Synergistic effects of binary ionic liquid-solvent systems on enzymatic esterification of esculin

Journal Pre-proofs Synergistic effects of binary ionic liquid-solvent systems on enzymatic esterification of esculin Jacob Nedergaard Pedersen, Shulai Liu, Ye Zhou, Thomas Balle, Xuebing Xu, Zheng Guo PII: DOI: Reference:

S0308-8146(19)31993-4 https://doi.org/10.1016/j.foodchem.2019.125858 FOCH 125858

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Food Chemistry

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29 May 2019 2 November 2019 3 November 2019

Please cite this article as: Nedergaard Pedersen, J., Liu, S., Zhou, Y., Balle, T., Xu, X., Guo, Z., Synergistic effects of binary ionic liquid-solvent systems on enzymatic esterification of esculin, Food Chemistry (2019), doi: https:// doi.org/10.1016/j.foodchem.2019.125858

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Synergistic effects of binary ionic liquid-solvent systems on enzymatic

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esterification of esculin

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Jacob Nedergaard Pedersena, Shulai Liua, b, Ye Zhoua, c, Thomas Balled, and Xuebing Xua, Zheng

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Guoa,*

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a

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bDepartment

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of Technology, Chaowang Rd 18, Hangzhou 310014, China;

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c

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Life Science, Jilin University, Changchun 130012, China;

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d

Department of Engineering, University of Aarhus, DK-8000 Aarhus C, Denmark; of Food Science, Institue of Ocean Research, Ocean College, Zhejiang University

Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of

Novozyms A/S DK, Krogshojvej 36, 2880 Bagsvaerd, Denmark;

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*Corresponding author:

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Zheng Guo, Tel: +45 8715 5528; Fax: +45 8612 3178. E-mail: [email protected]; [email protected]

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1

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ABSTRACT

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This work established a binary ionic liquid-solvent system for effective enzymatic esterification of

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naturally occurring phenolic glycosides (flavonoids); which could result in a dramatic enhancement

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of Novozym 435-catalyzed esterification of esculin, demonstrating a great synergetic effect. In

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essence, [OMIM][BF4]-toluene and [TOMA][Tf2N]-hexane binary systems both served > 90 mol%

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of conversions of esculin after 96 h of reaction at 60 °C. Typically, binary [TOMA][Tf2N]-hexane

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system enabled Novozym 435 with extremely high catalytic efficiency (kcat/Km =17.57×10-2 (Ms)-1),

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which was 55-fold higher than that Novozym 435 exhibited in t-butanol solvent (one of the best

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solvent systems for esterification reactions). It was also found that the superior matching in property

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and structure between IL and solvent was the decisive factor for the outperformance of

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[TOMA][Tf2N]-hexane binary system, in which [TOMA] and hexane facilitate the solubilization of

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esculin and fatty acids and [Tf2N]- anions and hexane offer protective effects for lipase at elevated

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temperatures.

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Keywords: Synergetic effect; flavonoids; ionic liquids (ILs); binary system; enzymatic

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esterification; lipase

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1. Introduction

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Phenolic glycosides (PHGs) are a vast class of naturally occurring compounds and commonly

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found as plant metabolites (Reichardt, Clausen & Bryant, 1988). Of particular interest is the group

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of biphenolic glycosides commonly known as flavonoids (Pietta, 2000). Flavonoids are known to

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show antioxidant properties via scavenging free radicals (Pietta, 2000; Afanas’ev, Dcrozhko,

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Brodskii, Kostyuk & Potapovitch, 1989), chelating redox active metals (Heim, Tagliaferro &

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Bobilya, 2002; Sugihara, Arakawa, Ohnishi & Furuno, 1999) or hindering lipid peroxidation

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(Sugihara, Arakawa, Ohnishi & Furuno, 1999; Torel, Cillard & Cillard, 1986). It was also found

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that flavonoids could probably lower the risk of coronary heart disease (Hertog, Kromhout,

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Aravanis, Blackburn, Buzina & Fidanza F. et al., 1995), and has been used to tentatively treat

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cancer despite that the anti-cancer effects were not completely confirmed yet (Birt, Hendrich &

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Wang, 2001; Le Marchand, Murphy, Hankin, Wilkens & Kolonel, 2000). The beneficial effects and

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the non-toxic nature of natural flavonoids make them appealing as antioxidants in oils and

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cosmetics and as bioactive food additives (Viskupicova, Danihelova, Ondrejovic, Liptaj & Sturdik,

44

2010).

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It has been shown however that the antioxidative effects of flavonoids and their derivatives in oil-

46

based solutions or emulsions are dependent on their lipophilicity (Rice-Evans, Miller & Paganga,

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1996; Shashank & Pandey, 2013). Fatty acid esterification of the glucose moiety was reported to

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enhance the lipophilicity of flavonoid and thus enable better incorporation of the antioxidant

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moieties into membranes and interface of lipid bilayers where oxidation is expected to occur

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(Katsoura, Polydera, Tsironis, Petraki, Rajačić, Tselepis & Stamatis, 2009; Mellou, Lazari, Skaltsa,

51

Tselepis, Kolisis & Stamatis, 2005; Kim, Choi, Lee & Ahn, 2003). Many organic solvents have

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been used in the enzymatic regioselective esterification of natural flavonoids (e.g. rutin) with fatty

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acids (Kontogianni, Skouridou, Sereti, Stamatis & Kolisis, 2003; Danieli, Luisetti, Sampognaro,

3

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Carrea & Riva, 1997; Mellou, Loutrari, Stamatis, Roussos & Kolisis, 2006). The polar organic

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solvents can dissolve the substrate, but the problem is that the solvent tends to cause inactivation via

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depriving the enzyme of bond water molecules that could stabilize the catalytic conformation (Yang

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& Pan, 2005).

