A process utilizing a thermomorphic deep eutectic solvent system within biocatalytic applications to recover the biocatalyst and the products

Technical field of the invention

The present invention relates to a process utilizing a thermomorphic deep eutectic solvent system within biocatalytic applications to recover the biocatalyst and the products. Another aspect of the present invention relates to a thermomorphic deep eutectic solvent system for performing a biocatalytic reaction comprising a deep eutectic solvent and a polar solvent.

Background of the invention

Only nine out of 51 routinely used solvents were classified as "recommended" based on a survey of global companies like Pfizer, Astra Zeneca, GSK, Sanofi or working groups such as the ACS Green Chemistry Institute Pharmaceutical Roundtable (GCI-PR). Organic solvents are ubiquitous in organic synthesis laboratories and for industrial synthetic chemistry, but unfortunately their use suffer from inherent toxicity and high volatility resulting in the release of harmful organic compounds into the environment. In addition, safety considerations must be well thought-out when working with organic solvents due to their flammability and immediate impact on laboratory personnel through inhalation and resorption. In the last decades, novel classes of "greener", safer and more sustainable solvents were developed and extensively studied, e.g. "molten salts", ionic liquids (ILs), super critical fluids (SCFs), and deep eutectic solvents (DESs).

Biotechnology, on the other hand, also addresses current and upcoming demands of our society for sustainable industrial production of chemicals as powerful substitutes for established chemical processes. This success story is proven by major industry examples of bio-based bulk chemicals such as (bio)ethanol, antibiotics, acrylamide and various intermediates for active pharmaceutical ingredients (APIs). Today, several hundreds of industrial biocatalytic processes are applied and we will witness that the number will be progressively rising in the future. Enzymes generally benefit from (i) exclusive chemo-, stereo- and/or enantioselectivities, (ii) mild reaction conditions, (iii) a broad substrate spectrum and (iv) their low intrinsic environmental impact as enzymes are considered to be fully biodegradable. Due to their cellular origin, enzymes operate best in water- enriched environments and they lose activity when water is replaced with alternative organic media. Additionally, many synthetically interesting substrates are not soluble in aqueous solutions and therefore, significantly lower productivities are observed. Consequently, researchers began to use nonconventional media in biocatalysis for higher enzymatic activities and higher solubility of substrates, and in this context DESs can be the solution for overcoming these limitations.

DESs, being knighted as "solvents for the 21st Century", or as "the organic reaction medium of the century", impress with their ecological, economical and practical advantages. First introduced in a pioneering work by Abbott et al. in 2003, DESs have been used in many research fields, and have recently also found their way into biocatalysis. In brief, many DESs are based on their biogenic origin (e.g. choline chloride, carboxylic acids, urea, citric acid, succinic acid, and glycerol) entailing (i) melting points below room temperature, (ii) high thermal stability, (iii) low volatility, (iv) diminished toxicity, (v) better biodegradability, (vi) straightforward preparation, (vii) large availability at acceptable costs and (viii) tailored solvent characteristics. Overall, DESs have a low ecological footprint and although many of these properties are also transferable to ILs, the inherent biodegradability, biocompatibility, and sustainability of DESs make them a promising alternative to ILs in some applications - but certainly not in all cases and far from entirety.

Generally, a DES is composed of a hydrogen bond donor (HBD), such as urea, citric acid or sorbitol and a hydrogen bond acceptor (HBA), such as choline chloride, glycine, or lactic acid. From this incomplete list, the hidden potential becomes clear: by combining the various components in different proportions, a new liquid phase by self-association is formed with a significant lower freezing point than that of the individual substances. According to the nature of the DESs' properties, they are fully, partly or not at all miscible with hydrophilic solvents like water or aqueous buffer solutions. Hydrophobic DESs were just very recently studied. In 2011, Bica et al. first reported on anesthetic lidocaine-based hydrophobic DESs. Nine years later, Longeras et al. reported an abrupt phase separation while increasing the temperature when lidocaine-based hydrophobic DES-water mixtures were used. Systems with a temperature-dependent phase change are so called aqueous two-phase (ATPS) thermomorphic multiphasic systems (TMSs) and were already reported in the literature for many applications. Briefly, a combination of different fractions of components such as buffer salts, water, and hydrophobic substances (e.g. ILs, polymers, organic solvents) form differently shaped miscibility gaps causing a macroscopic phase change from monophasic to biphasic or vice versa. Most commonly, miscibility gaps with an upper or lower critical solution temperature (UCST or LCST, respectively) are described in the literature. Beneficially, a reaction being performed at monophasic conditions is not restricted by mass transfer limitations and can therefore exploit its full potential with the desired solubility of substrates. Whereas, two-phasic conditions are beneficial for the downstream processing which can be tremendously improved since often the biocatalyst and the product distribute significantly different into the two phases upon the temperature change.

In 2011, Behr et a/, published a liquid immobilization concept for enzymes by TMSs. Here, a ternary mixture of water, hexanol, and methanol was used for the lipase-catalyzed hydrolysis of para-nitrophenyl palmitate. Amano lipase PS from Burkholderia cepacia was used as the biocatalyst and only a minor loss (2%) in product yields over five sequential recycling runs was reported. Recently, Langermann and co-workers described an IL-water-based TMSs for biocatalytic reactions with UCST phase behavior for homogeneous Candida antarctica lipase B (CalB)-catalyzed kinetic resolution of (rac)- 1-phenylethyl acetate. A high enzymatic activity and full conversion within the IL-based TMSs were observed and the biocatalyst was easily recycled after phase separation at lower temperatures and six consecutive reaction cycles were performed.

