Hydroxyl ammonium ionic liquids as media for biocatalytic oxidations

Article abstract of DOI:10.1039/c5gc02381e

In this work, neoteric and biodegradable ionic liquids (ILs) based on various hydroxyl ammonium cations and formic acid anions have been used as media for biocatalytic oxidoreductions catalyzed by different metalloproteins. The effect of these ILs on the biocatalytic behavior and structure of solubilized enzymes was investigated using cytochrome c (cyt c) as a model protein. The use of IL-based media enhances the tolerance of cyt c against the denaturing effect of H₂O₂ and increases (up to 20 fold) its catalytic efficiency compared to that observed in buffer. This beneficial effect strongly correlates with the concentration of ILs used, as well as the chaotropicity of their cations. UV-vis, circular dichroism and Fourier transform infrared (FT-IR) spectroscopic studies indicated that, the effect of ILs on the catalytic behavior of cyt c could be correlated with slight structural changes on the protein molecule and/or perturbations of the heme microenvironment. The use of hydroxyl ammonium-based ILs as reaction media increased (up to 4-fold) the decolorization activity of cyt c. All ILs used were recycled and successfully reused three times indicating the potential application of these novel ILs as environmentally friendly media for biocatalytic processes of industrial interest.

Full text of DOI:10.1039/c5gc02381e

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Page 1 of 42
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DOI: 10.1039/C5GC02381E
Hydroxyl ammonium ionic liquids as media for
biocatalytic oxidations
a
Athena A. Papadopoulou, a Andromachi Tzani,b Dimitrios Alivertis, Maria H.
b
Katsoura, a Angeliki C. Polydera, a Anastasia Detsi and Haralambos Stamatis a*
In this work, neoteric and biodegradable ionic liquids (ILs) based on various hydroxyl
ammonium cations and formic acid anion have been used as media for biocatalytic
oxidoreductions catalyzed by different metalloproteins. The effect of these ILs on the
biocatalytic behavior and structure of solubilized enzymes was investigated using cytochrome
c (cyt c) as a model protein. The use of ILs–based media enhances the tolerance of cyt c
against the denaturing effect of H2O2 and increases (up to 20 fold) its catalytic efficiency
compared to that observed in buffer. This beneficial effect strongly correlates with the
concentration of ILs used, as well as the chaotropicity of their cations. UV–vis, circular
dichroism and Fourier transform infrared (FTꢀIR) spectroscopic studies indicated that, the
effect of ILs on the catalytic behavior of cyt c could be correlated with slight structural
changes on the protein molecule and/or perturbations of the heme microenvironment. The use
of hydroxyl ammoniumꢀbased ILs as reaction media increased (up to 4ꢀfold) the decolorization
activity of cyt c. All ILs used were recycled and successfully reused three times indicating the
potential application of these novel ILs as environmentally friendly media for biocatalytic
processes of industrial interest.
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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23 toxicity and low biodegradability of commonly used second
24 generation ILs.13,14 Due to the above mentioned disadvantages,
1 Introduction
2
3
4
5
6
7
8
9
Ionic liquids (ILs), also called molten salts, are
25 over the last decade significant attention has been focused on
o
mixtures of cations and anions that melt below 100 C and
26 the development of novel ILs with enhanced green properties.
27 Recently, a third generation of ILs is emerging with structures
28 comprising of biodegradable and readily available nontoxic
29 ions such as natural bases, amino acids, sugars and naturally
30 occurring carboxylic acids.15ꢀ18
have received considerable attention over the last decade as an
environmentally friendly alternative to organic solvents. Due to
their interesting physical and chemical properties, such as
negligible vapour pressure, ability to dissolve various
hydrophobic/hydrophilic compounds and excellent chemical
and thermal stability, they have been widely used as “green”
31
Together with this third generation ILs, deepꢀ
32 eutecticꢀsolvents (DES) formed by mixture of bioꢀbased, nonꢀ
33 toxic, biodegradable and inexpensive salts (e.g. choline
34 chloride and urea or glycerol), represent also a promising
35 alternative option for using biodegradable ionic solvents in
36 biocatalysis and biotransformations.19ꢀ21
10 media for biocatalytic processes.1ꢀ3 The first studies of enzymeꢀ
11 catalyzed reactions in ILs were reported in so called second
12 generation ILs, which are mainly based on cations of
13 heterogeneous cyclic amines, such as substituted imidazoliums
14 and alkyl pyridiniums, as well as poor nucleophilic anions,
15 such as (BF4)ꢀ, (PF6)ꢀ, (CF3CO2)ꢀ,(CF3SO3)ꢀ.4ꢀ6
37
A family of third generation biocompatible ionic
38 liquids that are based on hydroxyl ammonium cation and
39 formic acid anion was described.22ꢀ27 These ILs display
40 significant scientific interest due to their low cost of
41 preparation and simple synthesis and purification methods,
42 since they can be easily formed by the stoichiometric
43 combination of a Brønsted acid with a Brønsted base.22,28
44 Furthermore, both cation and anion exhibit a considerably low
16
In the last years, numerous studies of second
17 generation ILs, in the context of biocatalysis, revealed that
18 many enzymes exhibit excellent selectivity and activity and
19 maintain very high thermal and operational stability in these
20 solvents.6ꢀ12 However, their use in large scale applications is
21 limited due to their difficult preparation and high cost.1
22 Moreover, concerns have arisen regarding the environmental
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VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C5GC02381E
45 toxicity and are biodegradable.14,18,29 For instance, formic acid
46 (methanoic acid), the simplest carboxylic acid that occurs
47 widely in nature and degrades readily in the presence of
48 oxygen, has low toxicity, hence it is used as a food additive and
49 as a preservative and antibacterial agent in livestock feed.29,30
50 Moreover, the presence of hydroxyl groups significantly
51 decreases the toxicity (up to 100 times lower compared to
52 imidazoliumꢀ or pyridiniumꢀ based ILs) and improves the
53 biodegradability of quaternary ammonium cations14,31 leading
54 to ionic solvents that are biodegradable, recyclable and not
55 harmful to the environment compared to conventional
56 solvents.18
88 formation of Compound II, while the reducing substrate is
89 oxidized to the radical product. The reaction cycle is completed
90 by the second reduction step, in which Compound II oxidizes
91 another molecule of the reducing substrate.36 Through kinetic
92 and stability studies, as well as the application of UVꢀvis,
93 ATRꢀFTIR and circular dichroism spectroscopic techniques,
94 we have investigated the effect of these neoteric ionic solvents
95 on the catalytic behaviour and structure of cyt c. Moreover, in
96 order to estimate the environmental impact of the above