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In recent years the field of ionic liquids (ILs) research has been well established and ILs are

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widely recognized as highly designable solvents for chemical and biochemical applications (Xu,

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Guo & Cheong, 2016; Cull, Holbrey, Vargas-Mora, Seddon & Lye, 2000; Brennecke & Maginn,

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2001; van Rantwijk & Sheldon, 2007; Xie and Wan, 2019). The excellent properties (e.g. thermal

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stability and negligible vapor pressure) of ILs render them less prone to environmental emissions

63

than volatile organic compounds (VOCs) that are usually employed as solvents for chemical and

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biochemical reactions (Xu, Guo & Cheong, 2016; Brennecke & Maginn, 2001; Itoh, Nishimura,

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Ouchi & Hayase, 2003). Furthermore, ILs can be commonly tuned for easy separation of products

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and/or catalyst as well as for ILs recycling for reuse (Xu, Guo & Cheong, 2016; Cull, Holbrey,

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Vargas-Mora, Seddon & Lye, 2000; Brennecke & Maginn, 2001), making them excellent solvents

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for industrial applications. In recent years many imidazolium- and pyridinium-based ILs have been

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employed as pure solvents for non-aqueous biocatalysis in general or for the acylation of flavonoids

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in particular. In the related studies, ILs tended to improve the regioselectivity (Katsoura, Polydera,

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Katapodis, Kolisis & Stamatis, 2007) as well as the rates (Guo, Kahveci, Özçelik & Xu, 2009; Lue,

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Guo & Xu, 2010) of reactions. Nevertheless, ILs yet showed some disadvantages in the enzymatic

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esterification of glycosides. Lower reaction rates were often observed in ILs compared to those in

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the optimal organic solvents. This could probably due to lower activity of the enzyme in ILs, which

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show ionic and polar properties and high viscosities that could result in mass transfer limitations

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(Kontogianni, Skouridou, Sereti, Stamatis & Kolisis, 2003; Danieli, Luisetti, Sampognaro, Carrea &

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Riva, 1997; Mellou, Loutrari, Stamatis, Roussos & Kolisis, 2006; Hu, Guo, Lue & Xu, 2009).

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To buffer the deleterious effect of ILs on enzyme activity, organic solvents have been

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supplemented in many IL-mediated enzymatic reactions. It was reported that more than 60% of

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yield could be harvested in enzymatic esterification of glucose in [BMIM][PF6] in the presence of

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40% t-butanol while no enzymatic reaction occurred in the pure ILs (Ganske & Bornscheuer, 2005).

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We have previously investigated lipase-catalyzed esterification of esculin in ILs that were buffered

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with organic solvents (Hu, Guo, Lue & Xu, 2009). Higher conversion at higher substrate

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concentrations was harvested in the binary system containing both ILs and organic solvents,

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indicative of possible synergetic effects of combination of ILs and organic solvents, but more

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interesting phenomena remained unknown. For instance, enzyme performance in the IL-solvent

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systems did not solely depend on the property of the pure ILs or organic solvents but on their

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similarity and compatibility. The advantageous effects of a binary IL-solvent system (enhanced

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volumetric productivity) over a single solvent system was also observed in other cases (Chen, Liu,

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Guo, Huang, Wang, Xu & Zheng, 2011). However, almost all reported studies are limited to

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descriptive reports. Little attempt was made in delineating the correlation of the synergy between

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ionic liquids and organic solvents that display different structures and properties. This would be of

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utmost importance to design more efficient binary system for biocatalysis.

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This study focuses on the design and optimization of enzymatic reaction system, not on

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evaluation of enzyme sources and properties. However, as reviewed elsewhere (Ortiz et al., 2019),

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Novozym 435 (Candida antarctica Lipase B), immobilized on a synthetic resin, is the most widely

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used lipase for organic synthesis, including esterification of flavonoids (Hu, Guo, Lue & Xu, 2009);

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which has demonstrated excellent thermostability and activity even in harsh conditions (Ganske &

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Bornscheuer, 2005). As pointed out by Oritz et al. (2019), despite of some limitations and problems

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in relation to certain applications, Novozym 435 will continue to be one of the most used

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biocatalysts provided no better alternative is developed. Therefore, Novozym 435 is selected as the

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only biocatalyst used to examine the IL-organic solvent system; whereas an intensive discussion of

103

enzyme itself is beyond the focus of the present study.

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The present work is a further study on lipase-catalyzed esterification of flavonoids in IL-solvent

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systems, attempting to uncover the mechanisms of synergetic effects in binary systems that has not

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been intensively investigated in the previously published work (Chen, Liu, Guo, Huang, Wang, Xu

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& Zheng, 2011). The synergetic binary systems were firstly determined by investigating enzymatic

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reactions in different binary IL-solvent systems (1:1, v/v), in which the pattern matching with

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properties (property matching, for short) of ILs and organic solvents was analyzed. The changes in

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reaction rate were then correlated with the changes of temperature and viscosity etc. The kinetic

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properties of the synergetic binary systems and corresponding single IL and organic solvent systems

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were measured, compared and analyzed to deduce why the synergy effects of ILs and organic

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solvents upon biocatalysis exists.