Thus, organic solvents are ubiquitous in organic synthesis laboratories and for industrial synthetic chemistry. Unfortunately, as mentioned above, their use suffer from inherent toxicity and high volatility resulting in the release of harmful organic compounds into the environment.

In addition, safety considerations must be well thought-out when working with organic solvents due to their flammability and immediate impact on laboratory personnel through inhalation and resorption. Thus, there is a demand for alternatives, which deals with the above-mentioned problems without losing the properties of the organic solvents used today.

Hence, a more green, safe, and sustainable solvent for use in the enzymatic reaction processes would be advantageous, and in particular a solvent, which solves the above-mentioned problems would be advantageous. Further, a system for separating the components of the enzymatic reaction would be especially advantageous.

Summary of the invention

Thus, an object of the present invention relates to a process utilizing a thermomorphic deep eutectic solvent (DES) system, with reduced toxicity and volatility compared to organic solvents, for biocatalytic reactions thereby increasing enzymatic activity and substrate solubility compared to enzymatic reactions in aqueous conditions.

Thus, an object of the present invention relates to a system for enzymatic reactions comprising a solvent with increased safety and sustainable properties, without compromising the high enzymatic activity and substrate solubility seen when using an organic solvent.

In particular, it is an object of the present invention to provide a system for enzymatic reactions that solves the above-mentioned problems of the prior art.

One aspect of the present invention relates to a process for performing a biocatalytic reaction, the process comprising at least the steps of: a. Catalysing a conversion of a starting material (SI) to a product (Pl) in the presence of a biocatalyst (Bl) in a solvent system, which is monophasic at a temperature Tl; b. Heating the solvent system to a temperature T2, or alternatively to a temperature T3 to accelerate the phase separation before reducing the temperature to T2, at which the solvent system is biphasic, wherein the biocatalyst (Bl) is present in one phase of the biphasic solvent system and the product (Pl) and unreacted starting material (SI) present in the other phase of the biphasic solvent system; c. Separation of the two phases of the solvent system obtained in step b) in order to separate the biocatalyst in one phase from the product and unreacted starting material in the other phase; wherein the solvent system comprises a deep eutectic solvent (DES) and a polar solvent.

Another aspect of the present invention relates to a solvent system for performing a biocatalytic reaction comprising a deep eutectic solvent (DES) and a polar solvent, which is monophasic at a temperature Tl, wherein a biocatalytic reaction can be conducted by catalysing the conversion of a starting material (SI) to a product (Pl) in the presence of a biocatalyst (Bl), and which is biphasic at a temperature T2, or alternatively at a temperature T3 to accelerate the phase separation before reducing the temperature to T2, enabling separation of the biocatalyst (Bl) present in one phase of the biphasic solvent system and the product (Pl) and unreacted starting material (SI) present in the other phase of the biphasic solvent system.

Brief description of the figures

Figure 1 depicts a hypothetical phase diagram of a multi-component system with a lower critical solution temperature (LCST) miscibility gap (Figure 1, left), and the concept of the LCST thermo-regulated multi-component deep eutectic solvent system (LCST-DES-TMS) (Figure 1, right). The temperature-controlled multicomponent solvent system switches between biphasic at higher temperatures (T>TCP) and monophasic at lower temperatures (T<TCP). An enzyme-catalysed reaction is carried out at lower temperatures in the monophasic system, and afterwards, the biocatalyst and substrates/products can easily be separated at biphasic conditions only by slightly increasing the temperature. The substrates/products accumulate in the upper DES phase, whereas the (catalytically active) biocatalyst accumulates in the lower aqueous phase and can be recycled.

Figure 2 illustrates the preparation of the DES (Figure 2, top), and a test reaction with benzaldehyde in the thermomorphic-DES-system (Figure 2, bottom). The DES was prepared by mixing oleic acid (OA) and lidocaine (LID) in a ratio of 1: 1 (mol/mol) and heated to 80 °C while gently stirring. As a test reaction, the reduction of benzaldehyde to benzyl alcohol using the cofactor NADH in the TMS- DES was investigated.

Figure 3 shows LCST-phase diagrams of lidocaine-based DES with ultrapure water (UPW) as the second component. The LCST critical point was determined to 25.5 ±0.5 °C at a mass fraction of WDES= 50%.

Figure 4 shows LCST-phase diagrams of lidocaine-based DES with different concentrations of potassium phosphate buffers (KPi) as the second component at pH 7.5 (Figure 4, left) and pH 6.0 (Figure 4, right). The LCST critical point was determined to 23.5 ±0.5 °C for WDES= 50% and 500 mmol-L ¹ KPi buffer at pH 7.5. Two local minima with two critical points were observed for the KPi buffers with higher concentrations e.g., for 500 mmol-L ¹ KPi buffer (pH 7.5), a first minimum at 22.0 ± 0.5 °C for WDES = 20% and a second minimum at 23.0 ± 0.5 °C for WDES = 75%. The use of KPi buffers with pH 6.0 unexpectedly resulted in binodals with an inverse shape at WDES = 0% to 50%.

