Influence of ionic liquids on lipase activity and stability in alcoholysis reactions

Abstract

Lipase activity and stability in ionic liquids containing N,N-dialkylimidazolium cations and different anions were investigated in alcoholysis reactions. Anions were the main factor affecting enzyme activity and stability, while hydrophobic and hydrophilic ionic liquids showed quite different phenomena. The initial enzyme activity in [Tf₂N]⁻ and [PF₆]⁻ based ionic liquids was much higher than that in ionic liquids containing anions such as [BF₄]⁻, [N(CN)₂]⁻ and [OAc]⁻. Novozyme 435 retained approximately 5%, 40% and 100% of the blank sample activity after being immersed in [BF₄]⁻, [PF₆]⁻ and [Tf₂N]⁻ based ionic liquids for 24 h. To reveal the influence of the ionic liquid properties, the initial enzyme activities were correlated with hydrophobicity (log P), polarity (E^N_T), hydrogen bond basicity (β) and viscosity. The initial enzyme activity increased with log P value and decreased with E^N_T value. In addition, the initial enzyme activity increased with β value in a narrow range (0.24–0.26) and then decreased continuously with the increase of β value. For [Tf₂N]⁻ based ionic liquids, the initial enzyme activity increased with viscosity, while the adverse relationship was found in [BF₄]⁻ and [PF₆]⁻ based ionic liquids. The enzyme secondary structure changes in ionic liquids were also investigated by FT-IR, which showed that the α-helix content decreased and the β-sheet content increased with the increase of immersion time.

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1. Introduction

Ionic liquids, considered a new and potential “green solvent”,^1,2 are widely used in enzymatic reactions. Most ionic liquids are hydrophobic and can dissolve small amounts of water.^1,3 Compared with traditional methods using organic solvents as reaction media, ionic liquids have some fine properties, such as low vapor pressure, preferable dissolution ability (dissolving polar and nonpolar substances), ability to form a two-phase system with other solvents, un-inflammability, and good thermal stability and chemical stability.² It is widely believed that lipases in ionic liquids have higher enzyme activity, stability and selectivity, which can increase the yield of the product and the conversion of materials. Moreover, ionic liquids can be designed as expected by selection of different cations, anions and substituents. Generally, based on cation composition, ionic liquids can be mainly divided into imidazolium, pyridinium, pyrrolidinium, phosphonium and ammonium ionic liquids.⁴

In the past few decades, ionic liquids have been applied as solvents to synthetize different products, in which lipases were used as catalysts. Hence, properties of lipase in ionic liquids are important and deserved to be investigated. Some conclusions have obtained that lipases in ionic liquids own higher enzyme activity, stability and selectivity.^5,6 Zhao et al.⁷ researched the effect of ionic liquid properties on Novozyme 435 under microwave irradiation, and they discovered that enzyme activity appeared loose bell-shaped relationship with hydrophobic (log P). They also found the viscosity affected the enzyme reaction rate, but not the primary factors to determine enzyme activity. Kaar et al.⁸ found that free lipase catalyzed the transesterification in [BMIM][PF₆] at an initial rate of 6.75 μM h⁻¹ mg⁻¹-enzyme, 1.5 times faster than the reaction in hexane. However, this lipase was unfortunately inactive in the other ionic liquids investigated ([BMIM][CH₃CO₂], [BMIM][NO₃], [BMIM][CF₃CO₂]). The results suggested that enzyme activity in ionic liquids is anion dependent. Sheldon et al.⁹ also considered that enzyme activity in ionic liquids is anion dependent and the enzyme activity had good correlation with log P.

Some studies have focused on diglyceride, monoglyceride and biodiesel production by enzymatic alcoholysis in ionic liquids, which obviously obtained good results.^10–12 However, the relationship between lipase activity, stability and ionic liquids properties was rarely reported in this kind of reaction up until now. Therefore, in this paper, the impact of ionic liquids containing imidazolium and different anions on lipase properties in alcoholysis reaction was investigated. Monoolein alcoholysis catalyzed by Novozyme 435 was chosen as a model reaction. It is expected that there is a correlation between lipase activity and properties of ionic liquids. Meanwhile, lipase secondary structure was measured by infrared spectroscopy (FT-IR) to assist to further analyses the stability.