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2. Materials and methods

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2.1. Materials

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Commercial immobilized lipase from Candida antarctica B (Novozym 435, ≥5,000 U/g) was

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obtained from Novozymes A/S (Bagsværd, Denmark). Palmitic acid (97%), esculin (97%),

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methanol, acetone, t-butanol, toluene, hexane, triethylamine, acetic acid, DMSO, and molecular

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sieves (3 Å) were purchased from Sigma-Aldrich (Broendby, Denmark). All the ILs were provided

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by

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methylimidazolium

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hexafluorophate), [OMIM][BF4] (1-methyl-3-octylimidazolium tetrafluoroborate), [OMIM][PF6]

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(1-methyl-3-octylimidazolium hexafluorophosphate), [MeOcPy][BF4] (3-methyl-1-octylpyridinium

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tetrafluoroborate), and [TOMA][Tf2N] (trioctylmethylammonium bis(trifluoromethylsulfonyl)

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imide) (Fig. S1).

Solvent

Innovation

Gmbh

(Köln,

tetrafluoroborate),

Germany)

including

[BMIM][PF6]

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[BMIM][BF4]

(1-butyl-3-

(1-butyl-3-methylimidazolium

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2.2. Enzymatic Esterification

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The enzymatic esterification of flavonoids in ionic liquids, organic solvents, and in binary IL-

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solvent systems was carried out in 10 mL glass vials with a screw cap. Esculin (15 mM) and

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palmitic acid (60 mM) were added into 2 mL of reaction media. The glass vials with screw caps

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were then incubated for 2 h at 40 °C with magnetic agitation at 150 rpm until the substrates were

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solubilized. The reaction was initiated by the addition of activated molecular sieves (3Å) and

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Novozym 435. A control experiment without enzyme was carried out in the same medium under

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identical conditions. The reaction was monitored by withdrawing samples at the set time intervals.

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The samples were diluted with DMSO and centrifuged at 4000 rpm for 20 min to remove any

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particulates. The supernatant was used for HPLC analysis. All experiments were performed in two

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replicates.

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2.3. Kinetic Study

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All kinetic assays were performed at 60 °C with agitation speed 150 rpm and an excessive and

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constant concentration of palmitic acid (120 mmol L-1). The concentration of esculin varied in the

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range of 7.5–60 mmol L-1. The kinetic parameters, the maximum reaction rate Vmax and the

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Michaelis constant Km were obtained by Lineweaver–Burk plot (the double reciprocal plot of

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reaction rate as a function of substrate concentration, 1/V vs 1/[S]). Ea was measured at 40–60 °C.

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2.4. Analysis of Reaction Mixtures

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HPLC analysis was carried out on a LaChrom, Merck system equipped with an evaporative light-

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scattering detector (PL-ELS 2100, Polymer Laboratories) as previously described (Hu, Guo, Lue &

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Xu, 2009). The column used was an Ascentis RP C8 column, 25 cm × 4.6 mm, 5 μm (Supelco

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Analytical, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark). The mobile phases used were

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methanol and 10 mM triethylamine solution (TEA buffer) buffered to pH of 4.0; The elution was

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programmed at a flow rate of 0.8 mL/min with the ratio of methanol to 10 mM TEA buffer changed

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from 0/100% to 100%/0 gradiently over 15 min, held at 100%/0 over 10 min, from 100%/0 to

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0/100% gradiently over 3 min, and held at 0/100% over 7 min. The column oven temperature was

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set at 40 °C. The detector settings were evaporator: 90 °C; nebulizer: 50 °C; and gas: 1.2 bar.

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Retention times at these conditions were around 10.05, 21.02, and 22.50 min for esculin, esculin

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ester, and palmitic acid, respectively. The quantity of product produced was calculated as a

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percentage of product peak area over the sum of esculin and esculin monoester peak area. The

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percentage area was regarded equal to the percentage mass, hence the esculin and product area

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percentages were divided by their own molecular weights to convert them into molar percentages

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(Hu, Guo, Lue & Xu, 2009). All analyses were carried out in triplicate. The relative standard

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deviation was measured below 3.6%.

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3. Results and discussion

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3.1. Introduction of organic solvents into ionic liquids results in a significantly improved reaction

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Novozym 435-catalyzed esterification of esculin with palmitic acid was respectively performed

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in both single and binary solvent system under identical conditions for comparison. As is shown in

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Fig. 1, esculin conversion in acetone reached 71 mol% after 48 h of reaction, indicative of high

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catalytic efficiency of Novozym 435 in acetone. However, conversion of esculin was below 11 mol%

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when using six neat ILs as the reaction media. The ascending order of esculin conversion was

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[TOMA][Tf2N] (10.7 mol%) > [BMIM][BF4] (3.8 mol%) > [BMIM][PF6] (2.3%) >

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[MeOcPy][BF4] (1.1 mol%) > [OMIM][PF6] = [OMIM][BF4] (0 mol%). In comparison with

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[BMIM][BF4] and [BMIM][PF6], even no reaction was investigated in both [OMIM][PF6] and

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[OMIM][BF4], indicating that the cation [OMIM]+ with longer carbon chain than [BMIM]+ could

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be unfavorable for the esterification of esculin.

