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Radical Scavenging by Acetone: A New Perspective to Understand Laccase/ABTS Inactivation and to Recover Redox Mediator Hao Liu 1, : , *, Pandeng Zhou 2, : , Xing Wu 1 , Jianliang Sun 1 and Shicheng Chen 3, * Received: 30 July 2015 ; Accepted: 28 October 2015 ; Published: 4 November 2015 Academic Editor: Derek J. McPhee 1 2 3

* :

State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China; [email protected] (X.W.); [email protected] (J.S.) School of Chemistry and Environmental Engineering, Hunan City University, Yiyang 413000, China; [email protected] Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA Correspondence: [email protected] (H.L.); [email protected] (S.C.); Tel.: +86-20-2223-6028 (H.L.); Fax: +86-20-2223-6078 (H.L.) These authors contributed equally to this work.

Abstract: The biosynthetic utilization of laccase/mediator system is problematic because the use of organic cosolvent causes significant inhibition of laccase activity. This work explored how the organic cosolvent impacts on the laccase catalytic capacity towards 2,21 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) in aqueous solution. Effects of acetone on the kinetic constants of laccase were determined and the results showed Km and V max varied exponentially with increasing acetone content. Acetone as well as some other cosolvents could transform ABTS radicals into its reductive form. The content of acetone in media significantly affected the radical scavenging rates. Up to 95% of the oxidized ABTS was successfully recovered in 80% (v/v) acetone in 60 min. This allows ABTS recycles at least six times with 70%–75% of active radicals recovered after each cycle. This solvent-based recovery strategy may help improve the economic feasibility of laccase/ABTS system in biosynthesis. Keywords: laccase; ABTS; inhibition; radical scavenging; recovery

1. Introduction Laccases (EC 1.10.3.2) have been intensively studied as a green catalyst for biosynthesizing polyphenols [1], heterocyclic compounds [2], graft copolymers [3], etc. When laccase is applied to oxidize a monomer with a high redox potential (e.g., exceeded 780 mV vs. NHE), redox mediators are required for promoting enzyme catalytic efficiency or initiating the oxidation [4]. An ideal mediator should be a recyclable small molecule that is stable in its oxidized or reduced form in reaction media [2]. ABTS (2,21 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is one of the most effective mediator because its radical has a sufficiently high redox potential (e.g., +1100 mV) and is very stable in aqueous solution [5]. However, laccase-catalyzed organic synthesis is preferentially conducted in homogeneous aqueous/organic (A/O) media, i.e., 50% (v/v) aqueous acetone, in order to dissolve hydrophobic substrates and improve the yield and molecular weight of polymer products [6,7]. In A/O media laccase activity towards ABTS and many other substrates is significantly decreased. The stability and recyclability of either enzyme or mediator are also inevitably influenced by the organic cosolvent. It remains unclear how the organic solvent impacts on the laccase-ABTS oxidation. Previous studies showed that the variation of medium polarity adversely changed the enzyme conformation Molecules 2015, 20, 1–7; doi:10.3390/molecules201119672

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and therefore reduced the affinity to substrates [7,8]. There is another possible interpretation that free radicals derived from laccase catalysis could be transformed into their original state in A/O media. However, very few reports are available on the roles of organic solvent in scavenging ABTS radicals. On the other hand, the relatively high cost of ABTS prevents its practical use unless it can be feasibly recovered. The only available process based on ammonium sulfate precipitation [9] is not practical because the subsequent separation is laborious and any residual salt may be harmful to the reuse of ABTS in biosynthesis. In this work we provided a new insight into ABTS radical transformation in A/O media. Thereafter, the feasibility of recovering ABTS using aqueous acetone was discussed. 2. Results and Discussion 2.1. Inhibition of Laccase-Catalyzed ABTS Oxidation by Acetone The Michaelis-Menten constants of laccase reacting with ABTS in aqueous/acetone (Aq/Ac) solutions were listed in Table 1. The apparent binding constant, Km , increased exponentially with increasing acetone content (0%–50%, v/v). Under the same conditions, the maximum rate of oxidation, V max , decreased exponentially with the increasing acetone concentration. The V max measured in 50% (v/v) Aq/Ac was two orders of magnitude lower than that in the reference aqueous solution (Table 1). The catalytic efficiency (V max /Km ) was also exponentially correlated with the acetone contents (Table 1). The V max /Km of laccase in 50% Aq/Ac dropped four orders of magnitude compared to the reference test. Kinetics studies of laccase were not determined in a media containing 60% (v/v) acetone or higher because the initial oxidation velocity was too minimal to be precisely determined and protein precipitation interfered with the test. Table 1. Michaelis-Menten constants of laccase in aqueous media of different acetone contents. Acetone Content (%, v/v)