97 mentioned ILs, their biodegradability has been assessed by
98 measuring the Biochemical Oxygen Demand (BOD).
99
57
In this work, four hydroxyl ammonium based ILs,
58 formed by different cations and the same anion (formic acid)
59 such as: 2ꢀhydroxyl ethylammonium formate (HEAF), 2ꢀ
60 hydroxyꢀNꢀmethylethanaminium formate (HMEAF), 2ꢀ
61 hydroxyꢀN,Nꢀdimethylethanaminium formate (HDMEAF) and
62 bis(2ꢀhydroxyethyl) ammonium formate (BHEAF), were used
63 as media for oxidoreductions catalyzed by various biocatalysts
64 such as cytochrome c (cyt c), peroxidase, tyrosinase, laccase
65 and alcohol dehydrogenase. The structures of the ILs used in
66 the present work are depicted in Fig.1. Our interest for these
67 solvents arises from the fact that these hydroxyl ammoniumꢀ
68 based ILs have been labeled as biodegradable, recyclable and
69 environmentally friendly media.18 Although the effect of
70 different second generation ionic liquids formed with synthetic
71 anions on the catalytic behaviour of cyt c has been recently
72 described32, to our knowledge, there is no published study
73 regarding the catalytic behaviour of biocatalysts, including
74 metalloproteins, in third generation environmentally friendly
75 ILs, as those described in the present work. Therefore, a
76 detailed investigation of the effect of such ILs on the catalytic
77 and structural behaviour of biocatalysts is of great interest. In
78 the present work,, this effect on the biochemical and structural
79 characteristics of metalloproteins was investigated using cyt c
80 as a model protein, since it is one of the most thoroughly
81 physicochemically characterized metalloproteins33,34 with
82 biotechnological interest.33ꢀ35 Cyt c is a hemoprotein that could
83 catalyse peroxidaseꢀlike reactions in the presence of an electron
84 acceptor such as hydrogen peroxide (H2O2). In this catalytic
85 cycle, the reaction follows a ping pong mechanism. Firstly,
86 hydrogen peroxide reacts with cyt c to yield an intermediate
87 called Compound I. Reduction of Compound I leads to the
100 Experimental Section
101 Materials
102 2ꢀ(methylamino) ethanol (Alfa Aesar), 2ꢀ(dimethylamino)ꢀ
103 ethanol (Alfa Aesar), diethanolamine (Merck), ethanolamine
104 (Sigma Aldrich), ethanol absolute (Sigma Aldrich) and formic
105 acid (Merck) were of the highest purity available (>99%) and
106 were used without further purification. Cytochrome c from
107 equine heart (>95% protein content), 552 U/mg solid (1 Unit
108 corresponds to the amount of enzyme that causes an increase in
109 absorbance at 470nm of 0.01 per minute at pH 7.0 and 25 °C in
110 a reaction mixture containing guaiacol and hydrogen peroxide),
111 peroxidase from horseradish HRP (E.C. 1.11.1.7.) ~ 66 %
112 protein content, 261 U/mg solid (1 Unit corresponds to the
113 amount of enzyme which produces 1mg of purpurogallin from
114 pyrogallol in 20 s at pH 6.0 at 25 oC) (type VI), tyrosinase from
115 mushroom Agaricus bisporus (EC 1.14.18.1), ~ 22 % protein
116 content, 3933U/mg solid (1 U unit corresponds to the amount
117 of enzyme that causes an increase in absorbance at 280nm of
118 0.001 per minute at pH 6.5 at 25 °C in a 3 ml reaction mixture
119 containing Lꢀtyrosine), laccase from Trametes versicolor (E.C.
120 1.10.3.2), ~ 8.5 % protein content, 10 U/mg solid (1 Unit
121 corresponds to the amount of enzyme which converts 1 ꢁmol
122 catechol per minute at pH 4.5 and 25 °C) and alcohol
123 dehydrogenase ADH from baker’s yeast (E.C. 1.1.1.1), >90%
124 protein content, 440 U/mg solid (1 U converts 1.0 ꢁmole of
125 ethanol to acetaldehyde per min at pH 8.8 at 25 oC) were
126 purchased from Sigma Aldrich and were used without further
127 purification. 2ꢀmethoxyphenol (guaiacol), 4ꢀmethylꢀcatechol
128 (>95%) and βꢀnicotinamide adenine dinucleotide (βꢀΝΑD+)
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129 (≥99%) were obtained from Sigma. 2,2'ꢀazinoꢀbis(3ꢀ
130 ethylbenzothiazolineꢀ6ꢀsulfonic acid) diammonium salt
131 (ABTS) and hydrogen peroxide (30% w/v) were purchased
132 from AppliChem and Fluka, respectively. H2O2 concentration
133 was determined spectrophotometrically at 240 nm (ε240=43.6
134 Mꢀ1 cmꢀ1).37
172 Capillary voltage was 23.0 Volts. Analysis was conducted only
173 on the positive (ESI+) mode because the instruments cutꢀoff
174 mass value is 50 and in the case of ILs studied, the HCOOꢀ ion
175 should appear at m/z 45.
176 2ꢀhydroxyꢀNꢀmethylethanaminium formate (HMEAF): δH
177 (300MHz; CDCl3) 2.66 (3 H, s, CH3ꢀ), 3.03 (2 H, t, J 2.7, ꢀOꢀ
+
178 CH2), 3.86 (2 H, t, J 3.0, ꢀCH2ꢀN), 7.98 (3 H, br s, ꢀNH2
179 OH), 8.55 (1 H, s, HꢀCOO).
&
135
136 Methods
137
138 Synthesis of ILs
180 δC (75 MHz, CDCl3): 32.67, 50,79, 56.74, 166.74.
181 FTꢀIR (ATR) vmax/cmꢀ1: 1340 ν (CN), 1469 νsym (COOꢀ), 1587
+
+
182 νasym (COOꢀ) & δ(NΗ2 ), 2775 ν (NΗ2 ), 3646 ν (ΟΗ).
139
Hydroxyl ammonium ILs were prepared by
+
183 MS (ESI): ES+ m/z: 76.1 (OHCH2CH2NH2 CH3, 100%)
140 neutralization of formic acid with different amines as described
141 in literature.18 0.1 mol of amine compounds (2ꢀ(methylamino)ꢀ
142 ethanol, 2ꢀ(dimethylamino)ꢀethanol, diethanolamine and
143 ethanolamine) were placed in a two necked roundꢀbottomed
144 flask equipped with a reflux condenser and a dropping funnel.
145 The flask was mounted in an ice bath due to the highly
146 exothermic nature of the acidꢀbase reaction. Increased heat
147 could lead to dehydration of the salt to the corresponding
148 amide. The formic acid (0.1 mol) was added drop wise to the
149 flask under nitrogen atmosphere and vigorous stirring with a
150 magnetic stirrer. Stirring was continued for 24 hours at room
151 temperature in order to obtain a viscous clear liquid. ILs were
152 dried at high vacuum at 40 oC with continuous stirring to
153 remove the water content until no further weight loss was
154 detected. The reaction yields for the synthesis of all ILs studied
155 were more than 97%. The ionic liquids, when not in use, were
156 stored at room temperature in wellꢀsealed glass vessels in a
157 desiccator.
184 Density (20oC): 1.1372 g/cm3
185 2ꢀhydroxyꢀN,Nꢀdimethylethanaminium formate (HDMEAF): δH
186 (300MHz; CDCl3) 2.74 (6 H, s, CH3ꢀ), 3.03 (2 H , t, J 6.0, ꢀOꢀ
187 CH2), 3.87 (2 H , t, J 5.0, ꢀCH2ꢀN), 8.58 (2 H, s, ꢀNH+ & OH),
188 9.48 (1H, s, HꢀCOOꢀ).
189 δC (75 MHz, CDCl3): 43.52, 56.42, 60.46, 168.99.
190 FTꢀIR (ATR) vmax/cmꢀ1: 1340 ν (CN), 1475 νsym (COOꢀ), 1600
191 νasym (COOꢀ), 2775 ν (NΗ+), 3632 ν (ΟΗ).
192 MS (ESI): ES+ m/z: 90.1 (OHCH2CH2NH2 (CH3)2, 100%)
193 Density (20oC): 1.0937 g/cm3
+
194 bis(2ꢀhydroxyethyl)ammonium
formate
(BHEAF):
δH
195 (300MHz; DMSO) 2.87 (4 H, t, ꢀOꢀCH2), 3.59 (4 H, t, ꢀCH2ꢀ
+
196 N), 6.03 (4 H, br s, ꢀNH2 & OH), 8.34 (1 H, s, HꢀCOOꢀ).
197 δC (75 MHz, CDCl3): 49.70, 57.38, 166.61.
198 FTꢀIR (ATR) vmax/cmꢀ1: 1342 ν (CN), 1450 νsym (COOꢀ), 1587
+
+
199 νasym (COOꢀ) & δ(NΗ2 ), 2798 ν (NΗ2 ), 3658 ν (ΟΗ).
200 MS (ESI): ES+ m/z: 106.2 (OHCH2CH2)2NH2 , 100 %)
201 Density (20oC): 1.1587 g/cm3
+
158
The densities of all ILs at 20οC were measured by a
202 2ꢀhydroxylethylammonium formate (HEAF): δH (300MHz;
203 DMSOꢀd6): 2.81 (2 H, t, J 5.2, ꢀOꢀCH2), 3.56 (2 H, t, J 5.2, ꢀ
204 CH2ꢀN), 7.41 (4 H, br s, ꢀNH3+ & OH), 8.41 (1 H, s, HꢀCOOꢀ).
205 δC (75 MHz, CDCl3): 49.70, 57.38, 166.61.