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Upon the addition of 50% (v/v %) acetone, significant but various levels of increases in

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conversion of esculin were observed in all the above ionic liquids. With the identical

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organic cations, [PF6]-based ILs favored higher conversion of esculin than [BF4]-based ones,

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suggesting that the more hydrophobic anion [PF6]- might display a better compatibility with

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substrates or being benign to lipase mediated reactions when mixed with acetone. On the other hand,

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when keeping anions unchanged, conversion of esculin in [BMIM]-based ILs was found to be

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higher than that in [OMIM]-based ones but similar with that in [MeOcPy]-based ones. Results

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indicated that the effect of the cations and the anions on the reaction might be independent and

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additive in the binary IL-solvent system.

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The highest yield of enzymatic transformation was harvested in [TOMA][Tf2N]-acetone solvent

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system with conversion of esculin reaching up to 80 mol% (48 h), which was even higher than that

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harvested in neat acetone solvent. The tri-octyl substitution of the quaternary ammonia cation

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probably has a beneficial effect towards solubilizing the substrate through hydrophobic interactions

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between the octyl-chains and the aliphatic part of palmitic acid. In addition, [Tf2N] is known to be

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benign to enzyme activity as observed in a number of previous studies (De Diego, Lozano, Gmouh,

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Vaultier & Iborra, 2005).

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Above all, results suggested that potential synergetic effects might exist between IL and organic

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solvent in the binary solvent system, and this kind of effects deserve further investigation.

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3.2. Synergetic effects resulted from more combinations of ILs and organic solvents

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To further explore the synergetic effect between ILs and organic solvents, we designed a scenario

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by evaluating Novozym 435 activity of catalyzing esterification of esculin in individual organic

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solvents (t-butanol, toluene and hexane), individual ILs (6 kinds mentioned above), and mixtures of

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organic solvents and ILs (1:1, v:v) at two different temperatures (40 and 60 °C) (Table 1).

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3.2.1. Effects of organic solvents or ionic liquids on enzymatic esterification of esculin in single

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solvent system

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Tert-butanol, hexane and toluene are among the widely used organic solvents for biocatalysis,

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either in food processing or chemical engineering. As shown in Table 1, reactions in the three

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solvents all showed less than 5 mol% of esculin conversions at 40 °C after 96 h. Significantly

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higher esculin conversions were observed in the case of tobuene and hexane when heating up to 60

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°C, indicative of temperature dependence of the biocatalysis in neat organic solvents. The improved

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conversion might be due to a higher solubility of esculin and improved reaction kinetics at elevated

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temperature.

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The conversions of esculin were generally low (1–14 mol%) in ILs-mediated reactions at 40 °C.

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Similarly, the conversions were improved remarkably when reaction temperature was elevated to 60

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oC,

imcreasing by 3- to 9-fold compared to those at 40 °C (Table 1, entries 7–12). [TOMA][Tf2N]

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was found to the optimal reaction medium among the selected ILs for enzymatic esterification of

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esculin both in 40 and 60 °C (Table 1, entries 13–18).

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3.2.2. Effects of synergetic effects between ILs and organic solvents on enzymatic esterification of

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esculin in binary solvent system

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Inspired by the synergetic effects found in the preliminary results that were shown in Fig. 1, we

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thus comprehensively examined enzymatic catalytic esterification of esculin mediated by the binary

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IL-solvent system (Table 1, entries 19–27). Due to the lower b.p. of acetone, its combination with

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ILs as binary system was not further investigated as the reaction temperature higher than 50°C is

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not applicable to acetone-based system. As is shown in Table 1, entries 1–18, toluene and

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[TOMA][Tf2N] served the highest conversion of esculin among their counterparts, respectively.

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Toluene was thus selected as additives into the six ILs, and [TOMA][Tf2N] was equally added to

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the three organic solvents for expected higher conversions.

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With viscosity sharply lowered, IL-toluene binary solvent systems of all six ILs served higher

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conversions of esculin (Table 1, entries 19–24), respectively, compared with toluene (Table 1, entry

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2) and the ILs (Table 1, entries 7–12). Specifically, [OMIM][BF4]-toluene (47.85 mol%, entry 21),

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[TOMA][Tf2N]-toluene (40.83 mol%, Table 1, entry 23) and [MeOcPy][BF4]-toluene (58.08 mol%,

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Table 1, entry 24) afforded an increase by 11.01-, 2.92- and 11.69-fold, respectively, compared with

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their pure IL counterparts.

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Upon addition of [TOMA][Tf2N] into the three organic solvents, esterification reactions at 60 °C

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in all [TOMA][Tf2N]-organic solvent binary solvent system exhibited higher conversions of esculin

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than those of reactions performed in both neat [TOMA][Tf2N] and the corresponding organic

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solvents at the same temperature, and surprisingly, reached up to 92 mol% ([TOMA][Tf2N]-

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hexane),which was higher than any previously reported results (Mellou, Lazari, Skaltsa, Tselepis,

230

Kolisis & Stamatis, 2005; Kim, Choi, Lee & Ahn, 2003; Kontogianni, Skouridou, Sereti, Stamatis

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& Kolisis, 2003; Danieli, Luisetti, Sampognaro, Carrea & Riva, 1997; Lue, Guo & Xu, 2010; Hu,

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Guo, Lue & Xu, 2009; Chen, Liu, Guo, Huang, Wang, Xu & Zheng, 2011).

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3.3. Factors contributing to the synergy of binary IL-solvent systems

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According to the results stated above, the structures of ILs, temperature, and property matching

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within the binary IL-solvent systems are the important factors in influencing reaction efficiency,

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and deserve further optimization.

