridinium-based ionic liquids, [EtPy][CF3COO] and [EtPy][BF4], were firstly reported to be biodegraded by an axenic culture of soil Coryne bacteria to low molecular organic acid products.79 Besides, a degradation pattern of the pyridinium ring in the C2 and C3 was proposed, which provided guidance suggestion for the following study on the degradation of this type of ILs. Recently, a systematical investigation on the biodegradation pathways of pyrininium, pyrrolidinium and ammonium-based ILs was reported by Deng et al.80 Apart from the ILs with a long-chain alkylpyridinium in C6, all the tested ILs had a degradation of 80-100% by a pure strain (Rhodococcus rhodochrous). However, in the cases of degradation by the activated sludge, the intermediate metabolites could be accumulated in the metabolic process, which exerted their different environment profile and fate. Although DESs have relative less toxicity, their environment footprints cannot be underestimated for the sustainable industrial exploitation. To date, there have been some reports 14


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on the toxicity of DESs.65,70, 81,82 Nevertheless, little knowledge was about the degradability of DESs. Notably, ChCl-based DESs with environmentally-friendly HBDs (glucose, glycerol, urea and so on) have been proved to be readily biodegradable.81,82 The superior biodegradability of the DESs, compared to traditional ILs, was mainly attributed to the relatively low ecotoxicity of the composed reagents (e.g. choline is an essential nutrient element for abundant organisms). Therefore, it is of great significance to better understand the relationship between toxicity and degradability

of

a

series

of

DESs

in

order

to

design

and

synthesize

more

environmentally-friendly DESs and to expand their practical use in biocatalysis. In a word, the biodegradability of ILs plays an important role in evaluating their environmental impact. In case of using ILs for whole-cell biocatalytic transformations, there may be conflict between the biotransformation application and environment friendliness. From an economic point of view, reusability of ILs is beneficial for large-scale biocatalytic processes, but (from the environmental point of view) it is advantageous if the ILs can be degraded by bacteria or fungi. We suggest that the best ILs would be selectively degraded by specific microbes; namely, the ILs cannot be metabolized by the microorganisms used to perform the biotransformation, but can be readily biodegraded by other microorganisms in the environment. For this reason, it is important to investigate the metabolic pathways via which common microorganisms used in biotransformations and those existing in the environment break down ILs, so that it will be

15



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possible to design green ILs that are resistant to degradation by the microorganisms used in biotransformation but are readily biodegradable in the environment.

WHOLE-CELL

BIOCATALYTIC

TRANSFORMATIONS

IN

ILS-CONTAINING

SYSTEM

As mentioned earlier, in many applications whole-cell biotransformations exhibit substantial advantages compared to free enzyme biocatalysis. Most of the studies, to date, on the use of whole-cell biocatalysts in ILs have focused on the production of chiral compounds, obtaining high yields and e.e. values in such reaction media. Especially, high substrate loading benefits from the introduction of hydrophobic ILs to a IL/buffer biphasic system. These biocatalytic processes mainly involve reduction, oxidation, hydrolysis and transesterification reactions (Table 4). Reduction reactions. The first literature about whole-cell biocatalysis in an IL-containing system was reported by Cull et al.2, who investigated the reduction of 1,3-dicyanobenzene (1,3-DCB) to 3-cyanobenzamide (3-CB) (Fig. 1a). It was shown that room-temperature ILs, such as [BMIm][PF6], can be successfully used in place of conventional solvents for the Rhodococcus R312-catalyzed biotransformation of 1,3-DCB in an aqueous/IL biphasic system. The substrate 1,3-DCB has very low aqueous solubility, but a high partition coefficient in the IL and organic phase. However, the initial rate of production of 3-CB in the water-[BMIm][PF6] system was 16


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slower than that in water-toluene system, due to the reduced rate of mass transfer of 1,3-DCB from the viscous IL phase. It was also shown that the specific activity of the biocatalyst in the water-[BMIm] [PF6] system was greater than in a water–toluene system. This suggested that the rate of production of 3-CB was limited by mass transfer of the substrate rather than by the activity of the biocatalyst. Moreover, in the water-toluene system, cells were found to aggregate near the interface, leading to formation of stable emulsions, which were not found with the solvent system containing [BMIm][PF6]. Hence, use of an IL in place of an organic solvent could simplify downstream separations. The biggest challenge and best solution is to prepare ILs that are of relatively low viscosity. Chiral alcohols are important intermediates for the synthesis of agrochemicals, liquid crystals, flavors, and pharmaceuticals especially. It has been reviewed that enantioselective reduction of ketones catalyzed by biocatalysts is an efficient and reliable route to obtain optically active alcohols.83 Hussain et al.84 investigated the steorospecific reduction of 6-Br-tetralone to the corresponding alcohol (S)-6-Br-β-tetralol in the presence of ILs using the yeast Trichosporon capitatum MY1890 and the bacterial strain Rhodococcus erthyropolis MA7213 (Fig. 1b). Five water-immiscible ILs and two water-miscible ILs were investigated as the second phase or used as additives in the process. For T. capitatum MY1890 bioconversions, 100% of conversion was obtained in the water-miscible hydrophilic IL [EMIm][TOS], which was much higher than that in the control water-ethanol (10% v/v) system. Of the water-immiscible ILs evaluated, 17



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[Oc(3)MeN][BTA] produced the best conversion (60%), but at a greatly reduced rate. For bioconversions carried out with R. erythropolis MA7213, [EMIm][TOS]–buffer was also the best co-solvent system, of which the conversion yield was doubled, and there was a four-fold increase in initial rate compared to the control. Overall, the yeast cells seemed to have better reaction performance than the bacteria. Where measured, enantiomeric excess values (e.e.) achieved in the presence of ionic liquids and controls containing 10% ethanol in place of the ionic liquid were similar and kept above 87 % (in favour of the S-enantiomer). The initial reaction rates of substrate conversion with the ILs were low, while the cell viabilities were higher than 80%.The low initial reaction rate was attributed to slower mass transfer rate of substrate from the IL phase to the aqueous phase, and the effect of the ILs on the cell membrane may inhibit entry of the substrate into cell and product egress. Additionally, the cells were observed to be dispersed within the medium, whereas in 10% ethanol solutions the cells aggregated to form a layer or film. Though a large proportion of studies demonstrated the feasibility of using ILs for whole cell bioreduction of ketones to their corresponding alcohols, no obvious correlation was apparent among the physiochemical properties of ILs, their influence on cell viability, and their effectiveness as media for whole-cell biocatalysis. Lou et al.85,86 investigated the enantioselective reduction of 4’-methoxyacetophenone (MOAP) to (R)-1-(4-methoxyphenyl)ethanol (R-MOPE) by immobilized Trigonopsis variabilis AS2.1611 cells and enantiocomplementary reduction of 18


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the same substrate to (S)-MOPE by immobilized cells of Rhodotorula sp. AS2.2241 cells (Fig. 1c). This was, to the authors’ knowledge, the first time reduction of MOAP to (R)-MOPE was successfully

achieved

in

an

IL-containing

co-solvent

system.