159 SVM 3000 Stabinger Viscometer (Anton Paar).
160
The chemical structure of the synthesized ILs was
161 determined by 1HꢀNMR, 13CꢀNMR, FTꢀIR and MS
162 spectroscopy. 1HꢀNMR spectra (300MHz) and 13CꢀNMR
163 spectra (75MHz) were recorded on a Varian Gemini 2000 (300
164 MHz) spectrometer. ILs were dissolved in DMSO and CDCl3.
165 J values are given in Hz.
206 FTꢀIR (ATR) vmax/cmꢀ1: 1338 ν (CN), 1400 νsym (COOꢀ),
+
+
207 δ(NΗ2 ), 1535 νasym (COOꢀ ) & δ(NΗ3 ), 2931ꢀ2863 ν (N+Η3),
208 3623ν (ΟΗ).
+
209 MS (ESI): ES+ m/z: 62.0 (OHCH2CH2)2NH3 , 100%)
166
FTꢀIR (ATR method) spectra were recorded by a
210 Density (20oC): 1.2059 g/cm3
167 JASCO 4200 spectrometer.
211
168
MS analysis was performed on a Varian 500 MS ion
169 trap mass spectrometer. Instrumental control and the data
170 processing were performed by the Varian MS workstation
171 software. The ionization type used was electrospray ionization.
212 Oxidation activity of metalloproteins
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DOI: 10.1039/C5GC02381E
213
The peroxidase activity of cyt c and HRP was
256 ionic liquids solutions) were reꢀadjusted (with HCl or NaOH)
257 to the required pH before being used in the biocatalytic
258 reactions. All reactions were performed at the optimal pH
259 required in each case. All experiments were performed in
260 triplicate. Control experiments without biocatalyst were also
261 carried out and no conversion of the substrates was observed in
262 all cases studied. All the reaction rates were calculated from
263 the slope of the linear portion of plots of absorbance versus
264 time. The relative activity was expressed in each case as the
265 ratio of activity in the presence of ILs to that observed in buffer
266 solutions.
214 determined by following the color formation during guaiacol
215 oxidation in the presence of H2O2. Reaction temperature was
o
216 set at 30 C and the increase of the absorbance at 470 nm was
217 monitored at an interval of 2 seconds for a time period of 30
218 seconds as described elsewhere.33. Reaction conditions were
219 adjusted according to the type of enzyme used for guaiacol
220 oxidation. In the case of cyt c, the oxidation reaction was
221 carried out in 50 mM sodium phosphate buffer pH 7.0 with 2
222 mM guaiacol, 20 mM of H2O2 and 13.8 U/mL of protein.38 The
223 concentration of ILs in the reaction medium ranged between 0ꢀ
224 75% (v/v). When HRP (0.026 U/mL, 50 mM sodium phosphate
225 buffer pH 6.5) was used as a catalyst, the concentration of the
226 substrate was 20 mM and the concentration of H2O2 was 0.2
227 mM.39
267
268 Kinetic study of cyt
269 determination
c
and activation energy (Ea)
228
In the case of tyrosinase, 4ꢀmethyl catechol was used
o
270
Guaiacol oxidation in the presence of H2O2 at 30 C
229 as a substrate and quinone formation was monitored at 390 nm.
230 The reaction was started by adding 23.6 U/mL of tyrosinase
231 solution prepared in 50 mM phosphate buffer pH 6.8,
232 containing 10 mM of the substrate at 27 oC.
271 was used as a model reaction for the determination of the effect
272 of various ILs on the kinetic constants of cyt c.36 In a typical
273 experimental procedure, guaiacol was added to a final
274 concentration of 2 mM, while H2O2 concentration was in the
275 range of 0.05ꢀ100 mM. The concentration of cyt c used in the
276 reaction was 25 ꢁg/mL. The oxidation of guaiacol was
277 monitored at 470 nm and the extinction coefficient for the
278 oxidation product was considered equal to ε= 26.6 mMꢀ1 cmꢀ1
233
Laccase activity was evaluated using ABTS as a
234 substrate. Reaction mixture contained 1 mM ABTS and 0.0084
o
235 U/mL enzyme in 100 mM acetate buffer pH 4.6 at 27 C and
236 the absorbance change was measured at 415 nm.40 For all
237 enzymatic oxidations, the amount of oxygen has been
238 considered in excess, since the reaction mixture was vortexed
239 for saturation with oxygen before adding the enzyme solution.
279 in all cases studied.42 The apparent kinetic parameters of
app
280 maximum velocity Vmax
and MichaelisꢀMenten constant
app
281 Km were determined through MichaelisꢀMenten equation for
282 initial reaction velocity. All the kinetic parameters were
283 determined by nonꢀlinear regression analysis using the program
284 Enzfitter (Biosoft, Cambridge, UK). Data reported are the
285 mean values of three independent experiments. For the
286 determination of the activation energy Ea for the oxidation of
287 guaiacol catalyzed by cyt c, reactions were performed in 1 mL
288 coꢀsolvent mixtures of 50 mM sodium phosphate buffer pH 7.0
289 and 30% (v/v) aqueous solutions of ILs containing 2 mM
290 guaiacol, 100 mM H2O2 and 25 ꢁg/mL of cyt c at a temperature
291 range from 20 to 60 oC. The activation energy Ea was
292 calculated from the Arrhenius plot through linear regression
293 analysis.
240
Activity of ADH was determined by measuring the
241 rate of reduction of βꢀNAD+ by ethanol at 340 nm. 10 U/mL
242 stock solution of enzyme was prepared immediately prior to
243 the reaction in 10 mM sodium phosphate buffer pH 7.5. In a
244 typical reaction mixture, 5 mM βꢀNAD+ and ethanol (3.2%,
245 v/v) were added in 50 mM sodium phosphate buffer pH 8.0.
246 The enzymatic reaction was initiated with the addition of 8 ꢁL
247 of ADH solution (0.08 U/mL) to the reaction mixture at 25 oC.
248 The increase in absorbance due to βꢀNADH formation was
249 monitored at 340 nm.41
250
In all cases studied, the reaction mixture (1mL) was
251 homogeneous and no precipitation was observed in the
252 presence of all ILs. Moreover, after incubation of ILs with
253 H2O2, no modification of their structure was observed by NMR
254 analysis in all cases studied. In order to avoid the ionic liquidsꢀ
255 induced interference to the pH, all the reaction media (bufferꢀ
294 Stability study of cyt c
295
Stability study of cyt c was performed by incubating
296 cyt c (13.8 U/mL) in aqueous solutions of ILs containing 1 mM
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297 H2O2 at 30 oC. The incubation mixture did not contain guaiacol
298 since the presence of reducing substrates in the reaction
335
The decolorization activity of cyt c was measured by
336 following color elimination of pinacyanol chloride (1,1′ꢀ
337 diethylꢀ2,2′ꢀcarbocyanine chloride) with H2O2 in buffer and in
338 the presence of various amounts of ILs. The reaction mixture
339 contained 130 ꢁΜ pinacyanol chloride and 80 ꢁg/mL cyt c.
340 The oxidation of the dye was started by adding 0.3 mM H2O2
341 at 27 oC under stirring at 300 rpm. At predetermined time
342 intervals 30 ꢁL aliquots were removed from the reaction
343 mixture and added to a 1:1 (v/v) mixture of methanol and 50
344 mM sodium phosphate buffer pH 7.0. The remaining
345 concentration of the dye was monitored by measuring the
346 absorbance at 603 nm using an extinction coefficient for
347 pinacyanol chloride equal to ε= 82,350 Mꢀ1 cmꢀ1.46
43,44
299 mixture could increase the stability of cyt c against H2O2.
300 After 15 min of incubation, 200 ꢁL of sample were removed
301 and transferred to a 96ꢀwell microplate in order to determine
302 the remaining peroxidase activity of cyt c using guaiacol (2
303 mM) as a substrate, as described before. All experiments were