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3.3.1. Effects of the structure and property of ILs and organic solvents

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As is shown in Fig. S2, [BMIM]-based ILs displayed the lowest viscosities among the six tested

239

ILs under all operating temperatures with [PF6]- anion contributing as more than two times as much

240

viscosity as [BF4]- anion, which was also observed in the case of [OMIM]-based ILs. With IL

241

anions unchanged, [OMIM]-based ILs similarly showed more than doubling viscosity, compared

242

with [BMIM]-based ones (Zheng et al., 2013). It was reported that the viscosity of ILs is dependent

243

on strength of intermolecular interactions, and thus positively correlated with the substituent chain

244

length of IL cations (Gardas & Coutinho, 2008; Bajić, Šerbanović, Živković, Jovanović &

245

Kijevčanin, 2014).

246

The other two ILs, [TOMA][Tf2N] and [MeOcPy][BF4], were very similar in viscosity. The octyl

247

substituents on both of the two ILs rendered them more viscous than the imidazolium ILs with a

248

shorter substituted chain. Regarding comparison between [MeOcPy][BF4] and [OMIM][BF4], it is

249

clear that the nature of the parent cationic structure could also exert certain effects on the IL

250

viscosity in spite of less choices than that of anions (Gardas & Coutinho, 2008; Bajić, Šerbanović,

251

Živković, Jovanović & Kijevčanin, 2014). [TOMA][Tf2N] showed a steeper decrease in viscosity

252

against temperature than the other ILs, the curves of which did not seem to intersect. This effect

253

shown in the case of [TOMA][Tf2N] might be due to high polarity of [Tf2N]- anion, which contains

12

254

two sulfonyl- and two triflourmethyl- moieties (Mandal, Ghosh, Banerjee, Kuchlyan & Sarkar,

255

2013).

256

In order to investigate the effects of organic solvents upon synergy in the binary solvent system-

257

mediated reaction more comprehensively, t-butanol, toluene and hexane were respectively used as

258

solvents (50%, v/v) with all the six ILs in the binary solvent system for esterification of esculin with

259

all the other reaction conditions remaining identical.

260

The Novozym 435-catalyzed esterification of esculin in various binary solvent mixtures was

261

performed at 40 °C for 96 h. T-butanol-based binary solvent systems showed relatively low

262

conversions of esculin (Fig. 2A), reaching up to 10 mol% in the case of [TOMA][Tf2N]-t-butanal

263

system, in which neat [TOMA][Tf2N] served 14 mol% of conversion under the same reaction

264

conditions. Results indicated a negative synergetic effects when between [TOMA][Tf2N] and t-

265

butanol in mediating enzymatic esterification. Furthermore, [OMIM]-based ILs served higher

266

conversion than [BMIM]-based ones when mixing t-butanol as co-solvent and there seems to be no

267

definite relation to the choice of fluorinated elemental anion. [MeOcPy][BF4]-t-butanol served the

268

lowest conversion of esculin, which was slightly lower than that was harvested in neat

269

[MeOcPy][BF4] ILs. The only difference between [OMIM][BF4] and [MeOcPy][BF4] is that

270

[OMIM] contains an imidazole moiety while [MeOcPy] harbors a pyridine ring. Accordingly, the

271

difference in reactivity that the two t-butanol-based systems served could be assigned to the size

272

and polarity of the ring structures in the IL cations.

273

As is stated above, when using toluene as the solvent, the six IL-toluene binary solvent systems

274

served distinctly different conversion of esculin with high variance, suggestive of significant roles

275

that ILs play in mediating the biocatalysis. The octyl/methyl substituted ILs [OMIM][BF4] and

276

[MeOcPy][BF4] systems both reached high conversions up to ~50 mol% (Fig. 2B). Conversely,

277

[BMIM]-based IL-toluene binary solvent system served the lowest two conversions of esculin.

13

278

Results indicated that the octyl substituents of ILs could promote esterification of esculin in non-

279

polar toluene. The increase in conversion is probably due to the lipophilic octyl-tail of the cations

280

that could promote the dissolving of esculin and palmitic acid substrates in the IL-toluene system.

281

With IL cations unchanged, [PF6]-based IL-toluene binary mixtures always served lower conversion

282

of esculin than [BF4]-based ones, suggesting that low compatibility between highly polar IL anions

283

and hydrophobic solvents might lead to negative synergetic effect upon IL-solvent binary system–

284

mediated reactions.

285

When using non-polar hexane as the co-solvent, binary [TOMA][Tf2N]-hexane mixtures served

286

the highest conversion of esculin at both 40 (30 mol%) and 60 °C (92 mol%) (Fig. 2C). Low

287

conversions were observed in all the other IL-hexane binary mixtures with [OMIM][BF4]-hexane

288

showing the highest conversion of only ~10 mol%. The distinct difference in synergetic effects

289

might be due to the likeness of the ‘tri-octyl tails’ of [TOMA] cation and the aliphatic hexane

290

promoting substrate dissolution as well as enzyme accessibility. Meanwhile, [TOMA][Tf2N]-

291

hexane binary mixtures showed little impact upon the water content of enzyme, which is a key

292

factor for enzyme activity and structural stability (Herbst & Peper, 2012). As an aliphatic compound,

293

hexane solvent displays high lipophilicity, thus forming shell-like structures around enzyme

294

hydrophilic surfaces. This “shell” could keep the bond water molecules in close contact with

295

enzyme and help maintain enzyme active conformations.

