Article pubs.acs.org/IECR
Using Ionic Liquid in a Biphasic System to Improve Asymmetric Hydrolysis of Styrene Oxide Catalyzed by Cross-Linked Enzyme Aggregates (CLEAs) of Mung Bean Epoxide Hydrolases Chun-Yang Yu,† Ping Wei,‡ Xiao-Feng Li,† Min-Hua Zong,*,‡ and Wen-Yong Lou*,†,‡ †
State Key Laboratory of Pulp and Paper Engineering and ‡Laboratory of Applied Biocatalysis, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China ABSTRACT: The asymmetric hydrolysis of racemic styrene oxide (SO) to (R)-1-phenyl-1,2-ethanediol using cross-linked enzyme aggregates (CLEAs) of epoxide hydrolases (EHs) from mung bean (mbEHs) was successfully conducted using ionic liquids (ILs) as cosolvents in biphasic systems. Of all the tested ILs, the best results were observed in the biphasic system containing [C4MIM][PF6] with better biocompatibility to the CLEAs of mbEHs. In the [C4MIM][PF6]/buffer biphasic system, it was found that the optimal volume ratio of IL to buffer, reaction temperature, buffer pH, and substrate concentration were 1:5, 40 °C, 7.5, and 120 mM, respectively. Under the optimized reaction conditions, the initial reaction rate, yield, product ee, and E value reached 3.35 mmol/min, 49%, 95.8%, and 151, respectively, which were much higher than the corresponding values reported previously. Furthermore, the CLEAs exhibited markedly enhanced operational stability in a [C4MIM][PF6]-based biphasic system as compared with an n-hexane-based biphasic system. Additionally, the CLEAs of mbEHs-catalyzed process with the IL [C4MIM][PF6] was shown to be feasible on a 500 mL preparative scale, demonstrating their great potential for biosynthesis of chiral ortho-diols.
1. INTRODUCTION Epoxide hydrolases (EHs) are ubiquitous in nature,1−3 and in recent years, use of EHs from different sources (microorganisms, plants, and animal tissues) for enantioselective hydrolytic ring-opening of epoxides to corresponding enantiopure ortho-diols has gained much attraction,4−9 because of their availability, high enantioselectivity and efficiency, mild reaction conditions, lack of requirement of cofactors, and low cost. Two novel EHs that have been discovered from mung bean10,11 were capable of effectively catalyzing enantioconvergent hydrolysis of p-nitrostyrene oxide to (R)-p-nitrophenyl glycol and showed great potential for synthesis of enantiopure chiral vicinal diols that are versatile intermediates for the preparation of pesticides, medicines, and fine chemicals.5,6,12 However, free EHs are usually sensitive to temperature or pH and difficultly recycled and reused. Therefore, immobilized enzymes have been frequently used to overcome these drawbacks.13,14 Compared with carrier-bound immobilization techniques, cross-linked enzyme aggregates (CLEAs) have a prominent advantageconferring the catalysts with high activities, since the activity dilution caused by inert carriers was avoided.15 Besides, the CLEAs technology appears to be superior to cross-linked enzyme crystals due to the simple preparation steps (precipitation and subsequent chemical crosslinking) and satisfactory activity recovery,16−18 thus obviating both the need for a highly purified enzyme and a complicated crystallization step. They also generally exhibit excellent stability.19 Most recently, the preparation and characterization of CLEAs of mung bean EHs (mbEHs) have been reported by our group, and CLEAs of mbEHs exhibited markedly enhanced stability and reusability compared to free mbEHs.20 As a result, CLEAs is becoming increasingly attractive for immobilization of various enzymes including EHs. © 2014 American Chemical Society