The

IL

used

was

[C2OHMIM][NO3], which was biocompatible with the cells and led to only a moderate increase in permeability of the cell membrane. Under optimized conditions, higher yield and product e.e. were afforded compared to those in aqueous buffer. The presence of IL in aqueous buffer allowed the cells to tolerate relatively high temperatures and substrate concentrations, and the immobilized cells manifested excellent operational and storage stability. In the enantiocomplementary system to reduce MOAP to (S)-MOPE, the water-immiscible IL [BMIm][PF6] was employed in an IL-aqueous biphasic system. Under the optimal conditions, the substrate conversion was up to 95.5% (compared to 68.6 % in aqueous buffer) and the product e.e. was >99%. The cells showed good reusability in the [BMIm][PF6]-aqueous biphasic system (8 batches with 90% of their original activity retained), which was much better than their performance in the monophasic buffer system (where only 25% of the original activity retained). The suitability of ILs for biphasic process design, has been explored in studies by Weuster-Botz and co-workers,87 where the enantioselective reductions of the prochiral ketones 2-octanone and 4-chloroacetophenone were used as model reactions, catalyzed by a whole-cell biocatalyst in the form of E. coli expressing alcohol dehydrogenase from L. brevis and formate dehydrogenase from Candida boidinii (Fig. 1d). Twenty-one ILs (encompassing seven different 19



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classes of cations and three different anions) were evaluated in a biphasic reaction systems. Good yields of (R)-2-octanol and (R)-1-(4-chlorophenyl)ethanol were obtained in biphasic systems containing [HMIm][PF6], or [NTf2] combined with any of the following cations: [(MOP)MPI] , [(MOE)MPL], [(EO2E)MPL] and [(NEMIm)EO2E], [BMIm], [HMIm], [BMPL] or [HMPL]. For example, the use of [HMPL][NTf2] led to the highest yield of 180 g (R)-2-octanol /L, chemical yield of 95%, and the product e.e. of >99%. Among the ILs tested, those containing the [PF6]and [NTf2]- anions generally gave better yields than the corresponding ILs with the [FAP]- anion. Only the monophasic aqueous system and two of the ILs tested ([(E2OH)MIm][NTf2] and [(P3OH)Pyr][NTf2]) gave yields of less than 2 %. Other enzymes such as the enantioselective ketoreductases from the cyanobacterium Synechococcus sp. strain PCC 7942, which can be expressed in active form in E. coli, may be suitable for performing effective reduction reactions in similar ionic liquid-containing systems.88 Oxidation reactions. In the first report of use of ILs to perform whole-cell oxygenase-catalysed reactions, Cornmell et al.19 investigated the bio-oxidation of toluene to toluene cis-glycol in a ILs-containing biphasic solvent system with E. coli expressing toluene dioxygenase from Pseudomonas putida F1 (Fig. 2a). The ILs were used in IL/aqueous biphasic systems, in which they acted as a substrate reservoir thus alleviating the substrate inhibition of the cells otherwise observed at high substrate concentrations. In the biphasic system containing [NMeOct3][NTf2] or [P6,6,6,14][NTf2] (IL:total volume phase ratio 0.23), the maximum toluene 20


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concentration tolerated was 75.2 mmol/L, which was an 8-fold increase compared with the control reactions without IL. The increased toluene concentration enabled by using either of these ILs resulted in a similar 2.5-fold increase in toluene cis-glycol concentration achieved in 50-ml scale flask biotransformations (to approximately 20 mM) compared with the single phase aqueous system. The product concentration with IL was also 10.5-fold higher than that achieved in a tetradecane:aqueous the biphasic system. Under these conditions the conversion of toluene to toluene cis-glycol was incomplete (27% vs. 88% with aqueous system). The possibility that this was due to oxygen limitation was tested by scaling the reaction in the aqueous:[P6,6,6,14][NTf2] system up to a 1.25 L, which resulted in a significant increase in product concentration (55.5 ± 22.5 mM) in the bioreactor compared with that in the flask cultures (20.7 ± 0.13 mM), consistent with the conclusion that productivity in the flask reactions was limited by availability of oxygen. In addition to the higher product concentrations afforded by use of the ILs, there were additional benefits in terms of absence of solvent emulsification, potentially improved safety and lower environmental impact. Fu et al.89 utilized whole cells of Armillaria luteo-virens Sacc to oxidize terpene betulin to betulinic acid, which has anti-HIV and anti-cancer activities as well as low toxicity to normal human cells,90 in IL-containing systems (Fig. 2b). In this study, four ILs, namely [BMIm][BF4], ([BMIm][PF6], [EMIm][BF4] and [OMIm][PF6], were investigated in separate co-solvent systems with aqueous buffer and hexane. Betulin-dependent oxygen consumption was used as a 21



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measure of betulin monooxygenase activity, which correlated with the production of the betulinic acid product. Under optimal conditions, a [BMIm][BF4]/hexane co-solvent system led to a yield of 11.14±1.51% after 18 h. This compared very favorably with an aqueous medium in which a yield of only 8.12 ± 0.88% was achieved after 72 h reaction time and also easily resulted in a loss of betulinic acid due to the extraction steps after reaction. Wu et al.91 also investigated the 11α-hydroxylation of 16α,17-epoxyprogesterone (EP) by Rhizopus nigricans in an IL-containing biphasic medium as a means of improving yields from this industrially important biotransformation (Fig. 2c). [BMIm][PF6] and [BMIm] [NTf2] were used as the second phase in the biotransformation of EP catalyzed by R. nigricans cells. Use of an [BMIm][PF6]-aqueous biphasic system greatly increased yield to 85 % in 24 h at 3 g/L feeding concentration (compared to less than 50 % achieved in a control aqueous system where the substrate was solubilised with ethanol). In contrast, the IL [BMIm][NTf2] afforded low yield of product and showed poor biocompatibility. Increasing the substrate concentration to 18 g/L and optimization of the phase ratio and in the [BMIm][PF6]-containing system led to an increased conversion of about 95%, and maintenance of the conversion to 87% after three reaction cycles with the aliquot of ionic liquid. Recently, Gao et al.92 reported the asymmetric sulfoxidation of a range of sulfides using a recombinant E. coli (P450pyrI83H-GDH) coexpressing P450pyrI83H monooxygenase and glucose dehydrogenase in an aqueous/ionic liquid biphasic system. The IL [P6,6,6,14]][NTf2] 22