Km (µM)

V max (µM¨ min´1 ¨ [E]´1 *)

V max /Km (L¨ mg´1 ¨ min´1 )

0 12.5 25 37.5 50

7.67 ˘ 0.44 25.07 ˘ 0.65 58.66 ˘ 2.24 91.99 ˘ 0.99 370.08 ˘ 20.55

42.86 ˘ 0.49 28.76 ˘ 0.45 8.13 ˘ 0.24 4.03 ˘ 0.08 1.94 ˘ 0.04

5.588 1.147 0.139 0.044 0.005

* [E] represents concentration of enzyme, mg¨ L´1 .

The effect of acetone on Km of laccase for ABTS, an ionic substrate, was similar to that reported on C. unicolor laccase and neutral substrates such as syringaldazine and 2,6-dimethoxyphenol; whereas, V max declined linearly with increasing acetone content in the reference article [10]. Instead, V max of C. hirsutus laccase determined using syringaldazine substrate varied exponentially with acetone concentration, while Km has a linear relationship with acetone concentration [11]. The difference may be attributed to different laccase sources and/or substrate properties. Moreover, the testing range of solvent contents may have significant impacts on the correlation results. A previous work by Rodakiewicz-Nowak et al. indicated that the profile of V max towards concentration of water-miscible solvent had been divided into three regions: (1) inhibition region with small decrease in catalytic rate; (2) denaturation region with a dramatic decrease in enzymatic activity; (3) residue region where few enzyme remains active [7]. Thus, the linear inhibition region was not observed possibly due to that the acetone content was too low to inhibit laccase activity considerably, i.e., protein was not denatured. Simple kinetic models like hyperbolic, non-competitive, or uncompetitive were not satisfied for describing the complicated mechanisms of solvent inhibition. Therefore, Rodakiewicz-Nowak et al. used a mixed inhibition model integrating binding constants of solvent molecules with both enzyme and enzyme—substrate complex [7]. It should be noted that hydration of enzyme, a prerequisite step for its catalytic action, was related to the water thermodynamic activity [12]. Organic solvents destructed the protein hydration 2

Molecules 2015, 20, 1–7

shell resulting in protein denaturation [11]. Solvents also influenced substrate-enzyme binding by altering pH, polarity, and/or hydrophobicity of the media [7,13]. Furthermore, the influence of solvent on intermediate was possibly involved in the catalytic process, i.e., destabilizing intermediate radicals and slowing down the conversion of substrates should be counted [14]. 2.2. Scavenging ABTS Molecules 2015, 20,Radicals page–page by Acetone We further evaluated capacity of organic solvent to transform the radicals to the reductive or 2.2. Scavenging ABTSthe Radicals by Acetone a new form byWe a kinetic UV-Vis spectroscopic analysis. Figure 1 showed that ABTS radicals (420 nm) further evaluated the capacity of organic solvent to transform the radicals to the reductive generatedMolecules 2015, 20, page–page  bya laccase nearly transformed into their original stateradicals (340 nm) in 80% (v/v) or new formcatalysis by a kineticwere UV-Vis spectroscopic analysis. Figure 1 showed that ABTS (420 nm) catalysis scavenging were nearly transformed their original (340 nm) in 80% (v/v)in the first Aq/Ac in generated 60 min. byInlaccase particular, of ABTSinto radicals in thestate initial stage (e.g., 2.2. Scavenging ABTS Radicals by Acetone  Aq/Ac in 60 min. In particular, scavenging of ABTS radicals in the initial stage (e.g., in the first 10 min) 10 min) was much faster than that in the subsequent stage (e.g., in the following 50 min).