Although the EH-catalyzed enantioselective hydrolytic ringopening of epoxides to enantiopure ortho-diols appeared to be very promising and competitive, most epoxides would spontaneously hydrolyze into corresponding diols without any selectivity in an aqueous phase. In addition, epoxides can be dissolved in aqueous solutions only at a very low concentration. The instability and low solubility of epoxides in aqueous phase may result in a remarkable decrease in the product yield,9,21 thus limiting the application of these enzymatic processes on a practical scale. In order to overcome these limitations, the asymmetric hydrolysis of styrene oxide (SO) catalyzed by crude mbEHs in organic solvent/buffer biphasic systems has been examined in our previous study.22 Although an organic solventcontaining biphasic system can partially inhibit the nonenzymatic hydrolysis of SO and thus enhance the product ee, organic solvents showed toxic effect on mbEHs, leading to a clear drop in the activity and stability of enzyme. Subsequently, addition of a small amount of hydrophilic ionic liquids (ILs) with relatively good biocompatibility into the n-hexanecontaining biphasic system reduced the amount of the nonenzymatic hydrolysis and significantly increased the product ee and the initial reaction rate of mbEHs-catalyzed hydrolysis of SO.23 However, it is still difficult to recycle and reuse the free mbEHs even from the IL-containing reaction system. Obviously, it is of great interest to combine the CLEAs of mbEHs with ILs to improve the asymmetric hydrolysis of epoxides, which, to our best knowledge, has been no reported so far. On the other hand, free EH-mediated hydrolytic ringReceived: Revised: Accepted: Published: 7923
November 5, 2013 April 12, 2014 April 16, 2014 April 16, 2014 dx.doi.org/10.1021/ie4037559 | Ind. Eng. Chem. Res. 2014, 53, 7923−7930
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defined as the amount of enzyme required to catalyze the release of 1 μmol PED per min under the given conditions. The activity recovery was calculated from the equation given below:
opening of epoxides in IL-containing systems remains unexplored largely, with only few published accounts.23−26 Herein, we report the utilization of various ILs in a two-phase system to efficiently improve asymmetric hydrolysis of racemic SO to (R)-PED, which is a valuable chiral intermediate for synthesis of nucleoside analogues with antiviral activity,27 catalyzed by CLEAs of mbEHs, and the effects of these ILs on the enzymatic reaction. Also, the efficient CLEAs of mbEHsbased process combined with ILs was evaluated on a preparative scale.
activity recovery (%) =
total activity of CLEAs (U ) total free enzyme activity used for CLEA production (U )
Under the above-described preparation conditions, the specific activity of CLEAs of EHs was around 67 U/g, and the activity recovery recorded about 92%. For measuring the initial reaction rate, samples were taken within the first 10 min of reaction time and the concentration of the formed product PED was determined by HPLC. The initial reaction rate was calculated from the following equation:
2. EXPERIMENTAL SECTION 2.1. Biological and Chemical Materials. Mung beans were purchased from a local supermarket. Racemic SO (98% purity) and (±)-PED (98% purity) were obtained from Guangzhou Qiyun Bioscience Co. Ltd., China. The 15 ILs used in this work, 1-(2′-hydroxyl)ethyl-3-methylimidazolium tetrafluoroborate ([C2OHMIM][BF4]), 1-(2′-hydroxylethyl)-3methylimidazolium trifluoromethanesulfonate ([C2OHMIM] [TfO]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2MIM][BF4]), 1-propyl-3-methylimidazolium tetrafluoroborate ([C3MIM][BF4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4MIM][BF4]), 1-(2′-hydroxylethyl)-3-methylimidazolium hexafluorophosphate ([C2OHMIM][PF6]), 1butyl-3-methylimidazolium saccharinate ([C4MIM][Sac]), 1pentyl-3-methylimidazolium tetrafluoroborate ([C5MIM][BF 4 ]), 3-allyl-1-methylimidazollium tetrafluoroborate ([AMIM][BF4]), 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C2MIM][Tf2N]), 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide([C4MIM][Tf2N]), 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM] [PF6]), 1-pentyl-3-methylimidazolium hexafluorophosphate([C5MIM][PF6]), 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]), and 1-heptyl-3-methylimidazolium hexafluorophosphate ([C7MIM][PF6]), were purchased from Lanzhou Institute of Chemical Physics (China) and were all of over 98% purity. The cholinium threonine ([Ch][Thr]), cholinium lysine ([Ch][Lys]), and cholinium proline ([Ch][Pro]) were prepared by our research group28 and were all of over 99% purity. All other chemicals were from commercial sources and were of analytical grade. 