304 performed in triplicate.
305 UV-vis spectroscopy
306
A doubleꢀbeam UVꢀvis spectrophotometer (UVꢀ1601
307 Shimadzu, Tokyo, Japan) was used in order to monitor the
308 effect of aqueous solutions of ILs on the absorption spectrum
309 of cyt c (25 ꢁg/mL) in a standard 1 cm path length quartz
310 cuvette. UVꢀvis absorption spectra of cyt c was recorded at 30
311 oC.
348 Recycle of ILs
349
The enzymatic decolorization of pinacyanol chloride
350 was further used in order to investigate the reusability of the
351 ILs. In this case, cyt c was immobilized on celite in a similar
352 manner as described elsewhere47, in order to facilitate the
353 recovery of the enzyme and the reuse of ILs. The reaction
354 mixture (0.5ml) contained 130 ꢁΜ pinacyanol chloride and 30
355 mg/mL of immobilized biocatalyst (containing 2 ꢁg of cyt c
356 per 1 mg of celite). The oxidation of the dye was started by
357 adding 0.3 mM H2O2. The reaction mixture was incubated at
312 ATR-IR Spectroscopy
313
Single pass attenuated total reflection Fourier
314 transform infrared (ATRꢀFTIR) spectra were recorded on a
315 Shimadzu FTꢀIR 8400 (Tokyo, Japan) infrared
316 spectrophotometer equipped with a deuterated triglycine sulfate
317 (DTGS) detector in the region of 400ꢀ 4000 cmꢀ1 using a
318 (ZnSe)ꢀattenuated total reflection accessory. 200 scans were
319 collected for each sample at 2 cmꢀ1 resolution and 1 cmꢀ1 time
320 interval. The concentration of cyt c was 5 mg/mL in buffer and
321 9 mg/mL in 30% (v/v) aqueous solutions of ILs. Reference
322 spectra under identical conditions without the presence of cyt c
323 were also recorded. Data analysis of the Amide I region and
324 band assignment were performed as described elsewhere.45
o
358 27 C under stirring at 300 rpm for 3 hours. At the end of the
359 incubation, 1 ml of water was added to the reaction mixture
360 and the immobilized biocatalyst was filtered off. The aqueous
361 filtrate containing the ILs was washed with ethyl acetate in
362 order to remove any amount of the residual substrates and
363 products and then the water was evaporated in vacuo. The
364 residual IL was dried under high vacuum at 40 oC until
365 constant weight. The structure and purity of the recycled IL
366 were verified with 1HꢀNMR, while the recycle process was
367 repeated up to three times.
325 Circular Dichroism Spectroscopy
326
Soret region CD spectra (350ꢀ450 nm) of cyt c (200
327 ꢁg/mL) in 0.5 mM sodium phosphate buffer pH 7.0 and in 60%
328 (v/v) aqueous solution of ILs were obtained using a Jasco Jꢀ815
329 spectropolarimeter (Tokyo, Japan) in a 1 cm path length quartz
330 cell. All spectra were obtained at 25 oC with a 2 nm bandwidth
331 and a scan speed of 10 nm/min. For every medium scanned, a
332 baseline was recorded and subtracted from the protein
333 spectrum. All scan measurements were performed in triplicate.
368 Biodegradability test
369
The biodegradability of four ILs has been assessed
370 by measuring the Biochemical Oxygen Demand (BOD).18,48
371 Biodegradation tests were carried out according to the
372 manometric respirometric method to determine the oxygen
373 demand for the biochemical degradation of each organic
374 substance after five days. A detailed description of the method
375 is described on the Supplementary data.
334 Dye decolorization
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417 activation of cyt c in the presence of these ILs was observed for
418 ABTS oxidation (data not shown), indicating that this
419 activation effect is independent of the substrate used. Although
420 cyt c activity has been studied in other biocompatible ILs, such
421 as alkyl ammonium and cholineꢀbased ILs, in those cases no
422 activation was observed, in contrast to the results obtained in
423 our study.50,52 It is interesting to note that, when equimolar
424 amounts of individual components of these ionic liquids
425 (hydroxyl amines and formic acid) were both added in buffer
426 solution (the amounts of the individual components
427 corresponded to the ones present in ILs and were adjusted
428 according to the desired concentration of ILs in buffer), no or
429 low catalytic activity of cyt c was observed (Fig.S.17.
430 Supplementary). This clearly indicates that, the beneficial
431 effect of ILs on the catalytic activity of cyt c is associated to
432 the formed salt and not to their individual components.
376
377 Results and Discussion
378 Effect of hydroxyl ammonium ILs on the activity of various
379 metalloproteins.
380
In the present study, the effect of four hydroxyl
381 ammoniumꢀbased ILs (ΗΕΑF, ΗΜΕΑF, ΗDΜΕΑF and
382 ΒΗΕΑF) on the activity of various metalloproteins such as
383 cytochrome c from horse heart (cyt c), horse radish peroxidase
384 (HRP), mushroom tyrosinase, laccase from Trametes versicolor
385 and alcohol dehydrogenase (ADH) from baker’s yeast was
386 investigated (Table 1). In most cases studied, the presence of
387 5% (v/v) hydroxyl ammonium ILs in the reaction medium
388 affected the activity of metalloproteins. The effect of ILs used
389 on the enzymatic activity depended on the biocatalyst used, as
390 well as on the nature of the cation of IL used. Particularly in
391 the case of HRP and ADH, the activity remained unchanged or
392 decreased, depending on the IL used. The oxidation activity of
393 laccase was significantly reduced in all ILs tested, which is in
394 accordance to that reported for imidazoliumꢀbased water
395 miscible ILs.49 However, it must be pointed out that, in the case
396 of cyt c, the presence of hydroxyl ammonium ILs in the
397 reaction medium significantly enhanced (up to 3.4 fold) its
398 peroxidation activity. An enhanced activity was also observed
399 for tyrosinase in the presence of HMEAF and BHEAF IL.
400
433
In order to gain a deeper insight into the influence of
434 the hydroxyl ammoniumꢀbased ILs on the peroxidase activity
435 of cyt c, the effect of the ILs on the apparent kinetic constants
app
app
436 Vmax and Km of cyt c for the oxidation of guaiacol with
437 H2O2 were determined. The effect of the nature and
438 concentration of ionic liquids on the catalytic efficiency,
439 expressed by the ratio Vmaxapp/Kmapp (in all cases studied the cyt
440 c concentration was the same), is presented in Fig. 3. As it can
441 be seen, the presence of ILs enhances, in most cases, the
442 catalytic efficiency of cyt c compared to buffer solution. The
443 highest catalytic efficiency was observed when HEAF was
444 used as coꢀsolvent, causing a more than 20ꢀfold increase in
445 catalytic efficiency at a concentration of this IL higher than
446 60% (v/v). The increased catalytic efficiency observed at high
447 concentrations of HEAF and BHEAF was the result of a
401 Effect of ILs on the peroxidative activity of cyt c
402
403
In order to further investigate the effect of hydroxyl
404 ammoniumꢀbased ILs on the catalytic behaviour of
405 metalloproteins, cyt c was chosen as a model protein. The
406 effect of the concentration of various hydroxyl ammonium ILs
407 on the peroxidase activity of cyt c using guaiacol as a substrate
408 is shown in Fig. 2. As it can be seen, the peroxidase activity of