Figure 5 shows the influence of additional substances to the final reaction system. The addition of either benzaldehyde (substrate), benzyl alcohol (product) or the cofactor NADH, results in a decrease in the cloud point of the system composed of 50 mmol-L ¹ KPI (pH 6.0) and a mass fraction of DES = 50%. A decrease of approximately 0.026 °C per concentration unit was observed for all compounds (Figure 5, left). The addition of bovine serum albumin (BSA) did not result in a substantial change in the cloud point of the system composed of 100 mmol-L ¹ KPi (pH 7.5) with a mass fraction of WDES = 50% (Figure 5, right).

Figure 6 shows the distribution of the BSA protein in the upper DES-enriched phase and lower aqueous-phase (AQ-phase) when run on a SDS polyacrylamide gel. At monophasic conditions, 800 mg-L ¹ BSA were dissolved in 50 mmol-L ¹ KPi (pH 6.0) and WDES = 50%, and afterwards, individual samples were taken from both phases at biphasic conditions and analyzed with SDS polyacrylamide gel electrophoresis. As expected, the blank samples without BSA did not have bands between 55-70 kDa in either the AQ- or DES-enriched phases (lane 5 and 6, respectively). In contrast, however, a band between 55-70 kDa was clearly visible in the AQ-enriched phase for the system with BSA (Figure 6, lane 8). A small protein band was also visible for the DES-enriched phase for the system with BSA (Figure 6, lane 9). Hence, the BSA protein is predominantly separated into the AQ-enriched phase at biphasic conditions. Lane 7 contains the PageRulerTM prestained protein ladder.

Figure 7 shows the experimental setup for recycling the horse liver alcohol dehydrogenase (HLADH)-enzyme in a system of 50 mmol-L ¹ KPi (pH 6.0) and a mass fraction of WDES = 50%. At the beginning of the reaction, 25 mmol-L ¹ benzaldehyde and 50 mmol-L ¹ NADH were added to a system containing 10 g DES and 9 mL of 50 mmol-L ¹ (pH 6.0). The system was cooled to 20 °C and stirred yielding a one-phase system (Figure 7, left). The reactor was then heated to 45 °C for 5 min and then cooled to 30 °C without stirring yielding a clear two- phase system (Figure 7, right). After 24 h settling, samples were taken from each phase at 30 °C . The system was cooled to 20 °C and 1 mL HLADH solution was added (400 mg-L ¹ final concentration) to start the reaction (Figure 7, left). The same temperature changes as described above were repeated, and samples were taken from each phase at biphasic conditions at 30 °C following settling for 24 h (Figure 7, right). The middle panel shows the system at 30 °C with stirring (Figure 7, middle). Following sampling, the old DES phase was discarded and the same amount of new DES was used along with new 10 mmol-L ¹ of benzaldehyde. In total, the enzyme in the AQ-enriched phase was recycled three times.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Aqueous two-phase system (ATPS)

A solvent system with a miscibility gap at a given temperature in which the two phases consist mainly of water and non-volatile components. Ionic liquids (IL)

A substance largely made of ions (salt) being in the liquid state with a melting point below 100 °C.

Lower critical solution temperature (LCST)

The local minimum of the binodal of a two-phase system.

Super critical fluids (SCFs)

Any substance at a temperature and pressure above its critical point, where distinct liquid and gas phases do not exist, but below the pressure required to compress it into a solid.

Thermomorphic multiphase system (TMSs) Mixtures of at least two solvents with a highly temperature-dependent miscibility gap.

Upper critical solution temperature (UCST)

The local maximum of the binodal of a two-phase system.

The present invention relates to a LCST-DES-TMS system for the integration of a reaction-extraction downstream processing approach (Figure 1). By the combination of a hydrophobic DES, composed of oleic acid (OA) and lidocaine (LID) (Figure 2; preparation of the DES), and an aqueous phosphate buffer phase, a temperature-controlled multi-component solvent system is formed which switches between monophasic at lower and biphasic at higher temperatures, that represents the LCST as described above. The inventors have found on a simple (catalytically active) protein liquid-immobilization within the aqueous phase combined with a product stream pre-concentration in a one-unit operation. In addition, an enzyme-catalyzed reaction is carried out at lower temperatures in the monophasic system, and afterwards, the biocatalyst and reactants/products can be separated easily at biphasic conditions only by slightly increasing the temperature (Figure 2; the reaction in the thermomorphic-DES-system). In all cases, upon phase separation the (catalytically active) protein accumulates mostly in the aqueous phase whereas the hydrophobic products and unreacted educts present in the likewise hydrophobic DES phase. The present invention solves several industrial challenges: (i) Reactions are running at low temperatures being beneficial for energy efficient synthesis, and for sensitive biocatalysts and organic components, (ii) the downstream processing is simplified by a selective extraction of biocatalysts and educts/ products in an one-unit operation, e.g. for the isolation of bulk chemicals from fermentative processes, and/or for the separation of the involved biocatalysts from the reaction broth, (iii) recovery and reuse of the biocatalysts (enzymes or whole-cells) through liquid-immobilization and (iv) bio-based DESs components are tailor made, renewable and biodegradable originating from industrial waste (e.g. glycerol to be used as HBD) or animal feed (e.g. choline chloride to be used as HBA).