2. Materials and methods

2.1 Materials and enzyme

Immobilized lipase from Candida antarctica (Novozyme 435) and were kindly donated by Novozymes (Beijing, China). [BMIM][BF₄], [HMIM][BF₄], [OMIM][BF₄], [BMIM][PF₆], [HMIM][PF₆], [OMIM][PF₆], [EMIM][Tf₂N], [BMIM][Tf₂N], [HMIM][Tf₂N], [OMIM][Tf₂N], [BMMIM][Tf₂N], [HMMIM][Tf₂N], [OMMIM][Tf₂N], [BMIM][N(CN)₂], [HMIM][N(CN)₂], [BMIM][OAc], [HMIM][OAc] were purchased from Chengjie Chemical Co. Ltd., Shanghai, China. Methyl oleate (analytical standard), methyl heptadecanoate (analytical standard) were purchased from Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China). 96% monoolein can be made of materials purchased from Qianwei oil Industry Co. Ltd. (Shanghai, China) by secondary molecular distillation. Hexane and acetone used for GC analysis were chromatographically pure. All other organic solvents were purchased commercially and were of analytical grade.

2.2 Enzyme activity in imidazolium ionic liquids

2.2.1 Effect of ionic liquids. The enzymatic glyceride alcoholysis reaction was conducted with agitation at 50 °C by reacting monoolein with methanol (mole ratio is 1) using 4% (w/w, relative to total reactants) Novozyme 435 as catalyst and 30% (w/w, relative to total reactants) ionic liquids as media. The initial rate was calculated based on the yield of methyl oleate at initial reaction time (10 min). All experiments were run at least in duplicate.

2.2.2 Effect of temperature. In order to saving the dosage of ionic liquids, the enzymatic alcoholysis reaction was conducted with agitation by reacting monoolein with methanol (mole ratio is 1) using 4% (w/w, relative to total reactants) Novozyme 435 as catalyst and 30% (w/w, relative to total reactants) ionic liquids as media. The reaction temperature was chosen at 30, 40, 50, 60 and 70 °C. The initial rate was calculated based on the yield of methyl oleate at initial reaction time.

2.3 Enzyme stability in ionic liquids

2.3.1 Pretreatment of enzymes. Appropriate lipase (Novozyme 435) was put into a beakers with appropriate ionic liquids (blank was without any ionic liquids), then the beakers were sealed and enzymes were cultivated in the condition of 50 °C for 1, 6, 12, 18 and 24 h. Enzymes were isolated via extraction of ionic liquids, diluted using the same amount of water, filtered through Buchner funnel by vacuum, dried for the same time. Untreated enzyme underwent the same process was used as blank.

2.3.2 Enzyme stability in alcoholysis reaction. The enzymatic glyceride alcoholysis reaction was conducted with agitation at 50 °C by reacting monoolein with methanol (mole ratio is 1) using 4% (w/w, relative to total reactants) Novozyme 435 (prepared in 2.3.1) as catalyst. The initial rate was calculated based on the yield of methyl oleate at initial reaction time (10 min), and the relative activity was based on the reaction used untreated enzyme (2.3.1) as catalyst.

2.4 GC determination of methyl oleate yield

The yield of methyl oleate was analyzed using a gas chromatograph (GC-2014, Agilent) equipped with a flame ionization detector (FID) by internal standard method. The internal standard was methyl heptadecanoate. The GC was fitted with an Agilent DB-FFAP column (30 m × 0.53 mm × 0.25 μm, length × I.D. × film thickness) and was operated with a 1 : 100 split ratio. The injector and detector temperatures were set to 250 °C. The oven program consisted of an initial temperature of 180 °C that was maintained for 1 min after which the temperature was increased at a rate of 15 °C min⁻¹ to a final temperature of 230 °C and maintained for 10 min.

