molecules Article
Molecular Mechanisms Underlying Inhibitory Binding of Alkylimidazolium Ionic Liquids to Laccase Jianliang Sun 1 , Hao Liu 1, *, Wenping Yang 2 , Shicheng Chen 3 and Shiyu Fu 1 1 2 3
*
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China;
[email protected] (J.S.);
[email protected] (S.F.) School of Mathematics, South China University of Technology, Guangzhou 510640, Guangdong, China;
[email protected] Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA;
[email protected] Correspondence:
[email protected]; Tel.: +86-20-2223-6028
Received: 19 July 2017; Accepted: 10 August 2017; Published: 15 August 2017
Abstract: Water-miscible alkylimidazolium ionic liquids (ILs) are “green” co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the TI Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C2 ~C6 ) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO4 − showed higher biocompatibility than did Ac− or Cl− , probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase. Keywords: ionic liquids (ILs); laccase; alkylimidazolium cations; competitive binding; kinetics; Hofmeister effects; molecular simulation
1. Introduction Laccase (EC 1.10.3.2) is an efficient biocatalyst for synthesizing dimers, polymers and composites from a diverse range of substrates including phenolics, aromatic amines, flavonoids, acrylamide and thiols [1,2]. Laccase-induced synthesis is often conducted in aqueous mixtures of organic solvents, e.g., acetone and methanol, to promote the yield and/or molecular weight of products [3,4]. Many laccases are tolerant to these solvents at a high content, e.g., 50% (v/v) [4,5]. However, the use of conventional volatile solvents threatens the environment [6]. Recently, ionic liquids (ILs) have been revealed as ideal alternative co-solvents due to their negligible vapor pressure, remarkable thermal stability and excellent recyclability [6,7]. Selected ILs could further lessen the inactivation of laccase by the template molecules during aniline polymerization [8], or by the oxidative form of redox mediators [9]. However, most water-miscible ILs for potential use in homogeneous synthesis have been identified as laccase inhibitors (Figure 1) except several particular species, e.g., [Emim]EtSO4 and [TMA]TfO, under specified conditions [8,10–12].
Molecules 2017, 22, 1353; doi:10.3390/molecules22081353
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Molecules 2017, 22, 1353 Molecules 2017, 22, 1353
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Figure Figure 1. 1. Scheme Scheme for for catalysis catalysis by by laccase laccase and and for for inhibition inhibition by by ionic ionic liquids liquids (ILs). (ILs).
Similar to those typical inorganic salts, ILs are constituted of a pair of cation and anion moieties. Similar to those typical inorganic salts, ILs are constituted of a pair of cation and anion moieties. Their biocompatibilities are greatly dependent on physicochemical properties [7]. For instance, Their biocompatibilities are greatly dependent on physicochemical properties [7]. For instance, Trametes Trametes versicolor (Tve) laccase was more frequently supported by hydrophobic ILs [10]. The versicolor (Tve) laccase was more frequently supported by hydrophobic ILs [10]. The inactivation inactivation effects of laccase by alkylimidazolium ILs aggravated with the increase of alkyl chain effects of laccase by alkylimidazolium ILs aggravated with the increase of alkyl chain length [13,14]. length [13,14]. On the contrary, there is evidence showing that alkyl sulfate anions with shorter chain On the contrary, there is evidence showing that alkyl sulfate anions with shorter chain length length have less inhibitory effects on Tve laccase activity [10]. For a better explanation of the distinct have less inhibitory effects on Tve laccase activity [10]. For a better explanation of the distinct effects of homologous series of ILs on enzyme stability, the Hofmeister effects hypothesis has effects of homologous series of ILs on enzyme stability, the Hofmeister effects hypothesis has proposed; in brief, the loss of enzyme activity tends to occur in the presence of chaotropic anions, e.g., proposed; in brief, the loss of enzyme activity tends to occur in the presence of chaotropic −>SO42−>PO [NTf2]−>[SCN]−>[CF−3SO3]−>Br−−>[BF4]−>Cl−>Ac 43−, and kosmotropic cations, e.g., Na+>K+> − >Br− >[BF ]− >Cl− >Ac− >SO 2− >PO 3− , and kosmotropic anions, e.g., [NTf ] >[SCN] >[CF SO ] 3 +>[N1,1,1,1] 3 4 4 Hofmeister effects +>[Emim] + [15,16]. [Hmim]+>[BmPy]++2>[Bmim] Anions exhibit4 stronger cations, e.g., Na >K+ > [Hmim]+ >[BmPy]+ >[Bmim]+ >[Emim]+ >[N1,1,1,1]+ [15,16]. Anions exhibit than cations [11]. The above Hofmeister series is an empirical rule developed on the basis of stronger Hofmeister effects than cations [11]. The above Hofmeister series is an empirical rule biochemical assays. It is not universally valid for predicting effects of ILs’ structures on any enzyme developed on the basis of biochemical assays. It is not universally valid for predicting effects of due to the wide variations of enzyme sources, substrate species and media features [15,16]. To ILs’ structures on any enzyme due to the wide variations of