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

The present invention describes a process using a thermomorphic deep eutectic solvent system within biocatalytic applications for recovery of the biocatalyst and the products.

In particular the invention describes a "Lower-critical-solution-temperature-DES- Thermomorphic multiphase system" characterized by being a monophasic system at low temperatures and a biphasic system when the temperature is slightly increased.

An enzyme-catalyzed reaction is carried out at lower temperatures in the monophasic system, and afterwards, the biocatalyst and reactants/ products can be separated easily at biphasic conditions only by slightly increasing the temperature.

In all cases, the (catalytically active) protein remains mostly in the aqueous phase whereas the hydrophobic products and unreacted educts stay in the hydrophobic DES phase.

A reaction being performed at monophasic conditions is not restricted by mass transfer limitations and can therefore exploit its full potential with the desired solubility of substrates. Whereas, two-phasic conditions are beneficial for the downstream processing, which can be tremendously improved since often the biocatalyst and the product distribute significantly different into the two phases upon the temperature change.

One aspect of the present invention relates to a process for performing a biocatalytic reaction, the process comprising at least the steps of: a. Catalysing a conversion of a starting material (SI) to a product (Pl) in the presence of a biocatalyst (Bl) in a solvent system, which is monophasic at a temperature Tl; b. Heating the solvent system to a temperature T2, or alternatively to a temperature T3 to accelerate the phase separation before reducing the temperature to T2, at which the solvent system is biphasic, wherein the biocatalyst (Bl) is present in one phase of the biphasic solvent system and the product (Pl) and unreacted starting material (SI) present in the other phase of the biphasic solvent system; c. Separation of the two phases of the solvent system obtained in step b) in order to separate the biocatalyst in one phase from the product and unreacted starting material in the other phase; wherein the solvent system comprises a deep eutectic solvent (DES) and a polar solvent.

Another aspect of the present invention relates to a solvent system for performing a biocatalytic reaction comprising a deep eutectic solvent (DES) and a polar solvent, which is monophasic at a temperature Tl, wherein a biocatalytic reaction can be conducted by catalysing the conversion of a starting material (SI) to a product (Pl) in the presence of a biocatalyst (Bl), and which is biphasic at a temperature T2, or alternatively at a temperature T3 to accelerate the phase separation before reducing the temperature to T2, enabling separation of the biocatalyst (Bl) present in one phase of the biphasic solvent system and the product (Pl) and unreacted starting material (SI) present in the other phase of the biphasic solvent system.

An embodiment of the present invention relates to the process or the solvent system, wherein the polar solvent is selected from the group consisting of water, a buffer and salt mixtures, preferably the polar solvent is water or buffer, more preferably a potassium phosphate (KPi) buffer.

Another embodiment of the present invention relates to the process or the solvent system, wherein the deep eutectic solvent (DES) consists of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA).

A further embodiment of the present invention relates to the process or solvent system, wherein the HBD is selected from the group consisting of oleic acid, stearic acid, palmitic acid, hexanoic acid, levulinic acid, octanoic acid, decanoic acid, dodecanoic acid, L- and D-glutamic acid, L-proline, L-arginine, n-butyl alcohol, 1-octanol, 1-dodecanol, oleyl alcohol, myristic acid, cis-9-octadecenoic acid, ricinoleic acid, 1-propanol, 1-butanol, hexyl alcohol, capryl alcohol, decyl alcohol, dodecyl alcohol, 1-tetradecanol, 1-hexadecanol, cyclohexanol, DL- menthol, ethylene glycol, 1,3-propanediol, glycerol, hydroquinone, 4- phenylphenol, 1,5-pentanediol, Ibuprofen, thymol, N,N'-dihexylthiourea, bisphenol Z, pyruvic acid, butyric acid, valeric acid, thymol, ketoprofen, diclofenac, camphor, borneol urea, citric acid or sorbitol, preferably the HBD is oleic acid.

Yet another embodiment relates to the process or solvent system, wherein the HBA is selected from the group consisting of lidocaine, glycine or lactic acid, tetrabutylammonium chloride, tetrabutylphosphonium chloride, tetrabutylammonium bromide, methyltrioctylammonium chloride, tetraheptylammonium chloride, tetraheptylammonium bromide, tetraoctylammonium bromide, tetraoctylammonium chloride, trihexyltetradecylphosphonium chloride, tetrahepthylammonium chloride, trihexyl (tetradecyl)phosphonium tetrafluoroborate, aropine, trioctylphosphine oxide, dodecyl-methyl-sulfoxide, /V-Methyl-ZV/ZV/ZV-trioctylammonium chloride, choline chloride, (-)-menthol, DL-menthol, carvacrol, coumarin, 1,2-decanediol, 1- napthol, 10-undecylenic acid, preferably the HBA is lidocaine.