296

3.3.2. Effect of reaction temperature

297

Based on the above screening results, [OMIM][BF4]-toluene, [TOMA][Tf2N]-hexane and

298

[TOMA][Tf2N]-t-butanol binary systems were selected for further optimization in terms of

299

temperature and substrate ratio.

300

Regarding temperature part, enzymatic reactions were performed over 96 h at 40, 50 and 60 °C,

301

respectively (Fig. 3). In general, results showed a positive correlation between reaction rate and

14

302

reaction temperature in the three binary solvent systems except that conversion of esculin in

303

[OMIM][BF4]-toluene exhibited optimal temperature of 50 °C. The ultimate conversions of esculin

304

after 96 h of reactions at 60 °C in [OMIM][BF4]-toluene, [TOMA][Tf2N]-hexane and

305

[TOMA][Tf2N]-t-butanol binary systems were 77, 92 and 68 mol%, respectively.

306

In the case of [TOMA][Tf2N]-hexane and [TOMA][Tf2N]-t-butanol binary systems (Fig. 3B &

307

3C), conversions of esculin showed significant improvement when increasing reaction temperature

308

from 40 to 50 °C, whereas much less increase in conversions of esculin was observed when heating

309

reaction mixtures from 50 to 60 °C. The high temperature-dependent reactivity in binary

310

[TOMA][Tf2N]- solvent systems indicate that [Tf2N]- might have a protective effect upon enzyme

311

structures at elevated temperatures, which was observed in previous studies (Hu, Guo, Lue & Xu,

312

2009).

313

In [OMIM][BF4]-toluene binary system, however, much less significant increase in conversion of

314

esculin was observed with temperature elevated from 40 to 50 °C, when compared with

315

[TOMA][Tf2N]-hexane and [TOMA][Tf2N]-t-butanol binary systems (Fig. 3A). When increasing

316

reaction temperature from 50 to 60 °C, negligible improvement of conversion was observed in the

317

early stages of reactions, and the conversion of esculin turned out lower than that at 50 °C after 72 h.

318

Results might be due to the deactivation effects that [BF4]- anions exerted on enzyme stability at

319

elevated temperatures (i.e. 60 °C) (Naushad, ALOthman, Khan & Ali, 2012).

320

3.3.3. Effects of palmitic acid-to-esculin ratio

321

The enzymatic esterification of esculin in [OMIM][BF4]-toluene, [TOMA][Tf2N]-hexane and

322

[TOMA][Tf2N]-t-butanol binary systems was further investigated in terms of substrate ratio

323

(palmitic acid to esculin). As is shown in Fig. 4, the reactions in the above three binary systems all

324

showed highest conversion when palmitic acid-to-esculin ratio was 4. Meanwhile, [TOMA][Tf2N]-

325

hexane served the highest conversions of esculin under all conditions of palmitic acid-to-esculin

15

326

ratio that ranged from 2 to 12. Results indicated that the ratio of substrates played the key role in

327

influencing conversion of enzymatic esterification.

328

Furthermore, [OMIM][BF4]-toluene served slightly higher conversion than [TOMA][Tf2N]-t-

329

butanol when palmitic acid-to-esculin ratio ranged from 2 to 8. However, increase in esculin

330

conversion was observed as the palmitic acid-to-esculin ratio increased from 6, and the order of

331

esculin conversion served by [OMIM][BF4]-toluene and [TOMA][Tf2N]-t-butanol was reversed

332

when the ratio increased beyond ~ 8. The uptrend and downtrend of conversions of esculin occurred

333

simultaneously when the substrate ratio was increased beyond 6 in [TOMA][Tf2N]-t-butanol and

334

[OMIM][BF4]-toluene, respectively, suggesting that the differences in effects of organic solvents

335

might be offset when decreasing their relative contents by adding the excess of the organic

336

substrates.

337

3.4. Kinetic properties of binary IL-solvent system-mediated enzymatic reactions.

338

Study of enzyme kinetics is a useful way that could reveal the possible catalytic mechanisms of

339

the enzyme under various conditions (Guo and Xu, 2006). To elucidate the mechanism of action of

340

the binary IL-solvent system effect on Novozym 435-catalyzed esterification of esculin with

341

palmitic acid, Vmax and Km constants were analyzed in the presence of neat t-butanol as the control

342

as well as binary mixtures (1:1, v/v) of [OMIM][BF4]-toluene, [TOMA][Tf2N]-hexane, and

343

[TOMA][Tf2N]-t-butanol (Table 2).

344

As is stated above, [TOMA][Tf2N]-hexane binary system served the highest conversion of

345

esculin among all the binary counterparts under identical conditions. Similarly, highest Vmax and Km

346

values were harvested in [TOMA][Tf2N]-hexane binary system, indicating the highest catalytic

347

efficiency and affinity towards substrates that Novozym 435 showed in [TOMA][Tf2N]-hexane

348

binary system with ultimate catalytic efficiency 38, 68, 55-fold of conversions served by

349

[OMIM][BF4]-tolune,

[TOMA][Tf2N]-t-butanol

16

and

t-butanol,

respectively.