2.2. Preparation of Crude mbEHs. The crude mbEHs were prepared according to the previously reported methods.11,23 2.3. Preparation of CLEAs of mbEHs and Assay of CLEAs’ Activity. The preparation of CLEAs of mbEHs was performed by slight modification of our reported method.20 In a typical experiment, saturated ammonium sulfate (80%, w/v) was used as precipitants and was added dropwise into 1 mL of free crude mbEHs (20 mg) in phosphate buffer (100 mM, pH 7.5). After the mixture was stirred for 30 min at 4 °C for complete precipitation of EHs, glutaraldehyde with a optimal concentration of 20 mM was used as a cross-linking agent and was slowly added to the mixture which was stirred at 4 °C for another 12 h. Then the suspension was centrifuged at 12000 rpm for 10 min at 4 °C. The pellet was washed for three times with phosphate buffer and finally stored in phosphate buffer (100 mM, pH 7.5) at 4 °C. The activity assay of CLEAs was carried out according to the previous method.20 The prepared CLEAs of mbEHs (30 mg) was added to 2 mL phosphate buffer (100 mM, pH 7.5) containing SO (10 mM). The reaction was conducted for 10 min at 35 °C and 220 rpm. One unit of enzyme activity was
initial reaction rate (Vo , mmol/min) = M t /t
where Mt (mmol) is the molar amount of the obtained PED in the initial 10 min and t (min) represents the reaction time (i.e., initial 10 min). 2.4. HPLC Analysis. The product concentration and the product ee were analyzed with Agilent 1100 HPLC equipped with a UV detector at 215 nm using CHIRALPAK OD-H column (4.6 mm × 250 mm, 5 μm, Japan). The mobile phase was a mixture of n-hexane and 2-propanol (95/5, v/v) at 0.4 mL/min. The retention times for (R)-PED and (S)-PED were 29.7 and 32.1 min, respectively. All the data reported here were averages of experiments performed in triplicate with less than 1% standard deviation. 2.5. General Procedure for Asymmetric Hydrolysis of Racemic SO to (R)-PED by CLEAs of mbEHs in ILContaining Biphasic System. Various water-miscible ILs (5% v/v) were added to the biphasic system (8 mL) consisting of n-hexane and phosphate buffer (100 mM, pH 7.5) with the volume ratio of 1:1. 0.79 U of CLEAs was added per milliliter of the aqueous phase. The reaction mixture was contained in a 20 mL Erlenmeyer flask capped with a septum and preincubated in a water-bath shaker at 220 rpm and 40 °C for 5 min. Then, the reactions were initiated by adding SO at 30 mM (the substrate concentrations were based on the total volume of the biphasic system, unless specified otherwise). Various water-immiscible ILs were added to aqueous phosphate buffer (100 mM, pHs 6.5−8.5) to form IL/buffer biphasic systems (8 mL) with different volume ratios (1:9− 1:1). 0.79 U of CLEAs was added per milliliter of the aqueous phase. The reaction mixture was contained in a 20 mL Erlenmeyer flask capped with a septum and preincubated at various shaking rates (180−260 rpm) and temperatures (30− 50 °C) for 5 min. Then, the reactions were initiated by adding SO at different concentrations (30−160 mM). For recording the reaction time course, aliquots (20 μL) were withdrawn every 30 min from each phase when the two phases were clearly separated by stopping the shaking for around 1 min. The product was extracted with 2 × 20 μL ethyl acetate prior to HPLC analysis. Throughout this study, the quantity of the product refers to the sum of that in each phase. 2.6. Reusability of CLEAs of mbEHs in IL-Containing Biphasic System. The reusability of CLEAs of mbEHs during the hydrolysis of SO was conducted in the [C4MIM][PF6]/ buffer biphasic system and also in the n-hexane/buffer biphasic system. Initially, the aliquot of CLEAs (0.79 U per mL of the aqueous phase) was added to the biphasic system (8 mL) of [C4MIM][PF6] (or n-hexane) and phosphate buffer (100 mM, 7924