In an embodiment, the HBD is levulinic acid and the HBA is tetrabutylammonium chloride, or the HBD is octanoic acid and the HBA is tetrabutylammonium chloride, or the HBD is decanoic acid and the HBA is tetrabutylammonium chloride, or the HBD is hexanoic acid and the HBA is tetrabutylammonium chloride, or the HBD is hexanoic acid and the HBA is tetrabutylammonium bromide, or the HBD is hexanoic acid and the HBA is methyltrioctylammonium chloride, or the HBD is palmitic acid and the HBA is methyltrioctylammonium chloride, or the HBD is oleic acid and the HBA is tetraheptylammonium chloride, or the HBD is decanoic acid and the HBA is lidocaine, or the HBD is DL-menthol and the HBA is lidocaine, or the HBD is thymol and the HBA is lidocaine, or the HBD is levulinic acid and the HBA is DL-menthol, or the HBD is Ibuprofen and the HBA is tetraheptylammonium chloride.

An embodiment of the present invention relates to the process or solvent system, wherein the molar ratio of HBA to HBD is 1:2 to 2: 1, preferably 1: 1, and the solution is heated to 65-95 °C, preferably 80 °C with gentle stirring.

A further embodiment of the present invention relates to the process or solvent system, wherein the molar ratio of DES to the polar solvent is 1:2 to 2: 1, preferably 1: 1.

An additional embodiment of the present invention relates to the process or solvent system, wherein T1 is below 26°C, and T2 is above 26°C, preferably T1 is 20 °C or less and T2 is 30 °C or more, and T3 is 40-50°C, preferably T3 is 45 °C. Another embodiment of the present invention relates to the process or solvent system, wherein the biocatalyst is an enzyme or microorganism.

Yet another embodiment of the present invention relates to the process or solvent system, wherein the enzyme is selected from the list comprising oxidoreductases like horse liver alcohol dehydrogenase (HLADH) enzyme, transferases, hydrolases, lyases, ligases, isomerases or transferases.

An embodiment of the present invention relates to the process or solvent system, wherein the at least one starting material (SI) is an organic or inorganic substance, such as benzaldehyde.

Another embodiment of the present invention relates to the process or solvent system, wherein the at least one product (Pl) is an organic or inorganic substance, such as benzyl alcohol.

An additional embodiment of the present invention relates to the process or solvent system, wherein the mass fraction of DES with fluid is WDES= 5- 100%, preferably WDES= 50%.

A further embodiment of the present invention relates to the process or solvent system, wherein the biocatalyst is present in the polar solvent phase of the biphasic solvent system and the product and unreacted starting material is present in the deep eutectic solvent (DES) phase.

Examples

Example 1 - Material and methods

The examples (2-7) were carried out using the materials and methods as described in example 1. All solvents, reactants, enzymes and starting materials were received from commercial suppliers in the highest available purity (Sigma-Aldrich, VWR) and used as received. Ultrapure water (UPW, 18.2 MQ-cm) was produced with a Milli- Q® Synthesis system by Millipore Corporation (now Merck Millipore, Darmstadt, Germany) and used throughout this study. Mass fraction (m/m) of the used DESs were always calculated for monophasic system conditions. As biocatalyst, recombinant horse liver alcohol dehydrogenase (HI_ADH) expressed in E. coli was purchased from Sigma-Aldrich (SKU: 55689-100MG) and used throughout this study. All experiments were carried out under atmospheric conditions if not stated otherwise. For tempering and shaking of the reaction vessels, a MKR 23 thermoshaker from Hettich (The Netherlands) was used.

Potassium phosphate Buffer solutions (KPi)

Potassium phosphate buffer solution (KPi buffer, 50 mmol-L ¹ , pH 7.5). 0.641 g dipotassium hydrogenphosphate (K2HPO4) and 0.180 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 100 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 7.5.

Potassium phosphate buffer solution (KPi buffer, 100 mmol-L ¹ , pH 7.5). 1.281 g dipotassium hydrogenphosphate (K2HPO4) and 0.360 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 100 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 7.5.

Potassium phosphate buffer solution (KPi buffer, 500 mmol-L ¹ , pH 7.5). 6.405 g dipotassium hydrogenphosphate (K2HPO4) and 1.800 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 100 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 7.5.

Potassium phosphate buffer solution (KPi buffer, 50 mmol-L ¹ , pH 6.0). 0.601 g dipotassium hydrogenphosphate (K2HPO4) and 2.932 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 500 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 6.0.

Potassium phosphate buffer solution (KPi buffer, 100 mmol-L ¹ , pH 6.0). 0.241 g dipotassium hydrogenphosphate (K2HPO4) and 1.173 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 100 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 6.0.

Potassium phosphate buffer solution (KPi buffer, 200 mmol-L ¹ , pH 6.0).

0.482 g dipotassium hydrogenphosphate (K2HPO4) and 2.346 g potassium dihydrogenphosphate (KH2PO4) were dissolved in UPW in a 100 mL volumetric flask and, if necessary, the pH was adjusted with potassium hydroxide to pH 6.0.

Preparation of the deep eutectic solvents

The DES (lidocaine:oleic acid) was prepared according to a modified literature description (Longeras, O. et al. 2020), and lidocaine (LID, 234.34 g-mol ^-1 ) and oleic acid (OA, 282.46 g-mol ^-1 ) in a mole fraction of 1: 1 were used. A sealed 20 mL screw cap vessel with nitrogen headspace was applied to prevent the oleic acid of any possible oxidation side reactions, and as an example, 6.01 g (21.3 mmol) of OA and 4.99 g (21.3 mmol) of LID were mixed and heated to 80 °C in an oil bath with gentle stirring, yielding 11 g (21.3 mmol) of DES as a light brown and highly viscous liquid (Figure 2). The eutectic point is estimated to -60 °C according to the literature (Longeras, O. et al. 2020 and Bica K. et al. 2011).