Absorption Absorption

was We further evaluated the capacity of organic solvent to transform the radicals to the reductive  much faster than that in the subsequent stage (e.g., in the following 50 min). or a new form by a kinetic UV‐Vis spectroscopic analysis. Figure 1 showed that ABTS radicals (420 nm)  1.2 generated by laccase catalysis were nearly transformed into their original state (340 nm) in 80% (v/v)  Mixing time Aq/Ac in 60 min. In particular, scavenging of ABTS radicals in the initial stage (e.g., in the first 10 min)  1.0 10 s was much faster than that in the subsequent stage (e.g., in the following 50 min).  10 min 0.8 1.2

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Figure 1. Time-dependent spectra of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Time-dependent spectra of 2,21 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 0.0 radicals in 80% (v/v) Aq/Ac.

Figure 1. radicals in 80% (v/v) Aq/Ac.

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(ABTS)

500

Attenuation of scavenging velocity withWavelength time is more(nm) clearly shown in Figure   2. Half of ABTS radicals were scavenged in the initial 7 min while 94.5% of radicals were consumed in 60 min. The Figure  1. scavenging Time‐dependent  spectra  of with 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic  (ABTS)  Attenuation ofvelocity velocity time is more clearly shown in acid)  Figure 2. Half decreasing was observed in the tested acetone content. The amount of recovered ABTS was of ABTS radicals in 80% (v/v) Aq/Ac.  radicals were scavenged min while 94.5% of acetone radicals were consumed in 60 min. The greatly dependentin onthe the initial acetone7content, indicating that contributed to the reductive of ABTS radicals.inSuch results in variation of absorption of decreasingtransformation velocity was observed thetransformation tested acetone content. The amount coefficient of recovered ABTS Attenuation of scavenging velocity with time is more clearly shown in Figure 2. Half of ABTS  ABTS at 420 nm (ε420nm) in Aq/Ac. Acetone contents should be taken into consideration when measuring radicals were scavenged in the initial 7 min while 94.5% of radicals were consumed in 60 min. The  was greatly dependent on the acetone content, indicating that acetone contributed to the reductive laccase activity in A/O media. Figure 3 indicates the standard derivation of ε420nm at 1 min was within decreasing velocity was observed in the tested acetone content. The amount of recovered ABTS was  transformation ofthe ABTS Such transformation results in variation of absorption coefficient ±2.5% in mediaradicals. with 0%–25% acetone, while ±6% wasthat  for 25%–50% Aq/Ac. greatly  dependent  on  the  acetone  content,  indicating  acetone  contributed  to  the  reductive 

Percentage of transformed ABTS (%) ABTS (%) Percentage of transformed

of ABTS at 420 nm (ε420nm ) in Aq/Ac. Acetone contents should be taken into consideration when transformation of ABTS radicals. Such transformation results in variation of absorption coefficient of  0 20% 60% measuringABTS at 420 nm (ε laccase activity in A/O media. Figure 3 40% indicates the 80% standard derivation of ε420nm at 420nm) in Aq/Ac. Acetone contents should be taken into consideration when measuring  100  at 1 min was within  1 min waslaccase activity in A/O media. Figure 3 indicates the standard derivation of ε within ˘2.5% in the media with 0%–25% acetone, while ˘6%420nm was for 25%–50% Aq/Ac. ±2.5% in the media with 0%–25% acetone, while ±6% was for 25%–50% Aq/Ac.  80

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Figure 2. Effects of acetone content on kinetic transformation of ABTS radicals.

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Figure 2. Effects of acetone content on kinetic transformation of ABTS radicals. 

Figure 2. Effects of acetone content on kinetic transformation of ABTS radicals.

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-1

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ε at 420 nm ( x 1000, M cm )

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Acetone content (%, v/v) Figure 3. Effects of mixing time on ε of ABTS radicals in Aq/Ac media.

Figure 3. Effects of mixing time on ε of ABTS radicals in Aq/Ac media.