2.5 Conformation analysis of lipase by FT-IR spectrometry

2.5.1 Enzymes deproteinization. Appropriate lipase (Novozyme 435) was mixed in dimethyl sulfoxide with agitation at 37 °C for 30 min. The mixture was pumped filtration and the filter mass was washed by dimethyl sulfoxide. Spherical resin recycled was soaked by 5% Triton X-100 with agitation at 37 °C for 30 min, the mixture was pumped filtration and the filter mass was washed by 5% Triton X-100, then washed by distilled water. Finally, the spherical resin was dried at 55 °C for 24 h. Carrier resin without protein was reserved.

2.5.2 The measurement of lipase secondary structure. The measurement of Novozyme 435 secondary structure was achieved using FT-IR spectrometry (Nicolet Nexus 470). IR spectra were measured at 25 °C. Conditions were 4 cm⁻¹ spectral resolution, 20 kHz scan speed, 128 scan co-additions, and the region was 500–4000 cm⁻¹. The spectrum acquisition (all samples were overlaid on a zinc selenide attenuated total reflectance (ATR) accessory) was from IR spectra, and the secondary structure elements based on the information of amide I region and the band assignment were manipulated using Omnic and Peakfit software.

3. Result and discussion

The major properties of ionic liquids are shown in Table 1 including hydrophobicity (log P), polarity (E^N_T), hydrogen bond basicity (β) and viscosity, and the initial enzyme activity in different ionic liquids was also presented in Table 1.

Table 1 The initial enzyme activity data of alcoholysis reaction in ionic liquids and physical properties of ionic liquids solvents^a

	Ionic liquids	Initial enzyme activity (mmol min^−1 mg^−1 E)	E^NT	log P	Viscosity^n (cP) (T in 25 °C)	H-Bond basicity (β)
a All reactions were conducted with agitation at 50 °C by reacting monoolein with methanol (mole ratio is 1) using 4% (w/w, relative to total reactants) Novozyme 435 as catalyst and 30% (w/w, relative to total reactants) ionic liquids as media; the initial enzyme activity were based on the mass of immobilized enzyme. Data from ^aref. 24; ^bref. 25; ^cref. 26; ^dref. 27; ^eref. 7; ^fref. 28; ^gref. 29; ^href. 30; ^iref. 31; ^jref. 32; ^kref. 33; ^lcalculated from octanol–water partition coefficient (Kow) in ref. 34; ^mref. 8; ^ndetermined by this study using the Cannon-Fenske Routine (CFR) viscometer at 25 °C.
1	[BMIM][BF4]	0.0245 ± 0.0017	0.673^a	−2.44^b	98	0.38^c
2	[HMIM][BF4]	0.0212 ± 0.00113			310
3	[OMIM][BF4]	0.0213 ± 0.00523	0.65^d	−1.34^e	440	0.41^d
4	[BMIM][PF6]	0.0914 ± 0.00948	0.669^c		380	0.24^f
5	[HMIM][PF6]	0.0494 ± 0.00608	0.66^d		560	0.58^g
6	[OMIM][PF6]	0.0724 ± 0.00467	0.633^h	−0.62^k	680	0.46^g
7	[EMIM][Tf2N]	0.0541 ± 0.00269	0.685^f	−1.05^k	32	0.24^f
8	[BMIM][Tf2N]	0.0809 ± 0.00707	0.645^f	−0.438^l	52	0.245^c,f
9	[HMIM][Tf2N]	0.1179 ± 0.0058	0.651^i	0.188^l	87	0.26^f
10	[OMIM][Tf2N]	0.0959 ± 0.00764	0.627^i	0.56^k	119	0.28^i
11	[BMMIM][Tf2N]	0.0743 ± 0.00721	0.552^h		93	0.24^c
12	[HMMIM][Tf2N]	0.0981 ± 0.00806	0.574^i	0.196^l		0.26^i
13	[OMMIM][Tf2N]	0.1230 ± 0.01075	0.525^h		150
14	[BMIM][N(CN)2]	0.0647 ± 0.00665	0.639^j		32	0.71^j
15	[HMIM][N(CN)2]	0.0558 ± 0.00608	0.63^j
16	[BMIM][OAc]	0.0008 ± 0.00014	0.57^j	−2.77^m
17	[HMIM][OAc]	0.0008 ± 0.00014