Gas chromatography (GO analysis

Gas chromatography (GC). A Nexis GC-2030 gas chromatograph from Shimadzu (Japan) with a flame ionization detector (FID) equipped with a CP-Chirasil-Dex CB column (CP7502, NLR0663300, 25 m x 0.25 mm within a coating thickness of 0.25 pm) from Agilent was used for analytics. Carrier gas helium (purity: 99.999%) with a flow rate of 0.67 mL-min ¹ was used for all measurements. Temperatures of the injector and detector were set to 275 °C and 250 °C, respectively. Temperature program : 3 min at 130 °C for equilibration, followed by hold time for 12 min at 130 °C after injection, followed by a heating rate of 30 °C-min ¹ to 190 °C and a hold time for 5 min. The sample was injected within split mode with a split ratio of 100 and a purge flow of 3 mL-min ^-1 . Calibration of the GC. Two different calibration methods were used. For the calibration of the samples from the DES phase, the neat substrate and product were diluted in a solution of dichloromethane (DCM) with 5 mmol-L ¹ of n-dodecane as internal tracer substance and measured with the GC. For the samples being extracted from the AQ layer, the neat substrate and product were first diluted in 50 mmol-L ¹ Kpi (pH 6.0) and then extracted with DCM/n-dodecane. The vials were centrifuged for one minute at 13400 rpm to promote the phase separation and the lower, DCM/n-dodecane phase was then analyzed with the GC.

Example 2 - Temperature related LCST phase behaviour of DES mixed with ultrapure water (UPW)

Aim

The aim of this study is to determine the temperature dependent binodal of DES and ultra-pure water (UPW).

Materia! and methods

To investigate the LCST phase behaviour of the DES with water, 20 samples with mass fractions from WDES= 5- 100% (m/m) within 5% (m/m) steps in UPW were prepared in GC vials to a final mass of one gram. All samples were prepared by mass on a fine scale and afterwards shaken at 1200 rpm and the temperature was stepwise increased within 0.5 K starting from 15 °C with an equilibration time of 5 minutes for each temperature. The experiment was continued until all samples had become a two-phase system (the liquid was turbid).

Results

The LCST critical point for DES with UPW was determined to 25.5 ±0.5 °C at a mass fraction of WDES= 50 (Figure 3).

Conclusion

In conclusion, the LCST critical point for DES mixed with UPW was determined to 25.5 ±0.5 °C at a mass fraction of WDES= 50 (Figure 3). Example 3 - Temperature related LCST phase behaviour of DES mixed with potassium phosphate buffer

Aim

Although enzymes need water to operate, it is well known that the application of buffer salts significantly increase the stability of enzymes due to a controlled pH and a controlled ionic strength of the solution cohering the enzyme's quaternary structure. Thus, the aim of this next study is to determine the temperature dependent binodal of DES and different potassium phosphate buffer (KPi) concentrations at pH 6.0 and 7.5.

Material and methods

To investigate the LCST phase behavior of DES with KPi buffer (all pH 7.5), 21 samples with WDES=0- 100% (m/m) within 5% (m/m) steps in the different KPi buffer concentrations (50 mmol-L ^-1 , 100 mmol-L ¹ or 500 mmol-L ^-1 ) were prepared in GC vials and investigated as described in example 2.

To investigate the LCST phase behavior of DES with KPi buffer (all pH 6.0), 21 samples with WDES=0- 100% (m/m) within 5% (m/m) steps in the different KPi buffer concentrations (50 mmol-L 100 mmol-L ¹ or 200 mmol-L ^-1 ) were prepared in GC vials and investigated as mentioned above.

Results

When combining the DES with potassium phosphate buffers at different concentrations, it is evident that the higher the concentration of the buffer salts, the lower the binodal of the respective system. The 50 mmol-L ¹ KPi at pH 6.0 was chosen for subsequent experiments since it was the most suitable for the final enzymatic reaction.

Conclusion

In conclusion, when combining the DES with potassium phosphate buffer at different concentrations, it was clear that the higher the concentration of the buffer salts, the lower the binodal of the respective system. The solution containing 50 mmol-L ¹ KPi at pH 6.0 was chosen for subsequent experiments since it was the most suitable for the final enzymatic reaction. Example 4 - Determination of the influence of educts, products, cofactors and proteins towards the binodal curve

Aim

Additional components can have a significant impact towards the binodal curve onto the system of interest. The aim of this study was therefore to determine the influence of educts, products, cofactors, and proteins towards the binodal curve.

Materia! and methods

To investigate a possible influence of proteins towards the LCST phase behavior of the DES with KPi buffer, Bovin Serum Albumin (BSA) was used as a model protein. A system of DES and 100 mmol-L ¹ KPi (pH 7.5) in a mass fraction of WDES = 50% was used with BSA concentrations from 0.024-1.563 mg-mL ^-1 . The vessels were shaken, tempered, and investigated as mentioned in example 2.