409 cyt c strongly depends on the nature of cations and the
410 concentration of ILs used, which is in accordance to that
411 observed previously for imidazolium, alkyl ammonium and
412 cholineꢀbased ILs42, 50ꢀ52. In most cases, the increase of the ILs
413 concentration significantly increases the peroxidase activity of
414 cyt c. A 9ꢀfold and 20ꢀfold activity enhancement was observed
415 respectively in BHEAF and HEAF at a concentration of ILs
416 equal to 60% (v/v), compared to buffer. Similar catalytic
app
app
448 simultaneous increase of Vmax
and decrease of Km
449 compared to that observed in buffer (Table S1). The low
app
450 apparent Km
values observed at high concentrations of
451 HEAF and BHEAF indicate that the affinity of cyt c towards
452 the substrate was increased, which may be correlated with
453 structural changes in the active site of cyt c and therefore,
454 changes in the microenvironment of heme.38,53 The increased
455 catalytic efficiency observed here is in accordance to that
456 reported for various enzymes in other ionic liquidꢀbased
457 systems.53ꢀ56 It was suggested that, the presence of ILs in the
458 reaction medium can increase the affinity of the enzyme to the
459 substrate, resulting in a higher catalytic efficiency compared to
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460 other media, such as organic solvents and aqueous
461 solutions.55,56
503 form of heme, as a final product, as well as in the formation of
504 radical species that could react and inactivate the heme
505 center.60ꢀ65
462
It has been proposed by several researchers that the
463 enzyme performance in hydrophilic ILs could be affected by
464 the kosmotropicity/chaotropicity properties of the ions of
465 ILs.57ꢀ59 Ions that are considered as kosmotropes promote water
466 structure, while chaotrope ones can suppress it.60 In the present
467 study, formic acid, used as the anion for the formation of all
468 ILs tested, is considered as a kosmotropic anion.61 On the other
469 hand, hydroxyl alkyl ammonium cations, which are more
470 hydrophilic than the chaotropic choline cation, could be
471 assumed to be highly chaotropic.43 This indicates that, the
472 presence of a kosmotropic anion and a chaotropic cation
473 improved the catalytic efficiency of cyt c which is in
474 accordance to that proposed for other enzymes in different
475 ionic liquids.57ꢀ59 It seems that, there is a correlation between
476 the chaotropicity of the cation and the catalytic efficiency of
477 cyt c in these media. More specifically, in the case of more
478 hydrophilic and thus more chaotropic BHEAF and HEAF
479 cations, the catalytic efficiency of cyt c was higher compared
480 to that observed for less chaotropic HMEAF and HDMEAF.
506
In order to investigate the stability of cyt c against
507 H2O2 in hydroxyl ammoniumꢀbased ILs, cyt c was incubated in
o
508 the presence of 1 mM H2O2 for 15 min at 30 C in buffer
509 containing various ILs (30ꢀ60% v/v) and the remaining
510 peroxidase activity was determined using guaiacol as a
511 reducing substrate (Fig. 4).
512
As seen in Fig. 4, the peroxidase activity of cyt c in
513 buffer was reduced by 40% after incubation with H2O2. The
514 tolerance of cyt c, in the presence of all ILs used, strongly
515 depends on the nature of ILs cations, while the effect of their
516 concentration is not so obvious. Namely, in the presence of
517 HMEAF and especially HDMEAF, the remaining activity of
518 cyt c in most cases was higher compared to that observed in
519 buffer, indicating that these ILs protect the protein from H2O2
520 deactivation. However, in the presence of HEAF or BHEAF,
521 the remaining peroxidase activity of cyt c after incubation with
522 H2O2 was significantly decreased compared to that observed in
523 buffer, for all concentrations tested.
481
In order to further investigate the effect of these ILs
524
It was reported that the stability of cyt c in the
482 on the peroxidase catalytic behavior of cyt c, the activation
483 energy (Ea) of cyt c for the oxidation of guaiacol in buffer, as
484 well as in the presence of 30% (v/v) aqueous solutions of
485 hydroxyl ammonium Ils, was determined over a temperature
486 range of 20 to 60 oC. The activation energies in various media,
487 calculated from the slope of the Arrhenius plots, are presented
488 in Table 2. As it can be seen, the presence of ILs in the reaction
489 mixture decreases the activation energy compared to that in
490 buffer solution and this decrease is more pronounced when
491 HMEAF and HEAF are used. This decrease in Ea value
492 observed here may be correlated to the effect of ILs on the
493 structure of the protein molecule and the formation of enzymeꢀ
494 substrate complex, as it was proposed for imidazoliumꢀbased
495 ionic liquids.56
525 presence of various hydrophilic ILs was strongly influenced by
526 the kosmotropicity/chaotropicity of the ions of ILs.50 However,
527 in our study, the effect of ILs on stability of cyt c does not
528 follow the Hofmeister series and therefore, the chaotropicity of
529 their cations. It seems that kosmotropicity/chaotropicity is not
530 the only key in determining the cyt c behavior in ILs.
531
It is interesting to note that, all the ILs used in the
532 present work are formed by hydroxyl ammonium cations. As it
533 has been proposed, these cations can mimic the molecular
534 structure of water with Hꢀbond accepting/donating
535 functionalities forming hydrogen bonds with the polypeptide
536 backbone of protein and thus affecting its structure and
537 function.59 It is worth noting that, these interactions should not
538 be too strong in order to avoid the dissociation of the hydrogen
539 bonds between the amino acids which could lead to the
540 disruption of the protein structure.66 Based on the basicity of
541 nitrogen of the amine residues of the ILs studied, the Hꢀ
542 bonding capability should increase by the following order:
543 HDMEAF <HMEAF < HEAF <BHEAF.67 As it can be seen in
544 Fig. 3, cyt c is more stable in aqueous solutions of HDMEAF
545 and HMEAF ILs, which could be possibly explained by the
496
497 Stability of cyt c against H2O2 in ILs
498
Ιt is well known that, hemeꢀcontaining enzymes,
499 such as peroxidases and cyt c, are inactivated by H2O2 in the
500 absence of reducing substrate in aqueous media.43,62 This
501 inactivation may be correlated to the modification of heme
502 resulting in the formation of a verdohemoprotein, an inactive
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546 decreased Hꢀbond ability and thus reduced interaction of these
547 ILs with the protein molecule.
588 composed by other hydrophilic ILs has also been previously
589 reported.50,51
548
Moreover, the increased stability of cyt c against
590
Since the most abundant element (about 40%) of the
549 H2O2 in HDMEAF could be attributed to the less hydrophilic
550 environment surrounding the protein created by this IL. The
551 less hydrophilic environment created by HDMEAF could limit
552 the diffusion of hydrophilic H2O2 towards the protein
553 microenvironment, thus reducing its denaturing effect. The
554 possible limited diffusion of H2O2 to the microenvironment and
555 thus to the active center of cyt c could probably also explain