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3.1 Enzyme activity in ionic liquids

The initial enzyme activity was calculated based on the yield of methyl oleate. Table 1 shows the initial enzyme activity in ionic liquids consisted of 5 different anions. The enzyme was unfortunately inactive in [OAc]⁻ based ionic liquids (16–17), however, the enzyme can play an effective role in other ionic liquids, such as ionic liquids containing [BF₄]⁻ (1–3), [N(CN)₂]⁻ (14–15), [PF₆]⁻ (4–6) and [Tf₂N]⁻ (7–12). In addition, initial enzyme activities in ionic liquids consisting of [Tf₂N]⁻ and [PF₆]⁻ were higher than those consisting of [BF₄]⁻ and [N(CN)₂]⁻. The initial enzyme activities were in a decreasing order of [Tf₂N]⁻ > [PF₆]⁻ > [N(CN)₂]⁻ > [BF₄]⁻ > [OAc]⁻ which was in agreement with previous reported conclusions.^8,13–15 Anion such as [BF₄]⁻ is more nucleophilic than [PF₆]⁻ and [Tf₂N]⁻, which may coordinate more strongly with positively charged sites in the lipase and thus cause conformation changes. However, very low initial enzyme activities were observed in ionic liquids with water miscible properties, such as [OAc]⁻, probably due to the influence of solvating properties on the lipase surface microenvironment.¹⁶ As seen in Table 1, the enzyme initial activity is closely related to anions of ionic liquids.

Table 1 also shows the initial enzyme activities in ionic liquids consisting of the same anions ([Tf₂N]⁻) and cations with different substituents. The initial enzyme activity increased gradually with the increase of chain length of the alkyl substituent of the imidazolium ring. The same phenomenon had been observed by A. P. de los Ríos et al.,¹⁷ they explained the increasing of chain length of cations lead to an increase in the hydrophobicity of the ionic liquids. However, initial enzyme activity in [OMIM][Tf₂N] decreased probably due to their higher viscosity than [HMIM][Tf₂N], which may prevent mass transfer in a certain extent. However, the rule could not be applied to another anion series such as [BMIM][PF₆], [HMIM][PF₆], [OMIM][PF₆], because high β value is not beneficial to the initial reaction, which will be explained in later part. Therefore, the effect on initial enzyme activity has little relationship to cations.¹⁶

Then, the initial enzyme activity data are plotted against properties of these ionic liquids.

3.1.1 Influence of hydrophobicity log P. Partition coefficients for ionic liquids were determined as a ratio of ionic liquid concentration in the octanol phase to the ionic liquid concentration in the aqueous phase. However, it is necessary to distinguish hydrophobicity with polarity, and hydrophobicity is often associated with the miscibility with water. Zhao et al.⁷ found a loose correlation (a bell curve) between initial activity and log P in simple transesterification. The increasing trend was affected by the anion's H-bond basicity, enzyme dissolution, anion ionic association ability, and cation hydrophobicity, while the decreasing trend is likely caused by substrate ground-state stabilization or hydrophobic interactions. Kaar et al.⁸ concluded that lipase was only active in hydrophobic ionic liquids, such as [BMIM][PF₆], but inactive in other hydrophilic ionic liquids including [BMIM][BF₄], [BMIM][NO₃] and [BMIM][CF₃COO].