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showed good biocompatibility to the E. coli and acted as a fine reservoir for the thioanisole. The problem of the substrate toxicity and inhibition to the sulfoxidation could be efficiently avoided by the KP buffer/IL biphasic system. As the same with other reports, the substrates concentration had a remarkable improvement, owing to their high distribution in the IL phase, which in turn caused the low substrate concentrations in the aqueous phase and further enhanced the enantioselectivity.

Hydrolysis reactions. Few publications have been reported about hydrolysis reactions in ILs-containing system with whole-cell as biocatalysts. Recently, a range of chiral chlorinated styrene oxide derivatives were prepared by marine cell-catalyzed hydrolyzation of styrene oxide.93 Two bacteria, Rhodococcus sp. YSMI04 and Roseobacter sp. TSBP12, with epoxide-degrading activity were isolated from the oil-spilled foreshore and demonstrated different and complementary enantioselectivity. However, this hydrolysis process was performed in an aqueous system. Chen et al.94 studied the production of glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) via selective hydrolysis of the glycosidic linkage in glycyrrhizin (GL) by whole-cell biocatalysis in an IL/buffer biphasic system based on [BMIm][PF6] (Fig. 3a). Three whole-cell biocatalysts were investigated: wild-type Penicillium purpurogenum Li-3 (w-PGUS) and recombinant strains (E. coli BL21 and P. pastoris GS115) expressing the β-D-glucuronidase gene from w-PGUS. The wild-type strain w-PGUS gave highest GAMG yield. After 60 h under optimal reaction 23



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conditions, a GAMG yield of 87.63% was achieved in the IL-containing biphasic system, which was a much higher yield than that in the monophasic buffer system (57.3%). When [BMIm][PF6] was reused in successive cycles, 85% of the original amount of IL was recovered after 8 batch reaction cycles. The product GAMG and the byproduct glycyrrhetic acid (GA) partitioned into the aqueous and IL phases, respectively, and so the desired product could be isolated from the aqueous phase as long as hydrolysis of the GAMG starting material was complete. Hence, the combination of a whole-cell hydrolytic biocatalysis and an IL-containing system provided a promising and economic way for the industrial production of GAMG by regioselective hydrolysis of GL. An E. coli whole-cell system co-expressing (S)-hydroxynitrile lyase from the cassava (Manihot esculenta) and an arylacetonitrilase from Pseudomonas fluorescens EBC191 has been studied for production of (S)-mandelic acid and (S)-mandeloamide from benzaldehyde and cyanide in ILs-containing media(Fig. 3b).95 [BMPL][NTf2] and [BMIm][PF6] were employed to form IL-containing biphasic systems, which systems allowed benzaldehyde to be dissolved in the IL phase to a concentration of 700 mM and thus to enable a high substrate conversion of 87–100%. In both biphasic systems benzaldehyde and cyanide were converted into (S)-mandeloamide and (S)-mandelic acid with e.e. > 94%. The use of the IL-containing biphasic systems also significantly reduced the inhibitory effect of benzaldehyde on the nitrilase activity. In contrast to other IL systems used previously with this reaction, where the organic substrates and the 24


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products were usually accumulated in the IL phase, here one of the substrates (HCN) and both products (mandelic acid and mandeloamide) predominantly partitioned in the aqueous phase.

Transesterification reactions. Generally, the transesterification process was catalyzed by isolated enzymes like lipase. Interestingly, IL-catalyzed transesterification reactions have been reported by some researchers.96,97 Arai et al.98 investigated the transesterification of a triglycerides from soybean oil with methanol (methanolysis), to produce fatty acyl methyl esters that could be used as biodiesel, catalyzed by fungal whole-cell biocatalysts in IL-containing biphasic systems (Fig. 4). Cells of the triacylglycerol lipase-producing wild-type Rhizopus oryzae (w-ROL) were investigated, together with three strains of recombinant Aspergillus oryzae expressing different lipases. The wild-type fungus w-ROL gave the highest yield of fatty acyl methyl esters (ME) in the [EMIm][BF4] and [BMIm][BF4]-based biphasic systems after 24 h of reaction. Although lipases were inactivated by an excess of methanol in conventional solvent systems, methanolysis proceeded even with a methanol/oil ratio of 4 in the IL-based biphasic system. Here the ILs functioned as a reservoir of methanol to alleviate deactivation of the enzyme. Whilst native w-ROL became deactivated in [BMIm][BF4] and lost their reusability, addition of glutaraldehyde made the biocatalyst more stable and recyclable. Compared to other conventional methods, e.g., alkali-catalyzed methanolysis, this whole-cell based bioprocess exhibited environmentally friendly and high conversion rate. It also demonstrated that ionic

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liquids are promising candidates for use in solvent systems for producing biodiesel via whole-cell biocatalysts.

WHOLE-CELL BIOCATALYTIC TRANSFORMATIONS IN DES-CONTAINING SYSTEM DESs, a new class of ILs, have been emerging as novel green solvents used in many respects and much attention has been paid to their applications in biocatalytic processes with successful results,106-110 owing to their nontoxic nature, good biodegradability and low cost. The first investigation about DES as an alternative solvent for biocatalysis was reported by Gorke et al..111 Up to now, few works have been published about the whole-cell biocatalysis in DES-containing systems.112,113 Maugeri et al.101, for the first time, reported the enantioselective reduction of ketone in a DES-water co-solvent system catalyzed by baker’s yeast (Fig. 5a). Though a relatively long reaction time was needed, an interesting phenomenon was observed, in which the enantioselectivity had a complete inversion with varying of the proportion of the DES added in the reaction system. It was speculated that the DESs could inhibit the enantioselective enzymes activity at different proportion thus leading to the formation of product with inverse configuration. Our research group has also evaluated the biocatalytic oxidation and reduction reactions catalyzed by the whole-cell of Acetobacter sp. CCTCC M209061 (Fig. 5b and 5c).104,105 High substrate loading and excellent catalytic performance were observed in the DES-containing system (urea and glycol as HBDs, respectively). The toxicity of substrate to the 26


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microbial cell could be weakened by the addition of certain DESs. Muller et al.114 reported the bioreduction of a series of ketones with recombinant E. coli cells in the presence of a relatively high concentration of DES (DES/buffer, 80:20, v/v) (Fig. 5d). In this case, diverse DESs showed different effects on the whole-cell biocatalytic process, and the DES ChCl/glycerol presented the best results with a markedly enhanced product e.e.. Obviously, DESs as a new class of green solvent manifested great potential for biocatalytic processes.