enzyme sources, substrate species and develop a better understanding, it is important to directly explore the laccase/salt molecular media features [15,16]. To develop a better understanding, it is important to directly explore the interactions and characterize the respective role of cations and anions. Previous authors have only laccase/salt molecular interactions and characterize the respective role of cations and anions. Previous reported the change in kinetics parameters of laccase by addition of ILs [5,10–14]. Advanced authors have only reported the change in kinetics parameters of laccase by addition of ILs [5,10–14]. investigation tools such as in silico simulation have been used for analyzing the molecular Advanced investigation tools such as in silico simulation have been used for analyzing the molecular interactions between laccase and substrate, in particular at the binding sites [12,17]. These tools, interactions between laccase and substrate, in particular at the binding sites [12,17]. These tools, however, have not been applied in the study of laccase/IL binding. This work investigated the however, have not been applied in the study of laccase/IL binding. This work investigated the inhibition mechanisms of water-soluble alkylimidazolium ILs on Myceliophthora thermophila (Mth) inhibition mechanisms of water-soluble alkylimidazolium ILs on Myceliophthora thermophila (Mth) laccase by using molecular docking and kinetics methods. The validation of the Hofmeister series, laccase by using molecular docking and kinetics methods. The validation of the Hofmeister series, the pH optima shift of laccase and radical scavenging of oxidation products in the presence of ILS the pH optima shift of laccase and radical scavenging of oxidation products in the presence of ILS were all comprehensively discussed. were all comprehensively discussed. 2. Results 2. Results 2.1. 2.1. Competitive Competitive Binding Binding of of Alkylimidazolium Alkylimidazolium Cations Cations The The roles roles of of cations cations were were revealed revealed by by comparing comparing the the kinetics kinetics parameters, parameters, i.e., i.e., initial initial velocity velocity (v (voo)) and inhibition constants (K i), and the docking sites of five alkylimidazolium chlorides ([Cnmim]Cl, n and inhibition constants (Ki ), and the docking sites of five alkylimidazolium chlorides ([Cn mim]Cl, =n 2, 4, 4, 6, 6, 8, 8, 10,10, Table 1).1). AllAll thethe tested ILsILs lowered laccase activity through competitive binding as = 2, Table tested lowered laccase activity through competitive binding indicated by the Lineweaver–Burk plots of 1/v o vs. 1/[S] ([S] was the concentration of substrate) as indicated by the Lineweaver–Burk plots of 1/vo vs. 1/[S] ([S] was the concentration of substrate) (Figure S1). All obtained plots obtained at varied concentrations of ILintersected ([I]) intersected the same (Figures2a 2aand andFigure S1). All plots at varied concentrations of IL ([I]) at the at same point point on the vertical axis. The apparent Michaelis constant (K m) increased with increasing [I] while on the vertical axis. The apparent Michaelis constant (Km ) increased with increasing [I] while the the maximum velocity (Vmax ) remained constant.Competitive Competitiveinhibition inhibitionconstants constants (K (Kic)) were thereby maximum velocity (Vmax ) remained constant. ic were thereby calculated Kic value decreased with the increase calculated from from Equation Equation (2) (2) and and shown shown in in Figure Figure 2c. 2c. Clearly, Clearly, K ic value decreased with the increase of alkyl chain length or n value. In particular, a dramatic inhibition was detected when n > 6. For [Omim]Cl (n = 8) or [Dmim]Cl (n = 10), the Kic was determined as 2.15 and 0.18 mM, respectively. Lower Kic corresponded to stronger inhibition capacity. The reaction velocities under regular
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of alkyl chain length or n value. In particular, a dramatic inhibition was detected when n > 6. For [Omim]Cl (n = 8) or [Dmim]Cl (n = 10), the Kic was determined as 2.15 and 0.18 mM, respectively. Molecules 2017, 22, 1353 14 Lower Kic corresponded to stronger inhibition capacity. The reaction velocities under3 ofregular conditions (See Section 4.4) were measured to verify the variation trend of K . The results in Figure 2d conditions (See Section 4.4) were measured to verify the variation trend of Kicic. The results in Figure showed that laccase almost inactivated by 50 mM of mM [Dmim]Cl. However, [Emim]Cl, 2d showed that was laccase was completely almost completely inactivated by 50 of [Dmim]Cl. However, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% of laccase activity, 3indicating that [Emim]Cl, [Bmim]Cl activity, Molecules 2017, 22, 1353 and [Hmim]Cl with shorter alkyl chains retained 65~75% of laccase 3 14 of 14 Molecules 2017, 22,22, 1353 of 14 Molecules 2017, 1353 3 of Molecules 2017, 22, 1353 3 of 14 Molecules Molecules Molecules 2017, 2017, 22, 2017, 1353 22, 1353 22, 1353 33of of 314 of314 of 14 Molecules 2017, 22, 1353 Molecules 2017, 22, 1353 3 3of 14 Molecules 2017, 22, 1353 of14 14 they are more promising for practical use compared [Dmim]Cl indicating that they are more promising for practicalto use comparedor to [Omim]Cl. [Dmim]Cl or [Omim]Cl.