Lipase activity data were plotted against log P value of some ionic liquids (Fig. 1). The initial enzyme activities in [BF₄]⁻, [PF₆]⁻ and [Tf₂N]⁻ based ionic liquids (1–13) suggested a good linear relation. As viewed from Fig. 1, the initial enzyme activity increased with log P value increasing, reached the highest activity in [HMIM][Tf₂N] with the log P value of 0.1179 mmol min⁻¹ mg⁻¹ E (9). Ionic liquids are more nucleophilic which can coordinate more strongly to positively charged sites in the lipase, causing lipase inactivation. Moreover, as we all know, lipase can retain better activity in media containing little or no water.

Fig. 1 Relationship between initial enzyme activity and ionic liquids' log P values.

3.1.2 Influence of E^N_T. Ionic liquids' polarity can be represented by E^N_T, which is solvatochromic polarity scale. Mutschler et al.¹⁸ researched the synthesis of methylglucose fatty acid esters in ionic liquids, and they found higher polarity of the ionic liquids correlates negatively to the conversions of product. Y. Liu et al.¹⁶ came to the conclusion that higher polarity of ionic liquids system leaded to poorer lipase conformation, which consequently decreased the enzyme activity.

As shown in Fig. 2(a), E^N_T value can be divided into three parts, including <0.6, 0.6–0.65 and >0.65. As a whole, the initial enzyme activity declined with the increase in E^N_T value. A significantly lower initial activity was observed when E^N_T value was greater than 0.65, thus higher E^N_T value was not beneficial to initial enzyme activity and may destroy the structure of enzyme. As mentioned above, anions have greater influence on the initial enzyme activity, so the initial enzyme activates in [Tf₂N]⁻ based ionic liquids were plotted against E^N_T value as shown in Fig. 2(b). With a few exceptions, the initial enzyme activities were obviously declined with the increase of E^N_T value. Initial enzyme activity in [BMMIM][Tf₂N] was much lower than the predicted value because the β scale was lower in these ionic liquids.

Fig. 2 Relationship between initial enzyme activity and ionic liquids' E^N_T values.

3.1.3 Influence of hydrogen bond basicity β. Hydrogen bond basicity β quantifies hydrogen bond accepting ability. So far, the relationship between β value and initial enzyme activity has not been reported. Bernson et al.¹⁹ discovered the strength of anion coordination was further dependent on the hydrogen bond basicity. Initial enzyme activity increased with hydrogen bond basicity β value increasing in narrow range (0.24–0.26). (Fig. 3) ([BF₄]⁻ based ionic liquids are excluded because low initial enzyme activity was observed). However, with β value continuously increasing (the β value is more than 0.26), the initial enzyme activity decreased sharply. Enzyme activity is likely related to the hydrogen bond acceptor strength of the anions: anion with low hydrogen bond basicity was beneficial for enzyme stability.⁷ Influences of hydrogen bond force mainly reflected two aspects. High hydrogen bond force may dissolve complex and high polar substances. On the one hand, ionic liquids owning higher hydrogen bond force may form hydrogen bond more easily, which destroys the structure of enzyme and decreases the enzyme activity. Moreover, high hydrogen bond force is the reason that ionic liquids may form hydrogen bond with monoglyceride, which affects the process of the reaction.¹² On the other hand, high hydrogen bond basicity and hydrophilic of ionic liquids drives the enzyme to dissolve in these media.^13,20 The dissolution of lipase in hydrophilic ionic liquids is an indication of strong interactions between the enzyme and solvent molecules. Such interactions may be unfavorable for active site and/or strong enough to disrupt the protein structure. This is a possible reason to explain the lower initial enzyme activity in [BF₄]⁻ based ionic liquids.

Fig. 3 Relationship between initial enzyme activity and ionic liquids' β values.

3.1.4 Influence of viscosity. Sheldon et al.²¹ considered that high viscosity of ionic liquids slowed down the change of protein structure, and could protect enzyme to keep original structure and activity. However, Zhao et al.⁷ had another conclusion that viscosity was not directly related to the enzyme activity. High viscosity might reduce the reaction rate because of mass transfer limitation.