Meanwhile,

350

[TOMA][Tf2N]-t-butanol binary system served higher Vmax, higher Kcat, neat t-butanol, suggestive of

351

higher turnover of biocatalysis in [TOMA][Tf2N]-t-butanol binary system. However, the

352

biocatalysis in [TOMA][Tf2N]-t-butanol binary system showed two times the Km value of the same

353

enzymatic reactions in neat t-butanol, leading to ultimately lower catalytic efficiency (Kcat/Km) of

354

[TOMA][Tf2N]-t-butanol

355

[OMIM][BF4]-toluene binary system served slightly lower Km and higher Vmax values in

356

comparison with neat t-butanol. The kinetic study suggested that the binary IL-solvent systems with

357

different synergetic effects could serve the enzymatic esterification by altering turnover or

358

affinity for substrate positively or negatively, need to be given further study.

binary

system

compared

with

neat

t-butanol.

Additionally,

359

To interpret the unique catalytic behavior of Novozym 435-catalyzed esterification of esculin

360

with palmitic acid in [TOMA][Tf2N]-hexane binary system, we propose the following assumptions:

361

1) The [TOMA][Tf2N]-hexane binary system displays a relatively high compatibility with low

362

measured viscosity (Table 1, entry 27); 2) The mixing between [TOMA][Tf2N] and hexane is

363

highly temperature-dependent, which was supported by experimental observation that

364

[TOMA][Tf2N]-hexane binary mixtures were transformed from three layers (solvent, interface and

365

IL) into a homogeneous phase when heating from room temperature to 60 °C (data not shown). The

366

phenomenon indicated a large energy barrier over 40–60 °C (high Ea value, Table 2), which could

367

be overcome by heating. According to the Eyring Equation, the transmission coefficient is getting

368

smaller at elevated temperature; facilitating an improved reaction rate. 3) Three octyl groups in

369

[TOMA] cation, along with hexane, could facilitate the dissolving of palmitic acid, and the

370

solubility of esculin could also be improved in [TOMA][Tf2N] ILs. Moreover, [Tf2N]- anions

371

reportedly provide a protective effect on the enzyme structure at high temperatures (Guo & Xu,

372

2006). Above all, all the factors together contribute to excellence of [TOMA][Tf2N]-hexane binary

373

system in mediating a high catalytic efficiency of Novozym 435 in esterification of esculin.

17

374

4. Conclusion

375

In Summary, for the first time this work presents a systematic study on the synergetic effects of

376

binary IL-solvent system in mediating lipase catalyzed esterification of flavonoids with fatty acids.

377

Property/structure matching between ILs and solvents is the key factor that contributes to the

378

synergy. The temperature plays a profound role in reducing viscosity of solvents, promoting

379

homogeneity of the binary systems as well as improving reaction kinetics. Two identified promising

380

systems, namely [OMIM][BF4]-toluene and [TOMA][Tf2N]-hexane served more than 90 mol% of

381

conversion of esculin in 96 h at 60 °C. Strikingly, [TOMA][Tf2N]-hexane binary system served

382

significant enhancement in turnover number kcat (1.82×10-3 s-1) by 6.28-fold, and in catalytic

383

efficiency kcat/Km (17.57×10-2 (Ms)-1) by 54.91-fold, compared with neat t-butanol (one of most

384

effective organic solvent systems). The knowledge obtained in this work should be useful for a

385

better understanding of the multiple interactions in binary IL−solvent mixtures, and also helpful for

386

the design and optimization of more systems, which are targeted to biocatalysis.

387 388

Declaration of interest

389

The authors declare no competing financial interest.

390 391

Acknowledgements

392

Ye Zhou acknowledges the Chinese Scholarship Council (CSC) for the financial support for his

393

study at Aarhus University. Financial support from AUFF-NOVA (AUFF-E-2015-FLS-9-12) is

394

gratefully acknowledged.

395 396

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397

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18. Katsoura M.H., Polydera A.C., Katapodis P., Kolisis F.N., Stamatis H. (2007). Effect of

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24. Mandal S., Ghosh S., Banerjee C., Kuchlyan J. & Sarkar N. (2013). Roles of viscosity, polarity,

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40. Zheng M.M., Wang L., Huang F.-H., Guo P.-M., Wei F., Deng Q.-C., et al. (2013) Ultrasound

498

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499

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500 501 502 503 504 505

Highlights 

An effective binary IL-solvent system is set for enzymatic conversion of esculin (92%);



Catalytic efficiency of binary [TOMA][Tf2N]-hexane system is 55-fold higher than t-

506

butanol.

507



Matching in property/structure between IL & solvent is a key factor for its outperformance;

508



Enhanced solubility of esculin by IL and reduced viscosity by co-solvent contribute to the

509

synergy;

510 511 512 513 514 515 516 517 518

Tables and Figures

519

Figure Captions

520

23

521

Fig. 1. Lipase-catalyzed esterification of esculin in neat solvents and IL-acetone binary solvent

522

systems. The conversions (40 ºC, 48 h) of esculin served by neat ionic liquids are shown in red

523

columns while results in the case of neat acetone and IL-acetone binary solvent systems are shown

524

in red columns.

525 526

Fig. 2. Time course of lipase-catalyzed eserification of esculin in IL-t-butanol (A), IL-tolunene (B),

527

and IL-hexane (C) binary solvent systems at 40 ºC.

528 529

Fig. 3. Time course of lipase-catalyzed eserification of esculin at different temperatures (40, 50, and

530

60 ºC) in [OMIM][BF4]-toluene (A), [TOMA][Tf2N]-hexane (B), and [TOMA][Tf2N]-t-butanol (C)

531

binary solvent systems.

532 533

Fig. 4. Effects of substrate ratio on lipase-catalyzed eserification of esculin in [OMIM][BF4]-

534

toluene, [TOMA][Tf2N]-hexane, and [TOMA][Tf2N]-t-butanol binary solvent systems.