400
15.92
.L/mol) .L/mol) ..L/mol) 1/v1/v (min (min (min L/mol) 1/v .L/mol) o1/v ooo (min 1/v (min .L/mol) ..L/mol) 1/vo1/v (min L/mol) (min 1/v (min o oo .L/mol) 1/vo (min . 1/vo (min L/mol) 1/vo (min.L/mol) 1/v (min.L/mol)
15.68 31.36 47.04[I]/mM
.L/mol) .L/mol) ..L/mol) 1/v1/v (min (min (min L/mol) 1/v .L/mol) o1/v ooo (min 1/v (min .L/mol) 1/vo (min.L/mol) ..L/mol) 1/v1/v (min L/mol) o (min (min 1/v o oo .L/mol) 1/vo (min 1/v (min.L/mol)
conditions (See Section 4.4) were measured to verify the variation trend . The results in Figure conditions (See Section 4.4) were measured toto verify the variation trend ofof Kof ic.icK The results ininFigure conditions (See Section 4.4) were measured verify the variation trend K . icThe results Figure conditions (See Section 4.4) were measured to verify the variation trend of K icic. The results in Figure conditions conditions conditions (See (See Section (See Section Section 4.4) 4.4) were 4.4) were measured were measured measured to verify to inactivated verify to verify the the variation the variation variation trend trend trend of K of ic.The .[Dmim]Cl. K of The ic[Dmim]Cl. .K The ic results .results The results results in Figure in Figure in Figure conditions (See Section 4.4) were measured verify the variation trend K results in Figure conditions (See Section 4.4) were measured toto verify the variation trend ofof K ic .ic[Dmim]Cl. results in Figure conditions (See Section 4.4) were measured to verify the variation trend of K ic .The The in Figure 2d showed that laccase was almost completely inactivated by 50 mM of However, 2d showed that laccase was almost completely by 50 mM of However, 2d showed that laccase was almost completely inactivated by 50 mM of However, 2d showed that laccase was However, 600almost completely inactivated by 50 mM of [Dmim]Cl. 600 2d 2d showed 2d showed showed that that laccase that laccase laccase was was almost was almost almost completely completely completely inactivated inactivated inactivated by by 50 by 50 mM 50 mM of mM [Dmim]Cl. of of [Dmim]Cl. [Dmim]Cl. However, However, However, 2d showed that laccase was almost completely inactivated by 50 mM of [Dmim]Cl. However, 2d showed that laccase was almost completely inactivated by 50 mM of [Dmim]Cl. However, 2d showed that laccase was almost completely inactivated by 50 mM of [Dmim]Cl. However, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% of laccase activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% ofoflaccase laccase activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% laccase activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% of activity, [I]/mM [I]/mM [Emim]Cl, [Emim]Cl, [Emim]Cl, [Bmim]Cl [Bmim]Cl [Bmim]Cl and and [Hmim]Cl and [Hmim]Cl [Hmim]Cl with with shorter with shorter shorter alkyl alkyl chains alkyl chains chains retained retained 65~75% 65~75% 65~75% of laccase of of laccase laccase activity, activity, activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% laccase activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains retained 65~75% ofof laccase activity, [Emim]Cl, [Bmim]Cl and [Hmim]Cl with shorter alkyl chains 65~75% of laccase activity, 500 500 indicating they are more promising for practical use compared to [Dmim]Cl or [Omim]Cl. 0that indicating that they are more promising for practical use compared toretained [Dmim]Cl or [Omim]Cl. indicating that they are more promising for practical use compared to [Dmim]Cl or [Omim]Cl. 0 retained indicating that they are more promising for practical use compared to [Dmim]Cl or [Omim]Cl. 7.47 indicating indicating indicating that that they that they are they are more are more promising more promising promising for for practical for practical practical use use compared use compared compared to [Dmim]Cl to [Dmim]Cl to [Dmim]Cl or [Omim]Cl. or [Omim]Cl. or [Omim]Cl. 7.58toto indicating that they are more promising for practical use compared [Dmim]Cl [Omim]Cl. indicating that they are more promising for practical use compared [Dmim]Cl oror [Omim]Cl. indicating that they are more promising for practical use compared to [Dmim]Cl or [Omim]Cl. 400
o
o
1/vo (min.L/mol)
31.84 600 600600 600 600600 600 600 300 300 47.76 600 600 600 600 [I]/mM [I]/mM 600 600 600 600 [I]/mM 600 600 600 600 [I]/mM [I]/mM [I]/mM [I]/mM 500 500 500500 500500 0[I]/mM 0 0 0 [I]/mM [I]/mM 0 0 [I]/mM 500 500 [I]/mM [I]/mM [I]/mM [I]/mM [I]/mM [I]/mM [I]/mM 0 0[I]/mM 200 200 500 500 7.47 500 500 500 500 500 500 500 500 500 500 0 0 7.58 007.47 07.47 0 7.47 0 0 007.58 07.58 0 7.58 400 400400 400 400400 15.68 15.68 15.68 15.92 7.47 7.477.47 15.92 15.92 7.47 400 400 7.47 7.47 7.58 7.58 7.58 7.58 7.58 7.58 15.68 15.92 100 400 100 31.36 400 400 31.36 31.36 400 31.84 400 400 400 400 400 400 400 15.68 15.68 15.68 400 31.84 31.84 15.68 15.92 15.92 15.92 15.68 15.68 15.92 15.92 15.92 31.36 31.84 300 300300 300 300300 47.04 47.04 47.04 47.76 31.36 31.36 31.36 300 47.76 47.76 31.36 31.36 31.36 31.84 31.84 31.84 300 31.84 31.84 31.84 47.04 47.76 0 300 300 300 300 300 300 47.04 47.04 300 300 300 300 3000 300 47.04 47.76 47.76 47.76 47.04 47.0447.04 47.76 47.76 -80 -60 -40 -20 200 0 20 40 60 -8047.76 -60 -40 200 -20 200 0 20 40 60 200 200200 200200 200 200 200 200 1/[S] (L/mmol) 1/[S] (L/mmol) 200 200 200 200 200 200 200 200 100 100 100 100 100 100 100 100 (a) Lineweaver-Burk plots 100 in100 the100 presence of [Emim]Cl (b) Lineweaver-Burk100 plots in100 the presence of KCl 100 100 100 100 100 100 100 0 0 0 0 0 0 5 0 0 -80 -60 -40 -20 0 20 40 60 -80 -60 -40 -20 0 -80-80 -60 -60-60 -40 -40-40 -20 -20-20 0 0 2020 40 4040 60 6060 -80-80 -60-60 -40 -40-40 -20 -20-20 0 0 2020 2040 4040 4060 6060 60 1.0 -60 -80 -80 0 000 0 200 0 000 0 200 1/[S] (L/mmol) 1/[S] (L/mmol) -80 -60 -80 -60 -40 -60 -40 -20 -40 -20 00 02020 20 0 20 40 20 40 60 40 60 60 -80 -80 -60 -80 -60 -40 -60 -40 -20 -40 -20 00 02020 20 0 20 40 20 40 60 40 60 60 -80 -60 -40 -20 -80 -60 -40 -20 1/[S] (L/mmol) 1/[S] (L/mmol) 1/[S] (L/mmol) 1/[S] (L/mmol) -80 -60 -40 -20 0 0-20 4040 6060 -60 -40 -20 0 0-20 4040 6060 -80 -80 -60 -40 -20 20 40 60 -80 -80 -60 -40 -20 20 40 60
1/[S] (L/mmol)
4
1/[S] (L/mmol)
Relative reaction velocity Relative reaction velocity Relative reaction velocity Relative reaction velocity Relative reaction velocity Relative reaction velocity Relative reaction velocity Relative reaction velocity Relativevelocity reaction velocity Relative reaction Relative reaction velocity
Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM) Inhibition constant (mM)
1/[S] 1/[S] (L/mmol) 1/[S] (L/mmol) (L/mmol) 1/[S] 1/[S] (L/mmol) 1/[S] (L/mmol) (L/mmol) 1/[S] (L/mmol) 1/[S] (L/mmol) 1/[S] (L/mmol) 1/[S] (L/mmol) 1/[S] 1/[S] (L/mmol) 0.8 (a) Lineweaver-Burk plots inpresence the presence of [Emim]Cl (b) (b) Lineweaver-Burk plots inpresence the presence of KCl (a)(a) Lineweaver-Burk plots in(L/mmol) the ofof [Emim]Cl Lineweaver-Burk plots inin the ofof KCl Lineweaver-Burk plots in the presence [Emim]Cl Lineweaver-Burk plots the presence KCl (a) Lineweaver-Burk plots in the presence of [Emim]Cl (b)(b) Lineweaver-Burk plots in the presence of KCl (a) Lineweaver-Burk (a) Lineweaver-Burk Lineweaver-Burk plots plots in plots the inpresence presence the in the presence presence of [Emim]Cl of [Emim]Cl of [Emim]Cl (b) (b) Lineweaver-Burk (b) Lineweaver-Burk Lineweaver-Burk plots plots in plots the inpresence presence the in the presence presence of KCl of KCl of KCl 5(a) (a) Lineweaver-Burk plots in the of [Emim]Cl (b) Lineweaver-Burk plots in the of 5 5 (a) Lineweaver-Burk plots in the presence of [Emim]Cl (b) Lineweaver-Burk plots in the presence of KCl (a) Lineweaver-Burk plots in the presence of [Emim]Cl (b) Lineweaver-Burk plots in the presence ofKCl KCl 5
Relative reaction velocity
Inhibition constant (mM)
1.0 3 1.01.00.6 1.0 5 555 5 5 1.0 1.0 1.0 1.0 1.0 1.0 4 424 4 0.80.8 0.8 0.8 0.4 4 444 4 4 0.8 0.8 0.8 0.8 0.8 0.8 3 3 3 0.60.6 0.6 3 0.6 0.2 31 333 3 3 0.6 0.6 0.6 0.6 0.6 0.6 2 2 2 0.4 0.4 0.4 2 0.4 0.0 20 222 2 2 0.4 0.4 0.4 0.4 0.4 0.4 1 0.20.2 0.2 11 01 0.2 2 4 6 8 10 12 0 2 4 6 8 10 12 1 111 1 1 0.2 0.2 0.2 0.2 0.2 0.2 Number of Carbon in alkyl chain of ILs Number of Carbon in sidechain of ILs 0 0.0 0 0 0.0 0.0 0 0.0 0.0 0.0 0.0 0.0 0 000 0 0 (c) Inhibition constants Measured relative reaction velocities 0.0 0.0 (d) 0 2 2 2 4 4 4 6 6 6 8 8 8 1010 10 1212 12 0 2 2 2 4 4 4 6 6 6 8 8 8 1010 10 1212 12 0 0 0 2 4 6 8 10 12 00 0 2 4 6 8 10 12 of Carbon in6 alkyl chain of ILs of 4Carbon in of Number of4of Carbon in alkyl chain of ILs Carbon in alkyl chain of ILs Number of in sidechain of8of ILs sidechain ILs 2of 2 4 4 4 6 6 8 8 10 8 10 10 12 12 12 0 0 2of 2Carbon 44 Carbon 6 8sidechain 10 810ILs 101212 10 12 2022 Number 4 6 8 10 12 0 2022 Number 6466in 8688 of 0 000 0Number 2 Number 6 8 10 12 0 2 Number 4 4of 6in 1010 4 6 8 10 12 0 12 12 12 Carbon in alkyl chain of ILs Number Carbon sidechain ILs
Figure 2. Effects of alkyl chain length in alkylimidazolium cations on kinetics of laccase/ABTS
Number Number Number of Carbon of Carbon ofin in Carbon alkyl inchain alkyl chain in alkyl of chain ILs ofin ILs ofalkylimidazolium ILs Number Number Number of Carbon ofon Carbon ofinin Carbon sidechain in sidechain inofsidechain of ILs oflaccase/ABTS ILs of ILs Number of Carbon alkyl chain of Number of sidechain of ILs Figure 2. Effects of alkyl chain length cations kinetics of Number ofInhibition Carbon in alkyl ofchain ILs Number of Carbon in alkyl chain ofILs ILs Number of Carbon in sidechain ILs Number ofCarbon Carbon in sidechain of ILs (c) Inhibition constants (d) Measured relative reaction velocities (c) Inhibition constants (d) Measured relative reaction velocities (c) constants (d) Measured relative reaction velocities Inhibition (d) Measured relative reaction velocities oxidation at(c) pH 3. (Note:constants Standard deviation bars were from triplicate measurements). (c) Inhibition (c) (c) Inhibition Inhibition constants constants constants (d) (d) Measured (d) Measured Measured relative relative relative reaction reaction reaction velocities velocities velocities (c) Inhibition constants (d) Measured relative reaction velocities Inhibition constants (d) Measured relative reaction velocities (c) Inhibition constants (d) Measured relative reaction velocities oxidation at pH(c) 3. (Note: Standard deviation bars were from triplicate measurements).