EFFECT OF ILS ON WHOLE-CELL BIOCATALYTIC PROCESS

As the foregoing examples illustrate, ILs can exert both positive and negative effects on whole-cell catalyzed biotransformation reactions. It is of significant worth to get deeper insight into how IL affect the biocatalytic process. In this section four main aspects of the influence of ILs on whole-cell biocatalysts will be discussed: (1) cell viability, (2) permeability, (3) partition coefficient of substrates and products, (4) activity of cell-associated enzymes. Effect on cell viability. The general toxicity of ILs to microbial cells has been covered in section 2 above. Here, we discuss current understanding about the specific mechanisms via which the toxicity of ionic liquids can impact upon the performance of whole-cell biocatalysts and how toxicity problems are evaluated in practice. Cell viability and membrane integrity are commonly used as indicators of IL toxicity. Clearly, biocompatibility of ILs must be established before their application in whole-cell biocatalytic processes.95 27



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Our group have investigated the biocompatibility of various ILs with Acetobacter sp. CCTCC M209061 by directly measuring the glucose metabolic activity retention (MAR) of the cells. MAR is the percent remaining metabolic activity after treatment with the IL (compared to a control treated with aqueous buffer). It is a measure of the cells’ tolerance to ILs and an easy indicator of cell viability cells.115 The MAR value was lower for cells in all the IL-containing systems (containing 5% v/v water-miscible IL in an IL:aqueous buffer monophasic system) than in aqueous buffer alone, showing that the ILs were all toxic to Acetobacter sp. CCTCC M209061 cells to some extent. Moreover, the MAR value in IL media containing the biotransformation substrate 4-(trimethylsilyl)-3-butyn-2-one (TMSBO; 6 mM) significantly decreased with the elongation of the alkyl chain on the IL cation. Addition of a hydroxyl group onto the cation [EMIm]+ improved the biocompatibility of IL toward the microbial cells. The reason might be that the hydroxyl-functionalized IL [EOHMIm][NO3] and water can form an OH-bridged protic environment which is favorable to the activity of the biocatalysts. BF4- and Cl-based ILs (at 10 and 23% v/v, respectively) showed low biocompatibility and caused significant decrease in cell viability, leading to the loss of the catalytic activity.19,116 However, it cannot simply be concluded that BF4- or Cl- is more toxic towards the cells because different kinds of IL used, IL content(v/v), the property of substrate and product, the time of exposure, the indicator microorganisms used and even different biomass concentrations may substantially influence the cell survival and metabolism. There is a pressing need for additional systematic studies on the 28


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combined effects of these factors on cell viability in order to facilitate industrial applications of ILs. Effect on cell membrane permeability. Cell membrane permeability is an important factor for the efficiency of whole-cell biotransformation processes.117 ILs may be able to increase cell membrane permeability and thus allow substrates and products to diffuse more readily into and out of the cells, resulting in acceleration of biocatalysis.85 Conversely, loss of membrane integrity might reduce cell viability, compromise cell metabolism and diminish biocatalytic reaction yields. Brautigam et al.118 reported the effect of imidazolium-based ILs on the cell membrane integrity (MI) of a recombinant E. coli strain engineered for catalyzing asymmetric reduction reactions. It showed that [PF6]- anion hold a marginally destructive influence on the MI of the cell, which decreased to 70% after being exposed to ILs for 5h, far lower than that in aqueous system (95%). In contrast, [NTf2]- anion caused greater damage to the cell membrane and the most damaging effect on the membrane was observed for the ILs containing the [E3FAP]- anion. Our group has also studied the effect of various ILs on cell membrane permeability with Acetobacter sp. CCTCC M209061.115 The changes in the A260 and A280 values, an indicator of the release of intracellular components (presumably mostly nucleic acids and proteins) into the medium, after removal of the cells. Different types of ILs had distinctly different effects on the permeability of the cell membrane . With BF4--based ILs, A260 and A280 values due to release of 29



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cellular components were relatively high, suggesting that these ILs greatly increased cell membrane permeability. However, in the presence of these BF4-based ILs, the cells had very poor catalytic activity. Thus it appeared that the BF4--based ILs damaged the cell membrane too seriously to maintain effective catalytic activity of the cells. Among all the ILs investigated, the greatest cell membrane integrity and the lowest A260 and A280 values were recorded for [EOHMIm][NO3] ,which also gave the best catalytic performance of the cells.. It appeared that a moderate increase in cell membrane permeability can enhance reaction efficiency whereas a large increase in permeability would put an irreversibly activity-lowering effect on the cell. It is possible that ILs increase the cell membrane permeability by interacting with the hydrophobic tail regions or charged head groups of membrane phospholipids. The speculation has been reinforced in another study by Yoo et al.119 The interaction between imidazolium-based ILs and the phosphatidylcholine (PC) lipid bilayer was shown by molecular dynamics simulation and free energy calculation. Interestingly, ILs behaved with the bilayer in a mechanism as ionic surfactants function, namely, the hydrophobic head could insert into the lipid bilayer. Long alkyl substituents of 1-alkyl-3-methylimidazolium chlorides IL could cause the membrane permeabilisation of A. nidulans and the heterogeneous charge distribution along the membrane surface might be related to this phenomenon.120 On the other hand, an increase in cell membrane permeability due to the ILs could be detrimental to biocatalysis owing to damage to the cell

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membrane that may lead to cell death and diminished availability of reducing equivalents for the reaction. 24, 117