To investigate a possible influence of the substrate benzaldehyde and the product benzyl alcohol towards the LCST phase behavior, a system of 50 mmol-L ¹ KPi (pH 6.0) in a mass fraction of WDES = 50% was used with concentrations of {10, 25, 50, 100 and 200} mmol-L ¹ of either the substrate or the product. The possible influence of the cofactor NADH was tested with concentrations of {5, 10, 25, 50, and 62.5} mmol-L ¹ of NADH. The vessels were shaken, tempered, and investigated as described in example 2.

Results

The addition of either benzaldehyde (substrate), benzyl alcohol (product) or the cofactor NADH, results in a decrease in the cloud point of the system composed of 50 mmol-L ¹ KPI (pH 6.0) and a mass fraction of DES= 50%. A decrease of approximately 0.026 °C per concentration unit was observed for all compounds (Figure 5, left). The addition of bovine serum albumin (BSA) in a typically applied (catalytically active) protein concentration did not result in a substantial change in the cloud point of the system composed of 100 mmol-L ¹ KPi (pH 7.5) with a mass fraction of WDES= 50% (Figure 5, right).

Conclusion

Benzaldehyde, benzyl alcohol and NADH decrease the cloud point with approximately 0.026 °C per concentration unit in the system composed of 50 mmol-L ¹ KPI (pH 6.0) and a mass fraction of WDES= 50°/O. In contrast, however, the addition of BSA did not result in a substantial change in the cloud point of the system composed of 100 mmol-L ¹ KPi (pH 7.5) with a mass fraction of WDES= 50%.

Example 5 - The distribution of proteins within the DES-TMS

Aim

The aim of the present study is to determine the protein distribution in the upper DES-enriched and lower aqueous (AQ)-phase of a biphasic DES system.

Material and methods

To analyze the distribution of proteins, BSA in particular, in the upper DES- enriched and the lower AQ-phase of a 30 °C-heated two-phase system, SDS polyacrylamide gel electrophoresis was used. One gram of a system of DES and 50 mmol-L ¹ KPi (pH 6.0) in a mass fraction of WDES = 50% was prepared and 0.8 mg BSA (800 mg-L ^-1 ) was added. Afterwards, the sample was cooled to 18 °C and shaken at 1200 rpm forming a monophasic homogenous system. Then, the samples were heated to 30 °C and equilibrated for 15 minutes without stirring generating a clear two-phase system. A sample of each phase layer was collected and analyzed via SDS polyacrylamide gel electrophoresis. PageRulerTM prestained protein ladder and GenScript SurePAGE, Bis-Tris, 10 x 8 gel at 140 V were used. 30 mL InstantBlue Coomassie Protein Staining was added to visualize the protein bands. Additionally, samples of the upper DES-enriched phase and of the lower AQ-enriched phase were analyzed without the addition of the BSA protein as a reference.

Results

As expected, the blank samples without BSA did not have bands between 55-70 kDa in either the AQ- or DES-enriched phases (lane 5 and 6, respectively). Hence, the blank samples do not have bands for BSA. In contrast, however, the BSA- distribution in a system with BSA was predominantly in the AQ-enriched phase as compared to the DES-enriched phase (lane 8 and 9, respectively). Thus, the BSA protein is predominantly separated into the AQ-enriched phase at biphasic conditions. Lane 7 contains the PageRulerTM prestained protein ladder.

Conclusion

Proteins such as BSA predominantly separates into the lower AQ-enriched phase during biphasic conditions in the DES-TMS.

Example 6 - The distribution of educts and products within the DES-TMS

Aim

The aim of this study is to determine the distribution of educts and products in the upper DES-enriched and lower AQ-phase of a biphasic DES system.

Materia! and methods

To analyze the distribution of benzaldehyde (substrate) and benzyl alcohol (product), a system composed of DES and 50 mmol-L ¹ KPi (pH 6.0) in a mass fraction of WDES = 50% was used. In a total of 1 mL volume, 25 mmol-L ¹ of either of benzaldehyde or benzyl alcohol was diluted at 20 °C. Then, the system was heated to 45 °C for five minutes and afterwards, the samples were settled at 30 °C for 24 h. A sample was taken from the lower, AQ-enriched phase and extracted with DCM/n-dodecane 1: 1 (v/v). The vial was centrifuged for one minute at 13400 rpm to promote the phase separation and the lower, DCM/n- dodecane phase was then analyzed with the GC. The upper, DES-enriched phase was diluted in DCM/n-dodecane 1: 1 (v/v) and analyzed with the GC.

Results

The reduction of benzaldehyde to benzyl alcohol catalysed by HLADH using the cofactor NADH was investigated in the DES-TMS (Figure 2). From table 1, it is evident that >95% of the hydrophobic substances (benzaldehyde, benzyl alcohol) accumulated into the hydrophobic, upper DES-enriched phase.

Table 1: Distribution of the substrate (benzaldehyde) and the product (benzyl alcohol) in a TMS-DES composed of 50 mmol-L ¹ Kpi (pH 6.0) and a mass fraction of WDES = 50% after separating the phases at temperatures above the cloud-point temperature. Quantity values were calculated as (concentration in the respective layer)/ (sum of concentration in both layers).

Conclusion

Hydrophobic substances including benzaldehyde and benzyl alcohol accumulate in the upper DES-enriched phase.