Figure 2. Effects of alkyl chain length in alkylimidazolium cations on kinetics of laccase/ABTS Figure 2.2.Effects Effects ofofalkyl alkyl chain length ininalkylimidazolium alkylimidazolium cations ononkinetics kinetics ofoflaccase/ABTS laccase/ABTS Figure Effects alkyl chain length alkylimidazolium cations kinetics laccase/ABTS Figure 2. of chain length in cations on of Figure Figure Figure Effects 2.at Effects 2. Effects of alkyl ofStandard of alkyl chain alkyl chain length chain length length in alkylimidazolium in alkylimidazolium in alkylimidazolium cations cations cations on kinetics on on kinetics kinetics of laccase/ABTS of of laccase/ABTS laccase/ABTS Figure 2.2. Effects of alkyl chain length in alkylimidazolium cations on kinetics laccase/ABTS Figure 2. Effects of alkyl chain length inionic alkylimidazolium cations on kinetics ofof laccase/ABTS Figure 2. Effects of alkyl chain length in alkylimidazolium cations on kinetics of laccase/ABTS Table 1. Chemical structures of liquids and substrates investigated in this work. oxidation pH 3. (Note: Standard deviation bars were from triplicate measurements). oxidation at pH 3. (Note: deviation bars were from triplicate measurements). oxidation at pH 3. (Note: Standard deviation bars were from triplicate measurements). oxidation at pH 3. (Note: Standard deviation bars were from triplicate measurements). Table 1.atat Chemical structures of ionic liquids and substrates investigated in this work. oxidation oxidation oxidation pH at pH 3. at (Note: pH 3. (Note: 3. (Note: Standard Standard Standard deviation deviation deviation bars bars were bars were from were from triplicate from triplicate triplicate measurements). measurements). measurements). oxidation pH Standard deviation bars were from triplicate measurements). oxidation at pH 3.3. (Note: Standard deviation bars were from triplicate measurements). oxidation at pH 3.(Note: (Note: Standard deviation bars were from triplicate measurements). Cations
Table 1. Chemical structures of ionic liquids and substrates investigated in this work. Table 1.1. Chemical structures ofof ionic liquids and substrates investigated inin this work. Table Chemical structures ionic liquids and substrates investigated this work. Table 1. Chemical structures of ionic liquids and substrates investigated in this work. Table Table Table Chemical 1. Chemical 1. Chemical structures structures structures of ionic of liquids ionic of liquids ionic liquids liquids and and substrates and substrates substrates investigated investigated investigated in this inwork. this in work. this work. work. Table structures ionic liquids and substrates investigated this work. Table 1.1.1. Chemical structures ofof ionic and substrates investigated inin this Table 1.Chemical Chemical structures of ionic liquids and substrates investigated in this work. Cations Cations Cations Cations Cations Cations Cations Cations Cations Cations Cations
[Emim]+
[Bmim]+ Anions + + + + [Emim] [Bmim] [Emim] [Bmim] [Emim] [Bmim] ++++ + ++ + [Emim] [Bmim] [Emim] [Bmim] + + + + +
[Hmim]+
+ + +[Emim] + +[Bmim] + Anions + [Emim] [Emim] [Bmim] [Bmim] [Emim] [Bmim] Anions [Emim] [Bmim] [Emim] [Bmim] Anions Anions Anions Anions Anions Anions Anions Anions Anions
Ac− − − − AcAc −− Ac Ac − −−−Ac−Ac− Ac
Ac Ac Ac − Ac
EtSO4−
+ + [Hmim] [Hmim] [Hmim] +++ [Hmim] [Hmim] + + +++ + [Hmim] [Hmim] [Hmim] [Hmim] [Hmim] [Hmim] +
[Omim]+ Substrates + + + [Omim] [Omim] [Omim] ++ + [Omim] [Omim] + + +
+ + + [Omim] [Omim] [Omim] [Omim] Substrates [Omim] [Omim] Substrates Substrates Substrates Substrates Substrates Substrates Substrates Substrates Substrates Substrates
[Dmim]+ + + + [Dmim] [Dmim] [Dmim] + ++ [Dmim] [Dmim] + + +++ + [Dmim] [Dmim] [Dmim] [Dmim] [Dmim] [Dmim]
2,2’-azino-bis-(32,6ethylbenzothiazoline-6dimethoxyphenol Guaiacol Syringaldezine 2,2’-azino-bis-(32,62,2’-azino-bis-(32,62,2’-azino-bis-(32,62,2’-azino-bis-(32,62,2’-azino-bis-(32,2’-azino-bis-(32,2’-azino-bis-(32,6ethylbenzothiazoline-6dimethoxyphenol Guaiacol Syringaldezine Syringaldezine sulphonic acid) (ABTS) dimethoxyphenol (2,6-DMP) 2,2’-azino-bis-(32,6ethylbenzothiazoline-6dimethoxyphenol Guaiacol ethylbenzothiazoline-6dimethoxyphenol Guaiacol Syringaldezine 2,2’-azino-bis-(32,62,2’-azino-bis-(32,6-2,6-2,6ethylbenzothiazoline-6Guaiacol Syringaldezine 2,2’-azino-bis-(32,6-
EtSO − 4− 4− EtSO 4− EtSO EtSO 44− − EtSO EtSO 4− 4−ethylbenzothiazoline-6ethylbenzothiazoline-6ethylbenzothiazoline-6ethylbenzothiazoline-6dimethoxyphenol dimethoxyphenol dimethoxyphenol Guaiacol Guaiacol GuaiacolSyringaldezine Syringaldezine Syringaldezine Syringaldezine sulphonic acid) (ABTS) dimethoxyphenol (2,6-DMP) EtSO 44−4−− ethylbenzothiazoline-6dimethoxyphenol Guaiacol sulphonic acid) (ABTS) (2,6-DMP) sulphonic acid) (ABTS) (2,6-DMP) EtSO 4−EtSO Guaiacol EtSO ethylbenzothiazoline-6dimethoxyphenol Guaiacol Syringaldezine sulphonic acid) (ABTS) (2,6-DMP) EtSO ethylbenzothiazoline-6dimethoxyphenol Guaiacol Syringaldezine Syringaldezine 4 sulphonic sulphonic sulphonic acid) acid) (ABTS) acid) (ABTS) (ABTS) (2,6-DMP) (2,6-DMP) (2,6-DMP) (2,6-DMP) sulphonic acid) (ABTS) (2,6-DMP) sulphonic acid) (ABTS) sulphonic acid) (ABTS) (2,6-DMP)