Transformation of ABTS radicals (ABTS·+) also occurred when other organic solvents were used + can be reduced to ABTS instead of acetone.ofABTS by oxidizing broad spectrum of lignin derived were Transformation ABTS radicals (ABTS¨ + ) also occurreda when other organic solvents + alcohols, aromatic aldehydes, ketones as well as many organic acids (e.g., acetate, glyoxylate, andlignin used instead of acetone. ABTS can be reduced to ABTS by oxidizing a broad spectrum of malonate) [15–17]. However, there was no direct evidence showing that acetone or other water-miscible derived alcohols, aromatic aldehydes, ketones as well as many organic acids (e.g., acetate, glyoxylate, organic solvent can be oxidized by ABTS radicals. Our results showed that ABTS·+ can be scavenged and malonate) [15–17]. However, there was no direct evidence showing that acetone or other by a variety of water-miscible organic solvents that follows a capacity order of 1,4-dioxane, acetone, water-miscible organic solvent can be oxidized by ABTS radicals. Our results showed that ABTS¨ + isopropanol, ethanol, methanol, and acetic acid (Table 2) indicating that the polarity of organic can be scavenged by a variety water-miscible organic solvents that follows a capacity order of solvents was a major influenceoffactor. However, the oxidation mechanisms (especially the resulting 1,4-dioxane, ethanol, methanol, and acetic (Table of 2)solvent-induced indicating that the products)acetone, should beisopropanol, further investigated for better understanding theacid phenomena polarity of organic solvents was a major influence factor. However, the oxidation mechanisms radicals scavenging.

(especially the resulting products) should be further investigated for better understanding the ·+ by various water-miscible organic solvents (80% volume percentage). Table Scavenging of ABTS phenomena of 2.solvent-induced radicals scavenging. Reduction Percentage Time for 50% Time for 90% Solventsof ABTS¨ + by various water-miscible organic solvents (80% volume percentage). Table 2. Scavenging at 45 min (%) Reduction (min) Reduction (min) Acetic acid 9.6 ± 0.6 ND * ND Reduction Time for 50% Methanol 39.3 ±Percentage 1.6 83 ± 2.9 346Time ± 4.5 for 90% Solvents at 45 min (%) Reduction (min) Reduction (min) Ethanol 50.5 ± 0.4 41 ± 1.8 209 ± 3.5 Isopropanol 84.5 15 ±ND 0.4 * 51 ± 1.3 ND Acetic acid 9.6 ±˘0.9 0.6 Methanol 39.3±˘1.1 1.6 ˘ 2.9 346 ˘ 4.5 Acetone 88.7 7 ±830.1 47 ± 0.4 Ethanol 50.5±˘0.9 0.4 ˘ 1.8 209 ˘ 3.5 1,4-Dioxane 94.1 2 ±410.1 13 ± 0.7 Isopropanol 84.5 ˘ 0.9 51 ˘ 1.3 * ND: Not detected. 15 ˘ 0.4 Acetone 88.7 ˘ 1.1 7 ˘ 0.1 47 ˘ 0.4 1,4-Dioxane 94.1 ˘ 0.9 2 ˘ 0.1 13 ˘ 0.7 2.3. Recovery of ABTS in Aqueous Acetone Media * ND: Not detected.

The capacity of acetone to scavenge ABTS·+ could possibly provide a new strategy to recover ABTS in reductive state (active one). ABTS·+ can couple with some of the reactants or products and longer incubations often lead toMedia less recovery [9,18]. UV-Vis spectra of recovered ABTS and 2.3. Recovery of ABTS inperiods Aqueous Acetone its regenerated radicals were demonstrated in Figure 4. Specific absorption at 340 nm of reductive + could possibly provide a new strategy to recover The acetone to cycle scavenge ABTS¨ but ABTScapacity appearedofafter the first of recovery, no specific peak at 420 nm or 730 nm appeared. ¨ + can was somestate weak absorbance 400–700 nm, whichwith correlated with ABTSor with slight and ABTSThere in reductive (active one). in ABTS couple some of thefaded reactants products magenta color. This suggests a new form of ABTS was generated. The reaction mixture was turned longer incubations periods often lead to less recovery [9,18]. UV-Vis spectra of recovered ABTS and its to be blue-green radicals after acetonein was removed by evaporation and by laccase.ABTS regenerated radicals were demonstrated Figure 4. Specific absorption at re-oxidized 340 nm of reductive However, the peak of specific absorbance at 420 nm decreased, i.e., around 20.8% of ABTS cannot be appeared after the first cycle of recovery, but no specific peak at 420 nm or 730 nm appeared. There recovered. Our recovery rate was much higher than that using the ammonium sulfate precipitation was some weak absorbance in 400–700 nm, which correlated with faded ABTS with slight magenta method (nearly 70% unrecoverable) [9].