Our initial enzyme activity data were plotted against viscosity values of some ionic liquids including [Tf₂N]⁻, [PF₆]⁻ and [BF₄]⁻ anions (Fig. 4). The relationship between the initial enzyme activity and viscosity was not coincident in different ionic liquids. For [Tf₂N]⁻ based ionic liquids, the initial enzyme activity increased with viscosity value increasing. However, for [BF₄]⁻ and [PF₆]⁻ based ionic liquids, the initial enzyme activity decreased. Viscosity value of [BF₄]⁻ and [PF₆]⁻ based ionic liquids were higher than [Tf₂N]⁻ based ionic liquids. Therefore, when viscosity value increased, the reaction rate deceased due to the mass transfer limitation. Viscosity value of [Tf₂N]⁻ based ionic liquids were relatively lower, and thus viscosity had no negative effect on the initial enzyme activity. As shown in Fig. 4, the initial enzyme activity in [HMIM][PF₆] was exceptive because higher β value hold back the action just like the statement pointed above.

Fig. 4 Relationship between initial enzyme activity and ionic liquids' viscosity values (the lines are to guide the general trend, not to make correlations).

3.1.5 The correlation analysis between ionic liquids properties and initial enzyme activities. As seen in Fig. 5, the correlation analysis between ionic liquids properties and initial enzyme activities was analyzed by Partial Least Squares Regression (PLSR). It is apparent to find that log P correlated positively to the initial enzyme activity while other three properties including E^N_T, viscosity and β correlated negatively to the initial enzyme activity in the whole. The contribution of log P to the initial enzyme activity was the most significant in these ionic liquids properties, followed by E^N_T, β and viscosity.

Fig. 5 The contribution analysis of ionic liquids properties on initial enzyme activity.

3.1.6 Influence of reaction temperature. From Fig. 6, it is noted that the initial enzyme activity gradually reduced by the order of [Tf₂N]⁻, [PF₆]⁻ and [BF₄]⁻ at all five temperatures from 30 to 70 °C. However, increasing the reaction temperature further to 70 °C did not obviously distinguish the initial enzyme activity among three different ionic liquids, which may be due to the volatilization of methanol at higher temperature. As seen in Fig. 6, the initial enzyme activity changed with different temperature and the conclusion can be seen in all three ionic liquids. It is proved that temperature can affect the initial enzyme activity. However, the effect is far smaller than the anions and their properties.

Fig. 6 Relationship between initial enzyme activity and temperature including three different anions ionic liquids.

3.2 Enzyme stability in ionic liquids

The enzyme stability for alcoholysis in ionic liquids was investigated as shown in Fig. 7. 9 kinds of ionic liquids including three anions were chosen to research the stability mainly due to important effects on enzyme activity of these anions. As seen in Fig. 7, after 18 h suspended in [PF₆]⁻ based ionic liquids at 50 °C, Novozyme 435 retained approximately 60% of the blank sample activity. What's more, Novozyme 435 lost no activity during the initial 1 h of incubation in [PF₆]⁻ based ionic liquids. However, the activity of Novozyme 435 in [BF₄]⁻ based ionic liquids lost faster. In the initial 1 h, Novozyme 435 retained approximately 20% of the blank sample activity. Enzyme activity was always high during the incubation in [Tf₂N]⁻ based ionic liquids, because the enzyme structure may be protected by ionic liquids in the process of water washing. Kaar et al.⁸ researched the lipase stability in [BMIM][PF₆] by Lipase Diagnostic kit, and discovered that after 48 h suspended in [BMIM][PF₆] at 50 °C, Novozyme 435 retained approximately 65% of its original activity. The hydrophobic ionic liquids coated and thus protected the layer of essential water surrounding the lipase.

Fig. 7 Stability of Novozyme 435 in 9 ionic liquids (three different anions) at 50 °C in glyceride alcoholysis reaction.