(ABTS) (2,6-DMP) According to enzymesulphonic kinetics acid) theory, competitive binding means that the inhibitors irreversibly According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly According toto enzyme kinetics theory, competitive binding means that the inhibitors irreversibly According enzyme kinetics theory, competitive binding means that the inhibitors irreversibly According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly occupy the specific binding sites for substrates and form an enzyme–inhibitor complex [18]. The According According According to enzyme to enzyme tobinding enzyme kinetics kinetics kinetics theory, theory, theory, competitive competitive competitive binding binding binding means means means that that the that the inhibitors the inhibitors inhibitors irreversibly irreversibly irreversibly According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly occupy the specific sites for substrates and form an enzyme–inhibitor complex [18]. The occupy the specific binding sites for substrates and form an enzyme–inhibitor complex [18]. The occupy the specific binding sites for substrates and form an enzyme–inhibitor complex [18]. The occupy theofspecific bindingcomplex sites for was substrates andwith formUV/Vis an enzyme–inhibitor complex3). [18]. The formation a laccase/IL verified spectroscopy (Figure A laccase/IL occupy occupy occupy the the specific the specific specific binding binding binding sites sites for sites for substrates for substrates substrates and and form and form an form an enzyme–inhibitor an enzyme–inhibitor enzyme–inhibitor complex complex complex [18]. [18]. The [18]. TheThe occupy the specific binding sites for substrates and form an enzyme–inhibitor complex [18]. The occupy the binding sites for substrates and form an enzyme–inhibitor complex [18]. The occupy the binding sites for substrates and form an enzyme–inhibitor complex [18]. The formation of a laccase/IL complex was verified with UV/Vis spectroscopy (Figure 3). A laccase/IL formation ofspecific aspecific laccase/IL complex was verified with UV/Vis spectroscopy (Figure 3).3). A laccase/IL formation of alaccase/IL laccase/IL complex was verified with UV/Vis spectroscopy (Figure Alaccase/IL laccase/IL formation of a complex was verified with UV/Vis spectroscopy (Figure 3). A complex sample was obtained by soaking Mth laccase in UV/Vis [Bmim]Cl/buffer solution, followed with formation formation formation of laccase/IL of a laccase/IL a laccase/IL complex complex complex was was verified was verified verified with with UV/Vis with UV/Vis spectroscopy spectroscopy spectroscopy (Figure (Figure (Figure 3). A 3). laccase/IL 3). A laccase/IL A laccase/IL formation complex was verified with UV/Vis spectroscopy (Figure 3). laccase/IL formation ofof aaalaccase/IL complex was verified with UV/Vis spectroscopy (Figure 3). AA laccase/IL formation of aoflaccase/IL laccase/IL complex was verified with UV/Vis spectroscopy (Figure 3). A laccase/IL
complex sample was obtained by soaking Mth laccase in [Bmim]Cl/buffer solution, followed with complex sample was obtained bybysoaking soaking Mth laccase inin[Bmim]Cl/buffer [Bmim]Cl/buffer solution, followed with complex sample was obtained soaking Mth laccase [Bmim]Cl/buffer solution, followed with complex sample was obtained by Mth laccase in solution, followed with complex complex complex sample sample sample was was obtained was obtained obtained by by soaking by soaking soaking Mth Mth laccase Mth laccase laccase in [Bmim]Cl/buffer in [Bmim]Cl/buffer in [Bmim]Cl/buffer solution, solution, solution, followed followed followed with with with complex sample was obtained by soaking Mth laccase in [Bmim]Cl/buffer solution, followed with complex sample was obtained by soaking Mth laccase in [Bmim]Cl/buffer solution, followed with complex sample was obtained by soaking Mth laccase in [Bmim]Cl/buffer solution, followed with
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Absorbance
According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly occupy the specific binding sites for substrates and form an enzyme–inhibitor complex [18]. Molecules 2017, 22,of 1353 4 of 14 The formation a laccase/IL complex was verified with UV/Vis spectroscopy (Figure 3). A laccase/IL complex sample was obtained by soaking Mth laccase in [Bmim]Cl/buffer solution, followed with lyophilization andthorough thorough washing with absolute ethanol. The resultant solid resultant wasre-dissolved totally relyophilization and washing with absolute ethanol. The solid was totally dissolved in water. The UV/Vis spectrum of the complex had both specific absorption bands of in water. The UV/Vis spectrum of the complex had both specific absorption bands of [Bmim]Cl [Bmim]Cl (at around 210 nm) and Mth laccase (260~300 nm). There was obvious red shift from the (at around 210 nm) and Mth laccase (260~300 nm). There was obvious red shift from the absorption absorption peakprotein of laccase at 272 nm the peak of laccase/[Bmim]Cl at suggests 292 nm, peak of laccase at 272protein nm to the peak of to laccase/[Bmim]Cl complex at 292complex nm, which which suggests the complexation of laccase with [Bmim]Cl absorbed energy. In addition, the the complexation of laccase with [Bmim]Cl absorbed energy. In addition, the re-dissolved complex re-dissolved complex still retained 65% of the initial activity (data not shown). still retained 65% of the initial activity (data not shown). 2.0 IL Laccase/IL complex Laccase 1.8 0.06 1.6 Red Shift 1.4 0.04 Laccase Laccase/IL 1.2 Complex 1.0 0.02 0.8 0.6 0.00 0.4 220 240 260 280 300 320 0.2 0.0 -0.2 200 220 240 260 280 300 320 340 360 380 400
Wave length (nm) Figure 3. 3. UV/Vis UV/Vis spectra Figure spectra of of [Bmim]Cl [Bmim]Cl (IL), (IL), laccase laccase and and laccase/[Bmim]Cl laccase/[Bmim]Clcomplex. complex.