556 the decreased activity of cyt c observed in HDMEAFꢀbased
557 reaction medium (Fig. 3).
591 secondary structure of cyt c is αꢀhelix,72 we also determined the
592 effect of ILs on αꢀhelix content (Table 3). The αꢀhelix content
593 was identified from the secondꢀderivative ATRꢀFTIR spectra
594 of the protein in various media, taking into consideration that
595 the bands at 1650–1660 cmꢀ1 were assigned to αꢀhelix.73,74 As it
596 can be seen, a slight increase in αꢀhelix content was observed
597 in aqueous solutions of HMEAF, HDMEAF and HEAF that
598 can be correlated to a more rigid structure of the protein.75 On
599 the other hand, the decrease in αꢀhelix content observed in
600 BHEAFꢀbased media could be attributed to a less rigid
601 structure of cyt c that could be correlated to the substantially
602 low stability of the protein observed in this IL (Fig. 4).
558
559 Structural characterization of cyt c using spectroscopic
560 techniques.
561
603
Conformational changes of the heme prosthetic
562
The effect of the hydroxyl ammonium ILs on the
604 group of cyt c in aqueous solutions of hydroxyl ammonium
605 ILsꢀbased media were further investigated using UVꢀVis
606 spectroscopy. As it can be seen in Fig. 5 and Fig. 6, the
607 oxidized (Fe(III)) form of cyt c in buffer has a characteristic
608 UVꢀvis spectrum consisting of a sharp Soret band at 409 nm, a
609 weaker, broad Qꢀband at 530nm and a very weak charge
610 transfer band from the sulfur atom of Met80 (the axial ligand)
611 with heme Fe(III) observed at 695 nm, which is in accordance
612 to that reported by other researchers.76ꢀ77
563 conformation of cyt c was investigated through ATRꢀFTIR,
564 UVꢀVis and circular dichroism (CD) spectroscopy. The
565 conformational changes of cyt c in the presence of hydroxyl
566 ammonium ILsꢀbased media compared to its structure in buffer
567 (50 mM sodium phosphate, pH 7.0) were investigated by ATRꢀ
568 FTIR spectroscopy. The analysis of the Amide I band at
569 approximately 1600–1700 cmꢀ1 (mainly due to the C=O
570 stretching vibration) makes it possible to obtain information on
571 the effect of ILs on the secondary structure of the protein.68ꢀ71
613
The incubation of cyt c in 60% (v/v) aqueous
572
Correlation coefficients (r) between the Amide I
614 solutions of HDMEAF, BEHAF and HEAF did not affect the
615 UVꢀVis spectrum of the protein (Fig.5) suggesting that the
616 polypeptide environment around the heme has been kept
617 intact.78 However, the UVꢀvis spectrum of cyt c in 60% (v/v)
618 aqueous solution of HMEAF was significantly changed (Fig.
619 5). More specifically, the Soret band of the protein was moved
620 at 413 nm, while at Qꢀband region a sharp αꢀband at 550 nm
621 and a sharp βꢀband at 520 nm appeared, indicating cyt c in
622 reduced state.79
573 spectra of cyt c dissolved in buffer and 30% (v/v) aqueous
574 solutions of ILs were evaluated according to previous studies.71
575 In particular, the correlation coefficient was calculated using
576 the formula
54
ͦ
ͦ
ǯ
r = Σͬ ͭ$/ ꢀͬ ͭ$ ,
$
$
577
578 where x and y are the absorbance values of the cyt c spectrum
579 dissolved in buffer and 30% (v/v) of ILs respectively, at the ith
580 frequency position for the range 1600ꢀ1700 cmꢀ1 (Amide I).
581 For identical spectra, a value of 1.0 will be returned.71 Table 3
582 shows the correlation coefficients and the differences on αꢀ
583 helix content of cyt c in the presence of various ILsꢀbased
584 media compared to buffer, as a result of ATRꢀFTIR analysis.
585 As it can be seen in Table 3, the relative structure of cyt c in
586 the presence of all ILs used is close to that in buffer. Similar
587 retention of the secondary structure of cyt c in various media
623
Moreover, in HMEAFꢀbased media, the charge
624 transfer band at 695 nm was significantly reduced compared to
625 that observed for the native protein (Fig. 6). This spectral
626 change indicates the partial perturbation or cleavage of the
627 coordination bond of Met80 with the heme iron.80 The loss of
628 the heme’s axial sulfurꢀcoordinated ligand has been correlated
629 with the progressive breaking of hydrogen bonds in the protein
630 interior and gradual exposure of amino acid residues and the
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631 porphyrin ring and hence loss of iron and the catalytic activity
632 of the protein.78,81
674 substrate, which is a symmetric trimethinecyanine dye with
675 industrial use.93 In this case, the initial concentration of H2O2
676 was kept low (0.3mM) in order to reduce its denaturing effect.
677 As it can be seen in Fig. 8 and Table 4, the decolorization
678 activity of cyt c, in the presence of all ILs studied, strongly
679 depends on the nature of the cation used, as well as the
680 concentration of the ILs in the reaction media. In the case of
681 HMEAF, the decolorization efficiency decreased, while the use
682 of BHEAF and HEAF enhanced the ability of cyt c to
683 decolorize the dye compared to that observed in buffer. More
684 specifically, the decolorization rate in the presence of HEAF
685 and BHEAF in the reaction mixture was up to 4ꢀfold and 5ꢀfold
686 higher, respectively, compared to that in buffer. It is worth
687 noting that, the decolorization yield was about 90% after 20
688 min of incubation in media containing various concentrations
689 of HEAF and BHEAF (15ꢀ60% v/v), while in buffer the
690 decolorization yield was less than 40%. The positive effect of
691 these two ILs increases with the increase of their concentration
692 in the reaction mixture. The high decolorization activity of cyt
693 c in reaction media containing BHEAF or HEAF could be
694 attributed to the enhanced peroxidation activity of cyt c
695 observed in these media (Fig.3). In order to further demonstrate
696 the green properties of these novel ILs, their recyclability and
697 reusability were also investigated, using the catalyzed by cyt c
698 decolorization of pinacyanol chloride as a model reaction (see
699 Experimental section). All ILs were reused in the same
700 decolorization reaction up to three times with comparable
701 decolorization yields to those observed for the initial reaction.
702 The beneficial effect of hydroxyl ammoniumꢀbased ILs on the
703 decolorization activity of cyt c together with their reusability
704 indicate that these ILs could be considered as promising
705 environmentally friendly media for biocatalytic decolorization
706 of dyes.
633
The structural changes of cyt c observed in HMEAFꢀ
634 based media were further investigated using CD spectroscopy.
635 Unfortunately, due to high absorbance of the IL in far and near
636 UV region, only the Soret region of the CD spectrum (350ꢀ450
637 nm) of cyt c could be investigated, which can provide further
638 insight on structural changes of the heme crevice.38,82,83 Fig. 7
639 shows the CD spectra of cyt c in 0.5 mM sodium phosphate
640 buffer pH 7.0 and in the presence of HMEAF (60% v/v). The
641 spectrum of cyt c, in its native conformation in buffer, exhibits
642 a negative peak at 416 nm and a positive peak at 402 nm, due
643 to a split Cotton effect.83 As it can be seen in Fig. 7, the
644 addition of HMEAF increases the positive band at 402 nm and