3.3 The secondary structure of Novozyme 435 analyzed by FT-IR spectrometry

The secondary structure of Novozyme 435 suspended in 9 ionic liquids for 0 h, 12 h and 24 h were analyzed to assist to explain the enzyme stability. The ability to retain the native conformation of enzyme is a characteristic of effective ionic liquids.^20,22 The secondary structure of enzyme can also be analyzed by FT-IR since proteins absorb infrared wavelengths due to the peptide bond vibrations. The amide I region (mainly due to the C=O stretching vibration) at approximately 1600–1700 cm⁻¹ is mostly used in protein secondary structure determination due to its sensitivity in conformational changes. Firstly, carrier resin lack the amide I region as shown in Fig. 8, and the influence of resin can be eliminated. Table 2 shows the secondary structure elements of Novozyme 435 in selected 9 ionic liquids. The α-helix content of original Novozyme 435 was 16.5%, and the β-sheet content was about 37%. With the increase of immersion time, the α-helix and β-sheet contents of Novozyme 435 changed a lot. The most obvious phenomenon is that α-helix content of Novozyme 435 decreased while β-sheet content increased. After the immersion in ionic liquids for 12 h, α-helix content of Novozyme 435 in [BF₄]⁻ based ionic liquids decreased most, followed by [PF₆]⁻ and [Tf₂N]⁻ and β-sheet content of Novozyme 435 increased in the order of [BF₄]⁻, [PF₆]⁻ and [Tf₂N]⁻. After the immersion in ionic liquids for 24 h, the rule is consistent with the previously described. The decrease in α-helix content of Novozyme 435 probably affects the lipase active site. The lower the α-helix content, the easier the substrates access the active site of lipase.^16,23

Fig. 8 Infrared absorption spectrum of carrier resin and ionic liquids untreated Novozyme 435.

Table 2 Quantitative estimation (%) of the secondary structure elements of treated Novozyme 435 calculated by FT-IR analysis in amide I region at 12 h and 24 h^a

	12 h		24 h
	α-Helix (%)	β-Sheet (%)	α-Helix (%)	β-Sheet (%)
a All experiments were run at least in duplicate and the percent errors were less than 5%.
[BMIM][BF4]	12.76	37.2	12.58	51.28
[HMIM][BF4]	10.82	57.72	10.17	58.01
[OMIM][BF4]	12.17	55.15	11.8	55.68
[BMIM][PF6]	11.8	48.19	11.42	53.48
[HMIM][PF6]	10.53	43.34	10.01	47.08
[OMIM][PF6]	15.42	46.26	15.35	47.17
[BMIM][Tf2N]	15.47	40.56	14.57	44.55
[HMIM][Tf2N]	16.24	36.73	15.7	42.77
[OMIM][Tf2N]	16.88	32.94	15.87	45.96

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4. Conclusions

Initial enzyme activity and stability were investigated in ionic liquids consisted of different anions in alcoholysis reaction. The anions were found to be the major factor to affect the initial enzyme activity and stability. Initial enzyme activity in hydrophobic ionic liquids was higher than hydrophilic ionic liquids. Several properties of ionic liquids such as log P, E^N_T, β and viscosity value were correlated with the initial enzyme activity. The initial enzyme activity increased with the increase of log P value and declined with the increase of E^N_T value. High β and viscosity values were not beneficial to initial enzyme activity. Temperature influenced the initial enzyme activity as well, but the effect was smaller than other factors. Enzyme stability was also related to anions. α-Helix and β-sheet contents of Novozyme 435 could assist to explain the stability in ionic liquids. With the increasing of immersing time, the α-helix content of Novozyme 435 decreased and the β-sheet content of Novozyme 435 increased slightly.

Acknowledgements

Support for this work was provided by the National Natural Science Foundation of PR China (31601433) and Jiangsu Provincial Natural Science Foundation (BK20140149).

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