Effect on partition coefficients of substrate and product. It has been proposed that equilibrium distribution coefficients of substrate and product are important, together with data about the toxicity of ILs, in predicting process efficiency of whole-cell biocatalysts in IL/aqueous

systems.121

Studying

the

reduction

of

4-chloroacetophenone

to

(R)-1-(4-chlorophenyl)ethanol (1-4-Cl-PE) catalysed by Lactobacillus kefir as a model reaction, they calculated that in systems containing organic solvents (such as an aqueous:decane biphasic system) yield would be diminished owing to the low distribution coefficient (log D) value for the product 1-4-Cl-PE (1.54) between the aqueous phase and decane, which meant that in a 20% solvent setup approximately 11.5% of the product was lost because it remained in the aqueous phase. With [BMIm][NTf2] as the co-solvent the log D value was substantially greater (2.03) and this loss would account for only 3.7%. Wang et al.122 carried out the bioreduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] in a series of IL-based biphasic systems. The partition coefficient for EAA in the [C4mim][PF6]/buffer biphasic system was much greater than that observed in the n-hexane/water biphasic system, suggesting that the IL [C4mim][PF6] was more suitable than n-hexane for the delivery of ATMS to the aqueous phase. This is probably responsible for the improved initial rate and yield observed when n-hexane was replaced by 31



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[C4mim][PF6] in the biphasic system. Notably, the partition coefficient of EAA decreased with the elongation of the side chain of cation as for the PF6- and Tf2N-based ionic liquid. However, a contrary result was afforded by Mao et al.,123 that higher distribution coefficients of 16α, 17-epoxyprogesterone were observed with increasing alkyl chain length. Thus, the effect of various ILs on the partition coefficient depends on the characteristic of substrate to some degree. Other studies have also indicated similar importance of the partition coefficient in reaction productivity.124,125

Greater partitioning of toxic substrates and products into the IL phase could reduce the effect of such toxic compounds on the cells, as well as alleviating any substrate or product inhibition observed in monophasic aqueous systems.3, 87 Partition coefficient in biphasic systems containing ILs is a function both of the IL and the chemical nature of the substrates and products. Larger partition coefficients, in favor of the increased partitioning of the substrate and product into the IL phase, correlated with greater biocompatibility of the IL with the cells and overall biocatalytic process efficiency.126 Although it is currently not possible reliably to predict the partition coefficients of substrates and products within IL-containing biphasic systems, appropriate IL-containing systems can in principle be selected for specific microbial catalytic reactions based on the property of the substrate and product. We suggest that further research into partition coefficients in IL-containing systems and development of better models to predict them will be an important direction for improving the efficiency of the whole-cell biotransformation process. 32


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Effect on enzymes within microbial cells. The effect of ILs on intracellular enzymes is inevitable, once the cell membrane integrity is destroyed by ILs, as demonstrated by previous reports.86, 127 In our early work, fluorescence microscopy analysis demonstrated that ILs could enter Rhodotorula sp. AS2.2241 cell and accumulate inside cell membrane,86 where the redox enzymes distributed, indicating that ILs might influence the catalytic performances of the intracellular enzymes. Subsequently, several researchers further demonstrated that ILs affected significantly the enzyme systems present in Aspergillus nidulans cells and consequently led to the change of the metabolism of intracellular chemicals.128-131 In the case, the metabolic pathways of carbohydrate and amino acid in Aspergillus nidulans were altered after the cells were exposed to ILs, possibly because of the expression change of the related enzyme genes.131 Similarly, it was also observed that the IL [Amim][BF4] affected the expression of enzyme genes present in Vibrio qinghaiensis sp.-Q67 cell,132 where several genes expressing luciferase, superoxide dismutase and catalase were up-regulated in a time-dependent hormesis manner. The above-mentioned observations clearly indicate that ILs could affect the enzymes present in microbial cells at a gene level. However, the specific effects and detailed mechanism of the ILs on the intracellular enzymes involved in the biotransformation remains largely unknown. More comparable and systematic experimental data as well as more accurate test methods are needed to understand the relationship between ILs and the cell metabolism and the enzymes involved in the whole-cell based biotransformation. 33



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CONCLUSIONS Application of ILs in whole-cell biocatalysis shows great potential for industrial applications. Avoiding expensive enzyme extraction procedures and co-factors make such approaches highly competitive compared to biocatalysis using purified enzymes. A substantial number of studies have shown that whole-cell based biocatalysis can exhibit high conversion rate, high stereoselectivity and favorable IL and biocatalyst re-use properties. Hydrophobic ILs are often used as substrate/product reservoir to circumvent the issues of low substrate aqueous solubility and substrate/product inhibition. However, there are some bottlenecks with regard to use of ILs in industrial applications, one of which is the toxicity of ILs towards microbial cells. Whilst important progress has been made in surveying the toxicity of ILs, additional work is needed to give a more nearly complete set of data for the toxicity of the extremely large number of ILs that are available. Another limitation to the application of ILs is the lack of understanding on the mechanism of the effect of ILs on whole cell-based biocatalytic processes. It has been shown that ILs can be accumulated into microbial cells and affect the permeability of the membrane. Further studies are now needed to determine how ILs interact with the cell membrane and influence reactions within the cell at the enzyme and gene control levels. Besides, in addition to the ongoing discoveries of novel and renewable ILs, the development of whole-cell biocatalysts designed with special characteristic through gene and protein engineering further provide the possibility to meet the process prospect in a sustainable way. Studies of this type will greatly 34


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enhance the uptake of ILs as media of whole-cell based biocatalysis in the research and commercial area.

AUTHOR INFORMATION Corresponding author * E-mail: [email protected] (Prof. W. Y. Lou); Tel.: +86-20-22236669. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We wish to thank the National Natural Science Foundation of China (21336002; 21222606; 21376096), the Program of State Key Laboratory of Pulp and Paper Engineering (2015C04), the Key Program of Guangdong Natural Science Foundation (S2013020013049), the Fundamental Research Funds for the Chinese Universities (2015PT002; 2015ZP009), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering for partially funding this work.