645 significantly vanishes the negative band at 416 nm. Similar
646 spectral changes have been previously reported due to
647 interactions of cyt
c
with other ILs and organic
648 solvents,33,51,52,84,85 as well as with denaturants, such as
649 guanidine, HCl or urea.86
650
The increase of the positive peak at 416 nm and the
651 disappearance of the negative peak at 402 nm observed here
652 could be attributed to the disordered orientation and
653 disturbance of the distance between the heme on the Met80
654 side and the aromatic residues Tryꢀ59 and Pheꢀ82 in the
655 polypeptide backbone of the heme crevice.87 Similar results
656 have also been observed when cyt c was dissolved in hydrated
657 pyridiniumꢀbased and neat imidazolium based ILs.51,85 The
658 findings from circular dichroism studies correlate well with the
659 UVꢀvis data described before, as well as with the reduced
660 peroxidase activity of cyt c in the presence of HMEAF
661 compared to the other ILs used in the present work (Fig. 3, 5,
662 6).
663
664 Decolorization of pinacyanol chloride by cyt c in ILs-based
665 media
707 Biodegradability assessment of ILs
708
In order to determine the biodegradability level of the
666
The accumulation of industrial dyes in wastewater
709 synthesized ILs, the biological oxygen demand (BOD) for the
710 biochemical degradation of each IL after five days was
711 determined. During the BOD test, the carbonaceous demand
712 (which refers to the conversion of organic carbon to carbon
713 dioxide) was taken into account and the results are reported as
714 carbonaceous BOD (CBOD).18. The results of the
715 biodegradation of the ILs are presented in Table 5.
667 has a profound environmental and health impact and, therefore,
668 their removal is a substantial challenge for the scientific and
669 industrial community.88 Several oxidative biocatalysts have
670 been used for the enzymatic elimination of dyes including cyt
671 c, laccases, peroxidases, etc.44,89ꢀ92 In order to investigate the
672 effect of the hydroxylꢀammonium ILs on the decolorization
673 activity of cyt c, we used pinacyanol chloride as a model
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716
The experimental results indicated that all ILs
758
717 studied present remarkable biodegradability potential, since a
718 percentage of more than 50% of the organic carbon was
719 biodegraded within five days. The relatively high
720 biodegradability of these ILs could be attributed to the
721 presence of hydroxyl groups on the ILs cations. It has been
722 reported that the biodegradability level of similar
723 ethanolamineꢀbased ILs depends mainly on the cation of the IL
724 molecule and on the groups that provide possible sites for
725 enzymatic hydrolysis, especially oxygen atoms (e.g. in the
726 form of hydroxyls) that present high degradation potential.14,94ꢀ
759 Acknowledgements
760 This research project has been coꢀfinanced by the
761 European Union (European Regional Development Fundꢀ
762 ERDF) and Greek national funds through the Operational
763 Program “THESSALYꢀ MAINLAND GREECE AND
764 EPIRUSꢀ2007ꢀ2013” of the National Strategic Reference
765 Framework (NSRF 2007ꢀ2013). Also the support from
766 bilateral Personnel Exchange Programme between Greece
767 and Germany (IKYDA 2015) is acknowledged. The
768 authors would like to thank the Atherothrombosis
769 Research Centre of the University of Ioannina for
770 providing access to the facilities.
771
772 Notes and references
96
a
727
773
Department of Biological Applications and Technology,
774 Laboratory of Biotechnology, University of Ioannina, University
775 Campus, 45110 Ioannina, Greece
728 Conclusions
b
776
School of Chemical Engineering, Laboratory of Organic
729
730
777 Chemistry, National Technical University of Athens, Heroon
778 Polytechniou 9, Zografou Campus, 15780 Athens, Greece
779 Eꢀmail: hstamati@cc.uoi.gr (H. Stamatis)
780 URL: http://biotechlab.bat.uoi.gr/index.php/en/
781 Tel.: +30 26510 97116; fax: +30 26510 97343
782 Electronic Supplementary Information (ESI) available
783
Herein, neoteric, low cost and biodegradable ILs,
731 based on the combination of a hydroxyl ammonium cation
732 and formic acid, have been prepared and used as media for
733 biocatalytic oxidation catalyzed by metalloproteins.
734 Kinetic and structural studies of cyt c indicate that the
735 presence of these ILs in the reaction mixture has a
736 considerable beneficial effect on the catalytic efficiency
737 and tolerance against hydrogen peroxide, while the protein
738 structure is slightly affected. The effect of these ILs on the
739 catalytic behaviour of cyt c strongly depends on the
740 structure of the hydroxyl ammonium cations used for their
741 formation. Moreover, the beneficial effect of hydroxyl
742 ammoniumꢀbased ILs on the biocatalyzed degradation of
743 an industrial dye, together with their efficient recyclability
744 and reusability, indicate the potential application of these
745 novel ILs as green media for biotransformations of
746 industrial interest. The use of immobilized enzymes97,98 or
747 the use of ILs as a support for enzyme immobilization99,100
748 is expected to facilitate the recovery and reuse of both the
749 biocatalyst and the ILs, enhancing therefore the green
750 character of such biocatalytic processes. Further
751 investigation on the effect of the nature and
752 physicochemical properties of cations and anions used for
753 the formation of such environmentally friendly ILs on the
754 catalytic behaviour of various industrial enzymes is in
755 progress in our lab.
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Journal Name
Figure Captions
Fig. 1. Structure of ILs used in the present study
Fig. 2. Relative peroxidase activity of cyt c for the oxidation of guaiacol in the presence of various amounts of hydroxyl
ammoniumꢀbased ILs. As 1.0 is indicated the peroxidase activity of cyt c in 50 mM phosphate buffer pH 7.0. Initial reaction rate
of the activity of cyt c in buffer is 10 ꢁM/min.
Fig. 3. Effect of the concentrations of hydroxyl ammoniumꢀbased ILs on the catalytic efficiency of cytochrome c for the
oxidation of guaiacol with H2O2. The black line represents the ratio of Vmaxapp/Kmapp of cyt c in buffer aqueous solution.
Fig. 4. Stability of cyt c in buffer and 30, 45 and 60 % v/v aqueous solutions of hydroxyl ammoniumꢀbased ILs, after incubation
for 15 min with 1 mM H2O2 at 30 oC. As 100% is indicated the activity at t = 0 min.
Fig. 5. UVꢀvisible spectra (300ꢀ600 nm) of cyt c in 50 mM phosphate buffer, pH 7.0 and in the presence of 60% (v/v) ILs. The
insets show the absorption spectra of the media (60% v/v ILs).
Fig. 6. Absorption spectra of cyt c at the charge transfer band (695 nm) in phosphate buffer 50 mM, pH 7.0 and in the presence of
60% (v/v) ILs.
Fig. 7. Soret region CD spectrum of cyt c in 50 mM phosphate buffer, pH 7.0 and in the presence of 60% (v/v) aqueous solution
of HMEAF
.
Fig. 8. Cyt cꢀcatalyzed decolorization of pinacyanol chloride with H2O2 in phosphate buffer 50 mM, pH 7.0 and in the presence
of various amounts (15ꢀ75 % v/v) of ILs a) HMEAF, b) HDMEAF, c) BHEAF and d) HEAF.
14 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