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two phases. When [C2MIM][BF4] was added, the initial reaction rate reached a maximum level of 0.71 mmol/min, but the yield was only around 23%, indicating that this IL is very toxic to CLEAs of mbEHs. Saccharin-based ILs have been reported as a nontoxic ILs, as saccharin is nontoxic, and has already been approved for human consumption as a nonnutritive sweetener.36 Unfortunately, the tested [C4MIM][Sac] exhibited negative effect on the catalytic performance of CLEAs (Table 1) and gave the relatively poor results of the initial reaction rate (0.07 mmol/min), yield (12.8%), product ee (41%), and E value (3), suggesting that [C4MIM][Sac] has toxic effect on the CLEAs of mbEHs. In the case of the anion [BF 4 ]-based ILs ([C 2 OHMIM][BF 4 ], [AMIM][BF 4 ], [CnMIM][BF4], n = 2−5), when the hydroxyl-functioned cation ([C2OHMIM]) replaced the cation [CnMIM] or [AMIM] in those ILs, the yield (35.6%), product ee (91.3%), and E value (45) of the enzymatic reaction were substantially improved. In order to further investigate whether this improvement was due to the effect of [C2OHMIM] or the combined action of [C2OHMIM] and [BF4], the ILs [C2OHMIM][TfO] and [C2OHMIM][PF6] were tested for the reaction. As shown in Table 1, when [BF4] was replaced by [TfO] or [PF6] as the anion of the IL, the initial reaction rate, yield, product ee, and E value decreased dramatically. Combined with the results using [CnMIM][BF4], it is obvious that the enhancement of the yield, product ee of (R)-PED and E value obtained with the existence of [C2OHMIM][BF4] in the n-hexane/phosphate buffer system results from the combined action of [C2OHMIM] and [BF4]. The choline amino acid ILs could be synthesized simply from choline hydroxide and amino acids by neutralization and, so, were expected to have low toxicity. Therefore, the three choline amino acid ILs ([Ch][Thr], [Ch][Pro], and [Ch][Arg]) were synthesized by our research group28 and were also examined here for the reaction. As can be seen in Table 1, the results were far from expectation. The CLEAs of mbEHs showed the drastic loss of activity in the [Ch][Thr], [Ch][Pro], or [Ch][Arg]containing systems, possibly due to the toxic and inhibitory effects of these three ILs on the CLEAs. Moreover, the catalytic properties of CLEAs might be remarkably influenced by the pH change of reaction system caused by water-miscible ILs. Thus, the pHs of the reaction systems containing the abovementioned water-miscible ILs at a concentration of 5% (v/v) were tested and compared with that in the IL-free system (pH 7.5). It was found that adding [Ch][Thr], [Ch][Pro], and [Ch][Arg] (5% v/v) led to a clear rise in the pH of the reaction system (from 7.5 to 8.7), while adding other water-miscible ILs (5%, v/v) to the reaction system resulted showed no significant variation in the pH (from 7.5 to around 7.2). The CLEAs’ activity was tested when the buffer pH varied from 7.5 to 7.2, and it was found that the activity of CLEAs showed no significant variation but was substantially reduced when the pH increased from 7.5 to 8.7. So it could be concluded that the effect of [Ch][Thr], [Ch][Pro], and [Ch][Arg] on the catalytic performance of CLEAs was also due to the IL-induced significant pH change of the reaction system. In all, adding water-miscible ILs into the n-hexane/buffer biphasic system led to the decrease in the initial reaction rate and E value for CLEAs-mediated hydrolysis of SO and were proved to be disappointing (Table 1). Therefore, n-hexane was replaced by several water-immiscible ILs as the second phase for the CLEAs of mbEHs-catalyzed asymmetric hydrolysis of SO. In our previous study,24 SO was found to be almost
pH 7.5; volume ratio: 1/5) containing 120 mM SO. Then, the reaction was performed at 40 °C and 240 rpm and was repeated for five batches (24 h per batch) under the same reaction conditions. Between batches, CLEAs of mbEHs were recovered from the reaction mixture by centrifugation, washed twice with phosphate buffer (100 mM, pH 7.5), and then added again to a fresh batch of reaction system. The relative activity of CLEAs employed for the first batch was defined as 100%.