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110. Monhemi, H.; Housaindokht, M. R.; Moosavi-Movahedi, A. A.; Bozorgmehr, M. R. How a protein can remain stable in a solvent with high content of urea: insights from molecular dynamics simulation of Candida antarctica lipase B in urea : choline chloride deep eutectic solvent. PCCP 2014, 16 (28), 14882-14893. 111. Gorke, J. T.; Srienc, F.; Kazlauskas, R. J. Hydrolase-catalyzed biotransformations in deep eutectic solvents. Chem. Commun. 2008, (10), 1235-1237. 112. Gutiérrez, M. C.; Ferrer, M. L.; Yuste, L.; Rojo, F.; del Monte, F. Bacteria Incorporation in Deep-eutectic Solvents through Freeze-Drying. Angew. Chem. Int. Ed. 2010, 49 (12), 2158-2162. 113. Mao, S.; Yu, L.; Ji, S.; Liu, X.; Lu, F. Evaluation of deep eutectic solvents as co-solvent for steroids 1-en-dehydrogenation biotransformation by Arthrobacter simplex. J. Chem. Technol. Biot. 2015, DOI: 10.1002/jctb.4691. 114. Müller, C. R.; Lavandera, I.; Gotor-Fernández, V.; Domínguez de María, P. Performance of Recombinant-Whole-Cell-Catalyzed Reductions in Deep-Eutectic-Solvent-Aqueous-Media Mixtures. ChemCatChem 2015, 7 (17), 2654-2659. 115. Xiao, Z. J.; Du, P. X.; Lou, W. Y.; Wu, H.; Zong, M. H. Using water-miscible ionic liquids to improve the biocatalytic anti-Prelog asymmetric reduction of prochiral ketones with whole cells of Acetobacter sp. CCTCC M209061. Chem. Eng. Sci. 2012, 84, 695-705.

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116. Zhang, B. B.; Lou, W. Y.; Chen, W. J.; Zong, M. H., Efficient Asymmetric Reduction of 4-(Trimethylsilyl)-3-Butyn-2-One by Candida parapsilosis Cells in an Ionic Liquid-Containing System. Plos One 2012, 7 (5), e37641. 117. Gangu, S. A.; Weatherley, L. R.; Scurto, A. M. Whole-Cell Biocatalysis with Ionic Liquids. Curr. Org. Chem. 2009, 13 (13), 1242-1258. 118. Bräutigam, S.; Bringer-Meyer, S.; Weuster-Botz, D. Asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems by use of recombinant Escherichia coli with intracellular cofactor regeneration. Tetrahedron: Asymmetr. 2007, 18 (16), 1883-1887. 119. Yoo, B.; Shah, J. K.; Zhu, Y.; Maginn, E. J. Amphiphilic interactions of ionic liquids with lipid biomembranes: a molecular simulation study. Soft Matter 2014, 10 (43), 8641-8651. 120. Gal, N.; Malferarri, D.; Kolusheva, S.; Galletti, P.; Tagliavini, E.; Jelinek, R. Membrane interactions of ionic liquids: Possible determinants for biological activity and toxicity. Biochim. Biophys. Acta 2012, 1818 (12), 2967-2974. 121. Pfruender, H.; Amidjojo, M.; Kragl, U.; Weuster-Botz, D. Efficient whole-cell biotransformation in a biphasic ionic liquid/water system. Angew. Chem. Int. Ed. 2004, 43 (34), 4529-4531. 122. Wang, X. T.; Yue, D. M.; Zong, M. H.; Lou, W. Y. Use of Ionic Liquid To Significantly Improve Asymmetric Reduction of Ethyl Acetoacetate Catalyzed by Acetobacter sp. CCTCC M209061 Cells. Ind. Eng. Chem. Res. 2013, 52 (35), 12550-12558.

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123. Mao, S.; Hua, B.; Wang, N.; Hu, X.; Ge, Z.; Li, Y.; Liu, S.; Lu, F. 11α hydroxylation of 16α, 17-epoxyprogesterone in biphasic ionic liquid/water system by Aspergillus ochraceus. J. Chem. Technol. Biot.. 2013, 88 (2), 287-292. 124. Lou, W. Y.; Chen, L.; Zhang, B. B.; Smith, T. J.; Zong, M. H. Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells. BMC Biotechnol. 2009, 9, 90. 125. Wang, W.; Zong, M. H.; Lou, W. Y. Use of an ionic liquid to improve asymmetric reduction of 4′-methoxyacetophenone catalyzed by immobilized Rhodotorula sp. AS2.2241 cells. J. Mol. Catal. B: Enzym. 2009, 56 (1), 70-76. 126. Zhang, B. B.; Cheng, J.; Lou, W. Y.; Wang, P.; Zong, M. H. Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems. Microb. Cell Fact. 2012, 11, 108. 127. Cornmell, R. J.; Winder, C. L.; Tiddy, G. J. T.; Goodacre, R.; Stephens, G. Accumulation of ionic liquids in Escherichia coli cells. Green Chem. 2008, 10 (8), 836-841. 128. Petkovic, M.; Hartmann, D. O.; Adamova, G.; Seddon, K. R.; Rebelo, L. P. N.; Pereira, C. S. Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New J. Chem. 2012, 36 (1), 56-63. 129. Hartmann, D. O.; Silva Pereira, C. A molecular analysis of the toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans. New J. Chem. 2013, 37 (5), 1569-1577.

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130. Hartmann, D. O.; Shimizu, K.; Siopa, F.; Leitao, M. C.; Afonso, C. A. M.; Canongia Lopes, J. N.; Silva Pereira, C. Plasma membrane permeabilisation by ionic liquids: a matter of charge. Green Chem. 2015, 17 (9), 4587-4598. 131. Martins, I.; Hartmann, D. O.; Alves, P. C.; Planchon, S.; Renaut, J.; Leitão, M. C.; Rebelo, L. P. N.; Silva Pereira, C. Proteomic alterations induced by ionic liquids in Aspergillus nidulans and Neurospora crassa. J. Proteomics 2013, 94, 262-278. 132. Yu, Z.; Zhang, J.; Liu, S. Biochemical and gene expression effects of 1-alkyl-3-methylimidazolium tetrafluoroborate on Vibrio qinghaiensis sp.-Q67. J. Hazard. Mater. 2015, 300, 483-492.