color. This suggests a new form of ABTS was generated. The reaction mixture was turned to be blue-green radicals after acetone was removed by evaporation and re-oxidized by laccase. However, 4 i.e., around 20.8% of ABTS cannot be recovered. the peak of specific absorbance at 420 nm decreased, Our recovery rate was much higher than that using the ammonium sulfate precipitation method (nearly 70% unrecoverable) [9]. 4

Molecules 2015, 20, 1–7 Molecules 2015, 20, page–page

ABTS could be recycled for at least six times under the experimental conditions as shown ABTS recycled forABTS at least six successfully times under the experimental as shown in 2015, 20, page–page in Molecules Figure 5. could Up tobe10% active was retained after sixconditions cycles. The recovered Figure 5. Up to 10% active ABTS was successfully retained after six cycles. The recovered ABTS ABTS exhibited magenta color with a broad absorption band ranging from 300 to 700 nm which ABTS could recycled at Many least six times under the experimental conditions as increased shown from in exhibited magenta color with afor broad absorption band ranging from 700 nm which increased with the be recycling times. authors also reported the300 redtoor purple byproduct Figure 5. recycling Up to 10% active ABTS was successfully retained after six cycles. The recovered ABTS with the times. Many authors also reported the red or purple byproduct from laccaselaccase-catalyzed ABTS oxidation and deduced it to be azodication (ABTS2+ ). This dication was 2+). This exhibited ABTS magenta color with broad absorption band ranging from 300 to 700 nm which increased catalyzed oxidation and adeduced it to be azodication (ABTS dication was characterized characterized as an EPR-silent form with UV absorption maxima at 260 and 294 nm [19]. However, with the recycling times. authors also reported purple byproductSolís-Oba from laccaseas an EPR-silent form withMany UV absorption maxima at 260the andred 294ornm [19]. However, et al., Solís-Oba et al., reported contrary findings that ABTS2+ is colorless. The red or purple byproduct 2+). This dication was characterized catalyzedcontrary ABTS oxidation to be azodication reported findingsand thatdeduced ABTS2+ isit colorless. The red (ABTS or purple byproduct could be associated could be associated to complex formed between the dication and some chemical species present in the as complex an EPR-silent form with UV maxima 260 andspecies 294 nmpresent [19]. However, Solís-Oba et To al., to formed between theabsorption dication and someat chemical in the solution [20]. solution [20]. To verify these conclusions, ABTS liquor after six recycling processes was ultrafiltered 2+ is colorless. reported contrary findings ABTS that ABTS The red orprocesses purple byproduct could be associated verify these conclusions, liquor after six recycling was ultrafiltered against a against a Millipore (10dication kDa). Thesome filter liquor was colorless while the retention to complex formedmembrane between the in the solution [20].liquor To Millipore membrane (10 kDa). The filter and liquor waschemical colorlessspecies while present the retention liquor exhibited 2+ exhibited obvious magenta color, which supports the formation of complex of ABTS and laccase. 2+ verify these conclusions, ABTS liquorthe after six recycling processes wasand ultrafiltered obvious magenta color, which supports formation of complex of ABTS laccase. against a Millipore membrane (10 kDa). The filter liquor was colorless while the retention liquor exhibited 2.5 supports the formation of complex of ABTS2+ and laccase. obvious magenta color, which Original radicals Recovered 1st cycle Reoxidized 1st cycle Original radicals Recovered 7th cycle Recovered 1st cycle Reoxidized 1st cycle Recovered 7th cycle

Absorption Absorption

2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 200 0.0

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200 300 400 500 600 700 800 900 Figure 4. Variation of UV-Vis spectra of recovered ABTS and its radicals. Wavelength (nm) ABTS and its radicals. Figure 4. Variation of UV-Vis spectra of recovered

Figure 4.50 Variation of UV-Vis spectra of recovered ABTS and its radicals.

No enzyme deactivation Deactivation

ActiveActive ABTSABTS (μΜ) (μΜ)

50 40

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40 30 30 20 20 10 10 0 0 0

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0 1 2 3 4 5 6 7 8 Figure 5. Attenuation of active ABTS radicals with recycling times.