In silico simulation showed that the active pocket near the TI Cu site (His431, Cys503, His508) in In silico simulation showed that the active pocket near the TI Cu site (His431, Cys503, His508) in Mth laccase could accommodate substrate molecules such as 2, 2′-azino-bis-(3-ethylbenzothiazolineMth laccase could accommodate substrate molecules such as 2, 20 -azino-bis-(3-ethylbenzothiazoline6-sulphonic acid) (ABTS) and three other phenolics (Figure 4a and Figure S2). The interactions between 6-sulphonic acid) (ABTS) and three other phenolics (Figures 4a and S2). The interactions between ABTS and binding sites were maintained by (1) hydrogen bonds at Thr187, Ser190, Phe194, Arg302 and ABTS and binding sites were maintained by (1) hydrogen bonds at Thr187, Ser190, Phe194, Arg302 Trp373, and (2) hydrophobic force at Gly191, Ala192, Pro193, Glu235, Phe371, Leu429 and His508. and Trp373, and (2) hydrophobic force at Gly191, Ala192, Pro193, Glu235, Phe371, Leu429 and His508. The active pocket around the TI Cu also functioned as the optimal binding site for alkylimidazolium The active pocket around the TI Cu also functioned as the optimal binding site for cations (Figure 4b–f). All cations could enter the pocket in the same orientation. The methylimidazolium alkylimidazolium cations (Figure 4b–f). All cations could enter the pocket in the same orientation. head possessing positive charge pointed at the hydrophobic outer edge of the pocket (brown color), The methylimidazolium head possessing positive charge pointed at the hydrophobic outer edge of the while the alkyl chain stretched into the hydrophilic inner channel (blue color). Alkylimidazolium pocket (brown color), while the alkyl chain stretched into the hydrophilic inner channel (blue color). cations complexed with laccase dominantly through hydrophobic interactions. The binding sites, Alkylimidazolium cations complexed with laccase dominantly through hydrophobic interactions. Thr187, Ser190/Gly191, Phe371, Trp373 and His508, were identical for all the tested inhibitors as well The binding sites, Thr187, Ser190/Gly191, Phe371, Trp373 and His508, were identical for all the as the substrate such as ABTS. Due to the lack of proton acceptor or donor groups in alkylimidazolium tested inhibitors as well as the substrate such as ABTS. Due to the lack of proton acceptor or donor structure, there was no hydrogen bond formed between laccase and IL cations. Electrostatic interactions groups in alkylimidazolium structure, there was no hydrogen bond formed between laccase and might exist and affect the orientation. Figure 4 showed that the positive head was always apart from IL cations. Electrostatic interactions might exist and affect the orientation. Figure 4 showed that His508, a base residue, probably due to the electrostatic repulsion. On the other hand, small cations the positive head was always apart from His508, a base residue, probably due to the electrostatic such as [Emim]+ could entirely enter the inner channel in the pocket, probably leaving sufficient space repulsion. On the other hand, small cations such as [Emim]+ could entirely enter the inner channel for ABTS binding near the TI Cu site. [Omim]+ and [Dmim]+ occupied Leu296/Leu297 near the pocket+ in the pocket, probably leaving sufficient space for ABTS binding near the TI Cu site. [Omim] edge and Leu429 around TI Cu, causing more server stereo-hindrance. As a result, the laccase activity and [Dmim]+ occupied Leu296/Leu297 near the pocket edge and Leu429 around TI Cu, causing was strongly or almost completely inhibited (Figure 2). Although Hofmeister effects hypothesis more server stereo-hindrance. As a result, the laccase activity was strongly or almost completely involves the cation series, our findings revealed the importance of stereo-hindrance effect especially inhibited (Figure 2). Although Hofmeister effects hypothesis involves the cation series, our findings the occupying of key amino acids in the IL-induced inhibition. revealed the importance of stereo-hindrance effect especially the occupying of key amino acids in the IL-induced inhibition.
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(a) ABTS
(b) [Emim]+
(c) [Bmim]+
(d) [Hmim]+
(e) [Omim]+
(f) [Dmim]+
Figure 4. Three dimensional conformations and binding sites from the docking of ABTS (a) and five Figure 4. Three dimensional conformations and binding sites from the docking of ABTS (a) and five alkylimidazolium cations (b–f) to the TI Cu active pocket of Mth laccase. alkylimidazolium cations (b–f) to the TI Cu active pocket of Mth laccase. -2.0
Binding energy (kJ/mol)
The binding energy was estimated from the docking top three trial conformations (Figure 5). Obviously, specific Specific -2.5 Blind docking docking for achieving energy minimization, although these docking gave more reliable results than blind two operations resulted in the-3.0 same binding sites for alkylimidazolium cations. The decrease of negative binding energy from C2 to C6 corresponded to the increase in the stability of enzyme–inhibitor complex, -3.5 consistent with the inhibition kinetics (Figure 2c). Binding energy slightly increased from C6 to C8, according to which less inhibition was expected. Many authors have also correlated the binding energy predictions -4.0 with the inhibition capacities of inhibitors [17,19]. However, due to the occupying of several key amino + or [Dmim]+ was rather significant. -4.5 acids, the inhibition by [Omim] -5.0
0
2
4
6
8
10
12
Number of Carbon in alkyl chain of ILs
Figure 5. Binding energy varied with the alkyl chain length of alkylimidazolium cations. (Note: The top three docking conformations were represented as filled, half-filled and empty symbols, respectively).
The binding energy was estimated from the top three trial conformations (Figure 5). Obviously, specific docking gave more reliable results than blind docking for achieving energy minimization, although these two operations resulted in the same binding sites for alkylimidazolium cations. The
(d) [Hmim]+
(e) [Omim]+
(f) [Dmim]+
Figure 4. Three dimensional conformations and binding sites from the docking of ABTS (a) and five 6 of 14 alkylimidazolium cations (b–f) to the TI Cu active pocket of Mth laccase.
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-2.0 Specific docking Blind docking
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Binding energy (kJ/mol)
-2.5 -3.0 -3.5 -4.0
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decrease of negative binding-4.5 energy from C2 to C6 corresponded to the increase in the stability of enzyme–inhibitor complex, consistent with the inhibition kinetics (Figure 2c). Binding energy slightly -5.0 0 2 4less inhibition 6 8 was 10 12 Many authors have also increased from C6 to C8, according to which expected. Number of Carbon in alkyl chain of ILs correlated the binding energy predictions with the inhibition capacities of inhibitors [17,19]. However, + or [Dmim]+ was rather due to the 5. occupying of several acids, inhibition by [Omim]cations. Figure Binding energy variedkey with the chain length of (Note: Figure withamino the alkyl alkyl chainthe length ofalkylimidazolium alkylimidazolium cations. (Note: The top top significant. three conformationswere wererepresented representedasas filled, half-filled empty symbols, respectively). three docking conformations filled, half-filled andand empty symbols, respectively). 2.2. Hofmeister Hofmeister Series of IL IL was Anions The binding energy estimated from the top three trial conformations (Figure 5). Obviously, 2.2. Series of Anions specific docking gave more reliable results than blind docking for achieving energy to minimization, Effects of ofsalt saltspecies speciesatatvarious variousconcentrations concentrations laccase activity were studied characterize Effects onon laccase activity were studied to characterize the although these two operations resulted in the same binding sites for alkylimidazolium cations. The the role IL anions. The results (Figure 6) demonstrated that anions play a role central rolesalt-induced in the saltrole of ILof anions. The results (Figure 6) demonstrated that anions play a central in the inducedinactivation, laccase inactivation, regardless of whether theiscation is organic or inorganic. laccase regardless of whether the cation organic or inorganic. Mixed Mixed triacid triacid bases, bases, i.e., phosphate and citrate, were the most compatible species, retaining at least 90% of activity i.e., phosphate and citrate, were the most compatible species, retaining at least 90% of activity at a concentration 200 mM equivalent molar concentration 600mM). mM).[Emim]EtSO [Emim]EtSO 4and and K 2SO SO 4 aatconcentration of of 200 mM (or(or equivalent molar concentration ofof600 K 4 2 4 ranked second, showing similar high kosmotropic (hydrated) features. Instead, acetate and chloride ranked second, showing similar high kosmotropic (hydrated) features. Instead, acetate and chloride ILs performed performed more more chaotropic chaotropic (weakly (weakly hydrated) hydrated) as as stronger stronger inhibitors. inhibitors. For For example, example, near near 50% 50% of of ILs laccase activity activity was was lost lost by by 100~130 100~130 mM mM acetate acetate or or chloride chloride ILs ILs at at pH pH 3. 3. KCl caused more more activity activity loss loss laccase KCl caused than alkylimidazolium chlorides. than alkylimidazolium chlorides.