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Table 1. The cations of ILs used for biocatalysis Name

Abbreviation

Structure +

Imidazolium

[IM]+

N

N

R1 R2

+

R2

Pyridinium

[Pyr]+

N

R1

+

Pyrrolidinium

[PL]+

N

R1

R2

+

R1

Quaternary ammonium

[Na b c d]+

N

R4

R2 R3

+

R1

Quaternary phosphonium

[Pm n o p]+

P

R4

+

R2

R1

Morpholinium

R2 R3

N

[CnMOR]+ O +

R

Quinolinium

N

[Cnquin]+

+

Piperidinium

[PI]+

N R2

Choline

[Ch]

HO

55


R1

N+


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 2. The anions of ILs used for biocatalysis Name

Abbreviation

Structure -

F

[BF4]-

Tetrafluoroborate

B

F

F

F

F

[PF6]-

Tetrafluoroborate

F

F P

F

F F

Bis[(trifluoromethyl)sul-fonyl] -imide

[NTf2]-

-

O

O

O

O

-

S

S CF 3

CF3

N _ O

O

[OTf]-

(trifluoromethyl) sulfonyl

S CF3

O

-

O O

[TOS]-

Tosylate

O

Alkyl sulfate

[CnOSO3]

-

O

S

_

O S

R1

O

O

-

O

2-Hydroxypropanoate

[lactate]

OH

O

_ O

[CnCOO]-

Linear alkanoate

O

R1 _

O O S

[sacch]-

Saccharinate

N

O

O

4-Methoxyphenyl sulfonate

[tosylate]

-

_ O

S O

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_

O O

1,4-Bis(2-ethylhexoxy)-1,4-di oxo-butane-2-sulfonate

O

[AOT]-

S

O

O O O

_ COO

Amino acid

[AA]-

C NH3 H R

-

Dicyanamide

[N(CN)2]

-

C

N

C

N

N O

O

Nitrate

NO3

-

N O N

Benzotriazole

[BTA]-

N

N H

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Table 3. Toxicity of some imidazolium-based ILs to microorganisms ILs

IC50(%v/v) Microorganisms

Indicator

Refs

[BMIM][PF6]

0.5

Escherichia coli

Growth inhibition

32

>20

Escherichia coli

Cell viability

20

>20

Saccharomyces cerevisiae

Cell viability

20

20


Growth inhibition

43

[BMIM][BF4] 15

Escherichia coli

Growth inhibition

44

15

Proteus vulgaris

Growth inhibition

44

1

Escherichia coli

Growth inhibition

45

7

Photobacterium phosphoreum

Inhibition of light emission 46

15

Escherichia coli

Growth inhibition

44

15

Proteus vulgaris

Growth inhibition

44

20


Growth inhibition

43

[HMIM][BF4] 16

Escherichia coli

Growth inhibition

44

16

Proteus vulgaris

Growth inhibition

44

[OMIM][PF6]

20


Growth inhibition

43

[DMIM][PF6]

20


Growth inhibition

43

[HMIM][PF6]

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Table 4. Examples of application of whole-cell transformation in ILs-containing system Entry

Reaction

IL

Microorganisms

Substrate

Comments

[BMIm][PF6]

Rhodococcus R312

1,3-dicyano-

The special activity of the

Refs

type 1

Reduction

2

biocatalyst in the water-IL benzene

system was higher than in water-toluene system

2

Reduction

[EMim][EtSO4]

Clostridium

nitrobenzene

sporogenes

Higher yield (79%) was

99

obtained compared with that with hexane as second phase

3

4

Reduction

Reduction

[N1,1,1,1][Cys]

[EMIm][TOS]

Trichoderma

3,5-bis(trifluoromet

The designed IL could

asperellum

hyl)

strengthen the coenzyme

ZJPH0810

acetophenone

regeneration

Trichosporon

6-Br-tetralone

Higher conversion was obtained

capitatum MY1890

100

84

for both the strains compared to ethanol co-solvent system

Rhodococcus erthyropolis MA7213

5

Reduction

[C2OHMIM][NO3]

Trigonopsis

4’-methoxy-

[BMIm][PF6]

variabilis AS2.1611

The IL with good

85-86

biocompatibility could acetophenone

moderately increase the cell

Rhodotorula sp.

membrane permeability, thus

AS2.2241

leading to high product yield and e.e.

6

Reduction

[HMIm] [PF6]

Recombinant E.coli

2-octanone

ILs with [PF6]-and [NTf2]-

87

anions gave better yields than [HMPL][NTf2]

4-chloro-

those with [FAP]- anion

acetophenone

7

Reduction

choline

Bake’s yeast

ethyl acetoacetate

Inversion of enantioselectivity was observed with varying of

59


101


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chloride/glycerol

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the proportion of the DES added

8

Reduction

[BMIm][NTf2]

Recombinant E.coli

3-chloro-1-phenyl-1

The substrate loading was

-propanone

improved from 30mM to 100

102

mM

9

Reduction

[BMIm][PF6]

S. cerevisiae

(Z)-3-halo-4-phenyl

-3-buten-2-one

10

Reduction

choline

Acetobacter sp.

chloride/urea

CCTCC M209061

3-chloropropiophen

The IL could efficiently

103

decreased the inhibitory effect of substrate

The substrate concentration had

104

a 3-fold improment

one

11

Oxidation

[NMeOct3][NTf2]

Recombinant E.coli

toluene

[P6,6,6,14][NTf2]

Both the substrate and product

19

concentration had a significant improvement of 2.5-fold and 10.5-fold, respectively, in the biphasic system

12

Oxidation

[BMIm][BF4]

Armillaria

terpene betulin

luteo-virens Sacc

The product yield had an

89

increase about 3% in a shorter

[BMIm][PF6]

reaction time of 18h

[EMIm][BF4] [OMIm][PF6]

13

Oxidation

[BMIm][PF6]

Rhizopus nigricans

16α,17-epoxyproge

Conversion up to 95% was

sterone

afforded, about 2-fold in an

91

aqueous system

14

Oxidation

[P6,6,6,14][NTf2]

Recombinant E.coli

thioanisole et al.

A remarkable improvement in substrates loading was observed in the KP buffer/IL biphasic

60


92

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system

15

Oxidation

choline

Acetobacter sp.

1-(4-methoxypheny

The substrate loading was

chloride/glycerol

CCTCC M209061

l)ethanol

substantially increased (55 mM

105

vs. 30 mM)

16

Hydrolysis

[BMIm][PF6]

Penicillium

glycyrrhizin

purpurogenum Li-3

A yield of 87.6% after 60h was

94

much higher than that in the buffer system (57.3%)

17

Hydrolysis

[HMPL][NTf2]

Recombinant E.coli

benzaldehyde

High substrate concentration of

cyanide

700 mM and conversion of

[BMIm][PF6]

18

Transeste

[EMIm][BF4]

95

87-100% were observed

Rhizopus oryzae

-rification

triglyceride and

High conversion rate was

methanol

afforded owing to the alleviated

[BMIm][BF4]

toxicity of methanol to the enzymes

61


98


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Figure captions Figure 1 The examples of whole-cell biocatalytic reduction reaction in ILs.