Recycling times

Deactivation ofFigure laccase (by heating) improved feasibility of ABTS recycling as shown in 5. Attenuation of active ABTSthe radicals with recycling times. Figure 5. Attenuation of active ABTS radicals with recycling times. Figure 5. A probable reason was that ABTS bound to laccase were released from enzyme. Thus, the Deactivation laccase (by heating) thewith feasibility of For ABTS recycling as shown in released ABTS wasofmore readily recoveredimproved after mixed acetone. control samples (without Deactivation of laccase (by heating) improved the feasibility of ABTS recycling as shown Figure 5. A probable reason wasbound that ABTS boundmay to laccase were released frominenzyme. the in boiling water treatment), ABTS to laccase be precipitated together acetone Thus, solution, Figure 5. led AABTS probable reason was that bound to laccase were released from enzyme. Thus, released was free more readily recovered afterAlmost mixed with acetone. For control samples (without which to fewer molecules forABTS recycling. 80% of laccase activity was recovered afterthe boilingABTS waterwas treatment), ABTS bound to can laccase may be precipitated together insupplementation. acetone solution, released more recovered after mixed with acetone. For control samples (without solvent evaporation. Thereadily residual laccase re-oxidize recovered ABTS without whichwater led to fewer free molecules for recycling. Almost of laccase activity was recovered after Therefore, the laccase/ABTS active state be readily by adding or removing acetone in boiling treatment), ABTS bound tocan laccase may controlled be80% precipitated together in acetone solution, solvent evaporation. The residual laccase can re-oxidize recovered ABTS without supplementation. the media, particular suitable for processAlmost control80% in biosynthesis. which led to which fewer is free molecules for recycling. of laccase activity was recovered after Therefore, the laccase/ABTS active state can be readily controlled by adding or removing acetone in 5 the media, which is particular suitable for process control in biosynthesis. 5 5

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solvent evaporation. The residual laccase can re-oxidize recovered ABTS without supplementation. Therefore, the laccase/ABTS active state can be readily controlled by adding or removing acetone in the media, which is particular suitable for process control in biosynthesis. 3. Experimental Section 3.1. Chemicals and Reagents Laccase (Novozym 51003, Tianjin, China) activity was determined against substrate ABTS at pH 4.0 [4]. ABTS was purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein concentration was determined by the Bradford method using BSA as a standard. Other chemicals were of analytical grade. 3.2. Laccase/ABTS Reaction Reaction of laccase and ABTS was performed in a quartz cuvette (1 ˆ 1 ˆ 4 cm3 ) at 30 ˘ 1 ˝ C. The reaction mixture consisted of 50 mM acetate buffer (pH 4.0), 0.005~0.2 mM ABTS and a certain volume percentage of acetone (see text). The reaction was started by adding a proper amount of laccase which was prescribed to oxidize less than 5% of ABTS in 3 min for measuring Michaelis-Menten constants (Km and V max ). UV-Vis spectrum was determined by using an Agilent 8453 UV-Vis spectrophotometer (Shanghai, China) at a ten-second interval. 3.3. ABTS Recovery Procedure For recovery study, 45 µmol ABTS was converted to blue-green radicals by laccase (100 U) at 30 ˝ C for 1 h. The reaction mixture was boiled for 10 min in order to inactivate laccase. Acetone was next added to the mixture with a final concentration of 80% (v/v). Acetone was removed by vacuum-rotary evaporation after incubation at 30 ˝ C for 60 min. The faded ABTS was re-oxidized with 100 U of fresh laccase. The re-generated ABTS radicals (ABTS¨ + ) with a representative absorbance peak at 750 nm were measured for calculating the recovery of active ABTS. The recovery process was repeated until no blue-green radicals could be generated. 4. Conclusions ABTS cationic radicals were transformed to the reductive form by acetone or other water-miscible organic solvents. We could recover up to 95% ABTS in 80% (v/v) acetone in 60 min. The laccase/ABTS active state was readily manipulated by adjusting acetone concentration in the media. In this study, we provided a simple method to recover an effective mediator that is particularly suitable for process control in biosynthesis. Acknowledgments: The present work was financially supported by the Foundation of State Key Laboratory of Pulp and Paper Engineering (No.2015QN03). Author Contributions: This research was initiated by H.L. at South China University of Technology and S.C. at Michigan State University. H.L. and P.Z. conducted the experimental work together with the assistant of X.W. and J.S. The authors agreed that the research results contributed as a part in P.Z.’s Ph.D. dissertation. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the ABTS radicals and ABTS dication are available from the authors. © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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