Example 7 - Reaction within DES-TMS

Aim

Up to this point, we have shown that the separation of protein (biocatalyst, e.g. HLADH) and educts (e.g. benzaldehyde) or products (e.g. benzyl alcohol) are possible by simply changing the temperature of the DES-TMS. The aim of the present study was therefore to investigate whether the biocatalyst could be retrieved and reused in a subsequent reaction.

Materia! and methods

For the biocatalytic reaction, DES was combined with 50 mmol-L ^-1 KPi buffer (pH 6.0) in a mass fraction of WDES = 50%. Ten g DES and 9 mL KPi (50 mmol-L ¹ , pH 6.0) were transferred to a tempered reactor vessel. 51 pL benzaldehyde (25 mM) and 0.66 g NADH (50 mM) were added. The system was cooled from 30 °C to 20 °C and stirred yielding a one-phase system. The reactor was then heated to 45 °C for 5 min and then cooled to 30 °C without stirring yielding a clear two- phase system. After 24 h settling, samples were taken from each phase of the clear two-phase system. A 200-pL sample was taken from the lower AQ-enriched phase and extracted with 200 pL of DCM/n-dodecane 1: 1 (v/v). The vial was centrifuged for one minute at 13400 rpm to promote the phase separation and the lower DCM/n-dodecane phase was then analyzed with the GC. Additionally, 200 pL of the upper DES-enriched phase was diluted into 200 pL DCM/n-dodecane 1: 1 (v/v) and analyzed with the GC. The system was then cooled to 20 °C (one-phase system), and 1 mL HI_ADH solution was added (8000 mg-L ¹ stock and 400 mg-L-1 final concentration). After 24 h reaction time, the system was heated to 45 °C for 5 min, and subsequently cooled to 30 °C. After 24 h, the two phases completely settled, and samples were taken from the upper and the lower phase as mentioned above.

After the biocatalytic reaction was carried out (see above), the enzyme was recycled by removing the AQ-enriched phase while discarding the DES phase. The reactor was cleaned, and the volume and mass of the AQ-enriched phase was determined and transferred back to the reactor. New DES was added in same mass as the AQ-enriched phase which varied for each recycling. Benzaldehyde (10 mmol-L ^-1 ) was added to the new system and the system was again tempered to 20 °C yielding a one-phase system. This procedure was then repeated three times.

Results

Figure 7 shows the experimental setup for recycling the HLADH enzyme in a system of 50 mmol-L ¹ KPi (pH 6.0) and a mass fraction of WDES= 50°/O whereas table 2 shows the measurements from this experiment. At the beginning of the reaction, 25 mmol-L ¹ benzaldehyde and 50 mmol-L ¹ NADH were added to a system containing 10 g DES and 9 mL of 50 mmol-L ¹ (pH 6.0). The system was cooled to 20 °C and stirred yielding a one-phase system. The reactor was then heated to 45 °C for 5 min and then cooled to 30 °C without stirring yielding a clear two-phase system. After 24 h settling, samples were taken from each phase at 30 °C (Table 2, Initial run). The system was cooled to 20 °C and 1 mL HLADH solution was added (400 mg-L ¹ final concentration) to start the reaction (Figure 7, left). Next, the system was heated to 45 °C to promote phase separation and subsequently cooled to 30 °C. The middle panel shows the system at 30 °C with stirring (Figure 7, middle). After settling for 24 h without stirring, samples were taken from each phase (Figure 7, right; Table 2, 0 runs). The old/used DES phase was discarded and the same amount of new/fresh DES was used along with new 10 mmol-L ¹ of benzaldehyde. In total, the enzyme was recycled three times (Table 2, 1-3 runs). This proof-of-concept experiment demonstrates:

(i) The reaction takes place, i.e. the reduction of benzaldehyde to benzyl alcohol.

(ii) The recycling of the enzyme is working otherwise no benzyl alcohol would be produced during the second and third subsequent reactions.

(iii) The extraction of substrate/product into the DES-enriched phase and of the enzyme into the AQ-enriched phase is successful (otherwise no second reaction would take place).

(iv) The TMS behavior is reproducible.

The decreased productivity of the enzyme HLADH may be either due to deactivation due to the long contact time within the system or/and that the NADH is consumed to a level that the reaction equilibrium is not favorable anymore. However, measurements of the NADH concentration were not performed.

Table 2: Overview of the concentrations of substrate and product after each recycling run in the scaled-up DES-TMS reaction system. Samples are measured on the GC and processed with the respective GC calibration method for either the AQ-enriched phase or the DES phase. Reaction time (temperature): 24 h (20 °C), phase separation time (temperature): 5 min (45 °C) followed by 24 h (30 °C), enzyme concentration: 400 mg-L ¹ , NADH starting concentration: 50 mmol-L ¹ .

Conclusion

This proof- of- co nee pt experiment shows that the extraction of substrate/product into the DES-enriched phase and of the enzyme into the AQ-enriched phase is successful and the subsequent recycling of the enzyme is working.

References

• Longeras, O. et al. "Deep Eutectic Solvent with Thermo-Switchable Hydrophobicity", ACS Sustainable Chem. Eng. 2020, 8, 12516-12520

• Bica K. et al. "Liquid forms of pharmaceutical co-crystals: exploring the boundaries of salt formation", Chem Commun (Camb). 2011, Feb 28;47(8):2267-9