Figure 2 The examples of whole-cell biocatalytic oxidation reaction in ILs.

Figure 3 The examples of whole-cell biocatalytic hydrolysis reaction in ILs.

Figure 4 The examples of whole-cell biocatalytic transesterification reaction in ILs.

Figure 5 The examples of whole-cell biocatalysis with DESs.

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a)

N

O

N

N

NH2

IL/buffer Rhodococcus R312 3-CB

1,3-DCB

b)

Br

IL/buffer

H

HO

HO

MeO

IL-containing system

Trigonopsis variabilis AS2.1611 cells MeO

Rhodotorula sp. AS2.2241 cells MeO (S)-MOPE

MOAP

(R)-MOPE O

OH

IL/buffer recombinant E. coli 2-Octanone

(R)-2-Octanol

Figure 1 Lou et al.

63


H

(R)-6-Br-beta-tetralol

O IL-containing system

d)

OH

(S)-6-Br-beta-tetralol

6-Br-beta-tetralone HO

Br +

Whole cells

O

c)

Br


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a)

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OH

IL/buffer E. coli

OH Toluene cis-glycol

Toluene

CH2

CH2 H 3C

b)

C

H

H

CH CH3 3

IL-containing system H3 C

Armillaria luteo-virens Sacc

HO

O C

H CH CH3 3

HO

HO

H

H

Betulinic acid

Betulin O O

c)

C

HH

CH2

H3 C H HO

H 3C

O O

HO

IL/buffer R.nigricans O

O

11-hydroxy-EP

EP

d)

O S

Me

S

IL-containing system

Me

recombinant E. coli

(R)-Phenyl methyl sulfoxide

Thioanisole

Figure 2 Lou et al.

64


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HO

a)

HO

O H

O

O OH

O OH OHOH

OH

O

+

H

H O OH

HO

COOH

H

O

IL/buffer Penicillium purpurogenum Li-3 O

O

HO

H

O H

COOH O

O

HO COOH

OH

GAMG (product)

GL (substrate)

b)

H O

H

OH

IL/buffer

CONH2

OH COOH

+

recombinant E.coli

Benzaldehyde

GA (by-product)

(S)-Mandeloamide

Figure 3 Lou et al.

65


(S)-Mandelic acid


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 66 of 73

CH2OOR1 CHOOR2

CH2 OH +

CH3 OH

IL/buffer Rhizopus oryzae

CH3OOR1

CH2OOR3

+

CHOOR2 CH2 OOR3

Figure 4 Lou et al.

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a)

O

O

OH

O

DES-water O

bake's yeast

O

Ethyl acetoacetate

(R)-or (S)-ethyl 3-hydroxybutyrate OH

O

b) Cl

Cl

ChCl/urea-water Acetobacter sp. cells

(S)-3-Chloro-phenylpropanol

3-Chloropropiophenone

c) HO O

Acetobacter sp. cells

rac-MOPE

d)

HO +

ChCl/glycol-water O

MOAP

O

H

S-MOPE OH

DES-buffer recombinant E.coli (S)-1-Phenyl-1-propanol

Propiohenone

O

O

Figure 5 Lou et al.

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Whole-cell biocatalytic processes with ionic liquids Pei Xu, Gao-Wei Zheng, Peng-Xuan Du, Min-Hua Zong, Wen-Yong Lou

The review has focused on use of ionic liquids for whole-cell biocatalytic processes and their effect behaviors on biocatalysis.

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Pei Xu is currently a Ph.D. student under the supervision of Prof. Wen-Yong Lou at the South China University of Technology (SCUT). His main research interests include the whole-cell biotransformation with untraditional solvents (ionic liquids and deep eutectic solvents) and the degradation of shrimp chitin waste with microbial fermentation. Currently, he has published three scientific papers, mainly in the area of asymmetric biocatalytic reduction and oxidation of chiral compounds.

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Gao-Wei Zheng is an associate professor at the East China University of Science and Technology (ECUST), China. He received his Ph.D. in Biochemical Engineering from the ECUST in 2011, and then joined the State Key Laboratory of Bioreactor Engineering of ECUST as an assistant professor, became associate professor in 2013. His research centres on organic synthesis via biocatalytic approaches. Particularly, he is interested in development of novel enzymes or creation of tailor-made enzymes in organic synthesis, redox reactions employing reductases and mutilenzymatic cascade reactions. Currently, he is the author and co-author of 28 journal papers and 6 patents, publishing mainly in the area of biocatalysis.

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Peng-Xuan Du was born in Anyang, China, in 1989. He obtained a master degree under the supervision of Prof. Min-Hua Zong in Fermentation Engineering (2013) from the South China University of Technology (SCUT). At the Lab of Applied Biocatalysis, Du has developed a biphasic system combining ionic liquids and deep eutectic solvents, used in the efficiently biocatalytic anti-Prelog reduction of prochiral ketones with whole cells.

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Page 72 of 73

Min-Hua Zong is a professor of biochemical engineering at South China University of Technology (SCUT). She received her Ph.D. in chemical engineering in 1988 from SCUT. From 1990-1991, she worked as a researcher at Kyoto University in Japan. She has been researching biocatalysis and biotransformation in non-aqueous media including ionic liquids for the past 25 years. Her current research interests have focused on enzyme catalysis in novel solvent media and immobilization of enzyme onto novel supporting materials. Currently, she is the author and co-author of over 200 journal papers and 20 patents.

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Wen-Yong Lou is currently a professor at South China University of Technology (SCUT). He received his Ph.D. in biochemical engineering in 2005 from SCUT. He was awarded the National Excellent Doctoral Dissertation Award in 2009, and obtained the National Science Fund for Excellent Young Scholars in 2012. His research interests have focused on biocatalysis in novel solvent media (ionic liquids, deep eutectic solvents), with particular attention to catalytic performances of microbial cells in ionic liquids and effect of ionic liquids on conformation of enzyme. He started research in the area of biocatalytic transformations in ionic liquids in 2001. He has published over 90 papers with 56 in the field of ionic liquids and deep eutectic solvents.

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Whole-Cell Biocatalytic Processes with Ionic Liquids - ACS Publications

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