Relative reaction velocity
1.0 0.8 0.6 0.4 0.2 0.0
KCl K2SO4 Buffer [Emim]SO4 [Emim]Ac [Bmim]Ac [Emim]Cl [Bmim]Cl 100
200
300
400 500 600700
Equivalent concentration of salts (mM)
Figure 6. 6. Salt effects on laccase laccase activity activity towards towards ABTS ABTS at at pH pH 3. 3. (Note: 30 mM buffer was used to to Figure maintain the the pH pH for for all all salts, salts, normalized normalized with with the the buffer buffer control). control). maintain
Intermolecular interactions between laccase and IL anions were built up for a better understanding of the Hofmeister series. Figure 7a,c showed that Ac− and EtSO4 − favorably bound with the same sites in Mth laccase, forming hydrogen bonds with Thr246 and Arg281 and hydrophobic interactions with His245. The optimal binding energy for Ac− and EtSO4 − were −3.42 and −4.06 kcal/mol, respectively. This site was also the most frequent one for Ac− among 200 docking runs (Figure 7a). However, together with several other predicted sites (Figure 7b), it was far away from the specific sites composed of four Cu ions. On the contrary, the most frequent binding sites for EtSO4 − were very close to the TI Cu active pocket (Figure 7d). This anion could thereby alter the ionic charge around the active (a) Ac¯¯, energy optimized, Ac¯¯, second most (c) EtSO4¯¯, energy 4¯¯, most frequent site, change the surrounding (b) static electric field and finally prevent the entry(d)ofEtSO alkylimidazolium most frequent (64 in 200 runs) frequent (35 in 200 runs) optimized (19 in 200 runs) (76 in 200 runs) cations into the pocket. Figure 7. Optimal docking conformations for Ac− and EtSO4−. (Note: Blue spheres are Cu ions; red structures are IL anions).
Intermolecular interactions between laccase and IL anions were built up for a better understanding of the Hofmeister series. Figure 7a,c showed that Ac− and EtSO4− favorably bound with the same sites in Mth laccase, forming hydrogen bonds with Thr246 and Arg281 and
Rel
0.2
[Emim]Cl [Bmim]Cl
0.0
100
200
300
400 500 600700
Equivalent concentration of salts (mM)
Figure Salt effects on laccase activity towards ABTS at pH 3. (Note: 30 mM buffer was used to7 of 14 Molecules 2017,6.22, 1353 maintain the pH for all salts, normalized with the buffer control).
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with the same sites in Mth laccase, forming hydrogen bonds with Thr246 and Arg281 and hydrophobic interactions with His245. The optimal binding energy for Ac− and EtSO4− were −3.42 and −4.06 kcal/mol, respectively. This site was also the most frequent one for Ac− among 200 docking runs ¯¯, most ¯¯, second (a)(Figure Ac¯¯, energy (b) Acwith most other predicted (c) EtSOsites 4¯¯, energy EtSO4far frequent 7a).optimized, However, together several (Figure 7b),(d) it was away from most frequent (64 in 200 runs) frequent (35 in 200 runs) optimized (19 in 200 runs) (76 in 200 runs) the specific sites composed of four Cu ions. On the contrary, the most frequent binding sites for EtSO4− were very Optimal close to the TI Cu conformations active pocket (Figure This anion could thereby alter ionic charge − 7d). Figure and EtSO 4−. (Note: Blue spheres arethe Cu red − . (Note: Figure 7. 7. Optimaldocking docking conformationsfor forAc Ac− and EtSO Blue spheres areions; Cu ions; 4 field around the active site, change the surrounding static electric and finally prevent the entry of structures are IL anions). red structures are IL anions). alkylimidazolium cations into the pocket.
Intermolecular interactions between laccase and IL anions were built up for a better 2.3.2.3. Shift of Laccase Optimal pHpHbybyILs Shift of Laccase Optimal ILs understanding of the Hofmeister series. Figure 7a,c showed that Ac− and EtSO4− favorably bound formation of of the laccase/IL complex would possiblybonds alter the the enzyme’s pH optima. Results formation laccase/IL complex would alter enzyme’s pH optima. Results with The theThe same sites in the Mth laccase, forming hydrogen with Thr246 and Arg281 and in Figure showedthat that theHis245. pH optima was not affected by4−any IL, in Figure 8a 8a showed the laccaseThe pHoptimal optimatowards towardsABTS ABTS was by tested any hydrophobic interactions with binding energy for Ac−not andaffected EtSO were −3.42tested and calibration ofof media pH. Loss activity was up to 30%, following order of of − among IL, after after calibration media pH. Loss oflaccase laccase activity was up following an order −4.06 kcal/mol, respectively. This site wasofalso the most frequent one forto Ac30%, 200an docking runs [Emim]EtSO 4