535 536 537 538 539

24

540 541

Table 1. Solvent dependency of lipase-catalyzed esterification of esculin with palmitic acid. a

b Reaction

Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Medium

16 17 18 19 20

acetone t-butanol toluene hexane t-butanol toluene hexane [BMIM][BF4] [BMIM][PF6] [OMIM][BF4] [OMIM][PF6] [TOMA][Tf2N] [MeOcPy][BF4] [BMIM][BF4] [BMIM][PF6]

c Viscosity Density Reaction 3 (g/cm ,20°C) (mPa s, 20 temperature °C) (°C) 0.784 0.295 0.781 3.35 0.862 0.55 0.671 0.29 0.781 3.35 0.862 0.55 0.671 0.29 1.212 104.20 1.379 352.21 1.104 427.70 1.245 908.20 1.101 633.70 1.290 535.54 1.212 104.20 1.379 352.21 1.104 427.70 1.245 908.20 1.101 633.70 1.290 535.54 n.d. 3.13

dConversion

40 40 40 40 60 60 60 40 40 40 40 40 40 60 60 60 60 60 60

of esculin (mol%) 70.35 1.26 2.22 n.d. 9.67 53.17 30.47 3.81 5.46 4.31 1.16 13.99 4.96 15.05 25.76 34.12 8.45 43.99 36.76

542

[OMIM][BF4] [OMIM][PF6] [TOMA][Tf2N] [MeOcPy][BF4] [BMIM][BF4] 40 10.02 +toluene 21 [BMIM][PF6] n.d. 2.86 40 6.21 +toluene 22 [OMIM][BF4] n.d. 2.85 40 47.85 +toluene 23 [OMIM][PF6] n.d. 2.68 40 11.67 +toluene 24 [TOMA][Tf2N] n.d. 1.33 40 40.83 +toluene 25 [MeOcPy][BF4] n.d. 3.79 40 58.08 +toluene 26 [TOMA][Tf2N] n.d. 1.33 60 77.27 +toluene 27 [TOMA][Tf2N] n.d. 5.74 60 67.52 +t-butanol 28 [TOMA].[Tf2N] n.d. 0.72 60 92.01 +hexane a In a typical reaction, 15 mM Esculin and 60 mM palmitic acid were dissolved in 2 mL medium

543

(organic solvent, ionic liquid or IL/Solvent mixture) with the presence of 150 mg activated 3Å

544

molecular sieves. The reaction was initiated by adding 15 mg Novozym 435 and conducted for 96h

545

with magnetic agitation at 150 rpm. 25

546

b

547

c Viscosity

548

were treated as ideal systems in the estimation of viscosity by the Grunberg correlation [45].

549

d

550

evaluation.

551

n.d.: not detectable.

For ionic liquid/organic solvent system, they were mixed at equal volume. values of ILs (at 20 °C) used were provided by the suppliers. The IL–solvent mixtures

All reactions were carried in duplicate, and the means of two determinations was used for result

552

26

553

Table 2. Some kinetic properties of the esterification of esculin with palmitic acid catalyzed by

554

Novozym 435 in different IL-solvent systems a. Medium [OMIM][BF4]toluene (1:1, v/v) [TOMA][Tf2N]hexane (1:1, v/v) [TOMA][Tf2N]/tbutanol (1:1, v/v) t-butanol

Vmax/ mM h1(g enzyme)-1 1.39

kcat (s-1)

Km/ mM

bE /kJ a mol-1

84.54

kcat /Km ((M×s)-1) 0.46×10-2

0.39×10-3

6.55

1.82×10-3

10.93

17.57×10-2

136.96

1.72

0.48×10-3

182.01

0.26×10-2

46.51

1.05

0.29×10-3

90.86

0.32×10-2

88.45

48.40

555 556

aAll

557

concentration of palmitic acid. The variation of concentration of esculin was in the ranges of 7.5-60

558

mmol L-1. b Ea was measured at 40–60 °C.

kinetic assays were done at 60 °C with agitation speed 150 rpm and an excessive and constant

559 560

27

561 562

Figure 1

563 564 565

Fig. 1. Lipase-catalyzed esterification of esculin in neat solvents and IL-acetone binary solvent

566

systems. The conversions (40 oC, 48 h) of esculin served by neat ionic liquids are shown in red

567

columns while results in the case of neat acetone and IL-acetone binary solvent systems are shown

568

in red columns.

569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586

28

587 588 589 590 591 592

593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617

Figure 2

Fig. 2. Time course of lipase-catalyzed eserification of esculin in IL-t-butanol (A), IL-tolunene (B), and IL-hexane (C) binary solvent systems at 40 oC.

29

618 619 620 621 622 623 624

625 626 627 628 629

Figure 3

Fig. 3. Time course of lipase-catalyzed eserification of esculin at different temperatures (40, 50, and 60 oC) in [OMIM][BF4]-toluene (A), [TOMA][Tf2N]-hexane (B), and [TOMA][Tf2N]-t-butanol (C) binary solvent systems.

630 631 632 633 634 635 636 637 638

30

639 640

Figure 4

641 642

Fig. 4. Effects of substrate ratio on lipase-catalyzed eserification of esculin in [OMIM][BF4]-

643

toluene, [TOMA][Tf2N]-hexane, and [TOMA][Tf2N]-t-butanol binary solvent systems.

644 645

31