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DOI: 10.1039/C5GC02381E
Fig. 1. Structure of ILs used in the present study

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DOI: 10.1039/C5GC02381E
Fig. 2. Relative peroxidase activity of cyt c for the oxidation of guaiacol in the presence of various amounts
of hydroxyl ammonium-based ILs. As 1.0 is indicated the peroxidase activity of cyt c in 50 mM phosphate
buffer pH 7.0. Initial reaction rate of the activity of cyt c in buffer is 10 µM/min
55x38mm (300 x 300 DPI)

Page 17 of 42
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DOI: 10.1039/C5GC02381E
Effect of the concentrations of hydroxyl ammonium-based ILs on the catalytic efficiency of cytochrome c for
app
the oxidation of guaiacol with H2O2. The black line represents the ratio of Vmaxapp/Km
of cyt c in buffer
aqueous solution
57x39mm (300 x 300 DPI)

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Stability of cyt c in buffer and 30, 45 and 60 % v/v aqueous solutions of hydroxyl ammonium-based ILs,
after incubation for 15 min with 1 mM H2O2 at 30 oC. As 100% is indicated the activity at t = 0 min.
57x39mm (300 x 300 DPI)

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DOI: 10.1039/C5GC02381E
UV-visible spectra (300-600 nm) of cyt c in 50 mM phosphate buffer, pH 7.0 and in the presence of 60%
(v/v) ILs. The insets show the absorption spectra of the media (60% v/v ILs).
119x172mm (300 x 300 DPI)

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Absorption spectra of cyt c at the charge transfer band (695 nm) in phosphate buffer 50 mM, pH 7.0 and in
the presence of 60% (v/v) ILs.
57x39mm (600 x 600 DPI)

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Soret region CD spectrum of cyt c in 50 mM phosphate buffer, pH 7.0 and in the presence of 60% (v/v)
aqueous solution of HMEAF
57x39mm (600 x 600 DPI)

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Cyt c-catalyzed decolorization of pinacyanol chloride with H2O2 in phosphate buffer 50 mM, pH 7.0 and in the
presence of various amounts (15-75 % v/v) of ILs a) HMEAF, b) HDMEAF, c) BHEAF and d) HEAF
118x82mm (300 x 300 DPI)

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Table 2. Activation energy Ea for the oxidation of guaiacol
catalyzed by cyt c in various ILs (30% v/v).
Activation Energy Ea ( kcal/mol)
Buffer
BHEAF
HDMEAF
HMEAF
HEAF
1.28
1.17
1.19
0.67
0.58

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DOI: 10.1039/C5GC02381E
Table 3. Correlation coefficient (r) between the ATRꢀFTIR
spectra of cyt c dissolved in 50 mM sodium phosphate
buffer, pH 7.0 and in 30% (v/v) aqueous solutions of ILs.
ꢁαꢀhelix estimation (%) is the difference between the
percentages of αꢀhelix content of cyt c in 30% (v/v) ILs
compared to that in buffer calculated by ATRꢀFTIR
analysis in Amide I region.
Ionic Liquid
r
ꢀαꢁhelix (%)
HMEAF
HDMEAF
BHEAF
HEAF
0.97
0.93
0.98
0.94
+0.80
+2.50
ꢀ0.68
+1.72

Page 25 of 42
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DOI: 10.1039/C5GC02381E
Table 4. Reaction rates of the cyt c-catalyzed
decolorization of pinacyanol chloride with H2O2 in
phosphate buffer 50 mM, pH 7.0 and in the presence of
various amounts of hydroxyl-ammonium ILs.
Decolorization rate (μΜ min-1 )
% v/v
ILs
0
HMEAF HDMEAF BHEAF HEAF
3.9
0.9
1.8
2.5
0.4
3.9
7.8
4.0
1.5
1.3
3.9
3.9
9.9
15
30
60
75
11.3
14.0
17.4
18.6
12.5
14.5
8.0

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DOI: 10.1039/C5GC02381E
Table 5. (%) Biodegradability assessment of the hydroxyl ammonium ILs.
Ionic Liquid
(%) Biodegradation *
2-HEAF
HMEAF
HDMEAF
BHEAF
58.9
55.0
57.4
52.2
* The percentage biodegradation is calculated by dividing the specific carbonaceous BOD (CBOD) by the ultimate
carbonaceous BOD (UCBOD). The CBOD value expresses the oxygen demand by microorganisms for degradation
of the ILs within five days (only carbonaceous stage). CBOD is the BOD value (mg O2/L) reading affected due to the
drop of pressure in the water sample bottle, minus the BOD value reading affected due to the drop of pressure in the
dilution water bottle (blank). The UCBOD value (mg O2/L), expresses the oxygen demand by microorganisms for
the ultimate degradation of the organic compound referring only to the conversion of organic carbon to carbon
dioxide, water, and new microbial cellular constituents.

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DOI: 10.1039/C5GC02381E
Hydroxyl ammonium ionic liquids are a biodegradable, non-toxic family of third generation ionic liquids with a beneficial effect
on the catalytic efficiency of metalloproteins such as cytochrome c.

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Electronic Supplementary Information
Hydroxyl ammonium ionic liquids as media for
biocatalytic oxidations
α
Athena A. Papadopoulou, α Andromachi Tzani, b Dimitrios Alivertis, Maria H. Katsoura, α Angeliki
α
C. Polydera, α Anastasia Detsi b and Haralambos Stamatis *
α Department of Biological Applications and Technology, University of Ioannina, University Campus,
45110 Ioannina, Greece
b Department of Chemical Sciences, Laboratory of Organic Chemistry, National Technical University of
Athens, Heroon Polytechniou 9, Zografou Campus, 15780 Athens, Greece
Corresponding Author: E-mail: hstamati@cc.uoi.gr (H. Stamatis)
URL: http://biotechlab.bat.uoi.gr/index.php/en/
Tel.: +30 26510 97116; fax: +30 26510 97343
1

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DOI: 10.1039/C5GC02381E
Biodegradability test
Biodegradation is the natural process for the removal of organic substances from the
environment. The determination of the biodegradability level of organic substances such as ILs
is essential in order to estimate their environmental impact. Biodegradability assessment of ILs
have been examined by measuring the Biochemical Oxygen Demand (BOD) [1],[2].
In this work, biodegradation tests were carried out according to a manometric method so
as to determine the oxygen demand for the biochemical degradation of each organic substance
after five days. VELP BOD manometric apparatus was used to measure the BOD of the IL
inoculated samples. This method is based on the steady decrease of the pressure in a closed
system as a result of oxygen consumption. The carbon dioxide which is produced is bounded by
a strongly alkaline medium (KOH pellets above the solution) so as not to interfere with the final
measurements. The nutrients prepared are:
•
Ferric chloride hexahydrate: 0.25 g FeCl3·6H2O to a final volume of 1 L with distilled
water.
•
•
Calcium chloride anhydrous: 27.5 g CaCl2 to a final volume of 1 L with distilled water.
Magnesium sulfate heptahydrate: 22.5 g MgSO4·7H2O to a final volume of 1 L with
distilled water.
•
Phosphate salts solution (buffer): 8.5 g KH2PO4, 21.7 g K2HPO4, 33.4 g Na2HPO4·7H2O
and 1.7 g NH4Cl to a final volume of 1 L with distilled water.
This method consists of filling each BOD flask with specific amount of IL, 135 mL
aqueous solution of nutrients and 15 mL microorganisms. The seed source of microorganisms
was mixed liquor which was taken from a secondary sedimentation tank of urban waste water of
Psyttaleia sewage treatment plant in Greece. A blank solution was also prepared, containing only
nutrients and mixed liquor.
In general, two stages of degradation take place during the BOD test, carbonaceous and
nitrogenous but in this work only the carbonaceous demand taken into account and the BOD
values will be reported as CBOD (degradation of the organic carbon). Inhibition of nitrogenous
bacteria was achieved by a thiourea solution (2 g thiourea to a final volume of 1 L with distilled
water) which was also added to BOD samples (0.5 mL of the solution in each flask). The
samples were kept at 20 ± 1°C in darkness in tightly closed bottles for an incubation period of 5
days.
2