3. RESULTS AND DISCUSSION 3.1. Evaluation of Various ILs for Asymmetric Hydrolysis of Racemic SO with CLEAs of mbEHs. A number of investigations have shown that effects of various ILs on biocatalytic reactions varied widely, and in many cases, a significant enhancement in chemical yield and product ee was observed in the presence of ILs compared to conventional solvents.29−31 Also, it is well-known that the catalytic properties of enzymes in IL-containing reaction systems are closely related to the cation and anion types of the used ILs.32,33 Hence, we initially evaluated a wide range of water-miscible or waterimmiscible ILs used in a two-phase system for the asymmetric hydrolysis of SO to (R)-PED catalyzed by the CLEAs of mbEHs, and examined the influence of the cation and anion of the ILs on the CLEAs-based hydrolysis. Several water-miscible ILs as cosolvents were initially added into the aqueous phase of the n-hexane/buffer biphasic system that had proven to be a better reaction medium,22 and their effects on the CLEAs of mbEHs-mediated asymmetric hydrolysis of SO were examined. As evident in Table 1, for the biphasic systems involving [CnMIM][BF4] (n = 2−5), the initial reaction rate, yield of (R)-PED, product ee, and enantiomeric ratio (E value)34 decreased with the elongation of the alkyl chain of the IL cation, which might be due to the increased viscosity of the IL with increasing n value,35 thus limiting the mass transfer of substrate and product between the Table 1. Effect of Various Water-Miscible ILs on Asymmetric Hydrolysis of SO Catalyzed by CLEAs of mbEHsa water-miscible ILs no ILs (control) [C2OHMIM] [BF4] [C2OHMIM] [TfO] [C2MIM][BF4] [C3MIM][BF4] [C4MIM][BF4] [C5MIM][BF4] [AMIM][BF4] [C2OHMIM] [PF6] [C4MIM][Sac] [Ch][Thr] [Ch][Lys] [Ch][Pro]
Vo (10−1 mmol/ min)
yield (%)
product ee (%)
E valueb
8.8 6.0
45.8 35.6
93.5 91.3
69 45
4.8
7.3
15.6
2
7.1 6.0 2.5 1.2 2.5 1.5
23.0 19.7 16.1 4.7 8.3 6.9
89.1 78.0 68.0 26.0 83.0 43.0
23 10 7 2 12 3
0.7 1.8 1.1 0.7
12.8 13.0 1.4 1.0
41.0 92.0 71.0 38.0
3 25 5 3
a Reaction conditions: 8 mL of n-hexane/phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/1) containing various ILs (5% v/v), 40 °C, 220 rpm, 30 mM SO, 0.79 U/mL CLEAs of mbEHs. bThe enantiomeric ratio (E value) was calculated by the following equation: E = ln[1 − C(1 + eediol)]/ln[1 − C(1 − eediol)],34 where C was the conversion extent of substrate and eediol was the enantiomeric excess (ee) of the formed product diol.
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scattered in the IL phase and the majority of the product (R)PED was in the aqueous phase, facilitating timely separation of the product from the substrate and thus enhancing the initial reaction rate and yield of the undergoing reaction. To a certain extent, this avoided the nonenzymatic hydrolysis of SO and consequently enhanced the value of E and the product ee Hence, it is of great interest to study the effects of various water-immiscible ILs on the biohydrolysis reaction with CLEAs of mbEHs. From the data summarized in Table 2 by using six waterimmiscible ILs (with [CnMIM] (n = 2−7) as the cations and Table 2. Effect of Various Water-Immiscible ILs on Asymmetric Hydrolysis of SO Catalyzed by CLEAs of mbEHsa water-immiscible ILs
Vo (10−1 mmol/ min)
yield (%)
product ee (%)
E value
[C2MIM][Tf2N] [C4MIM][Tf2N] [C4MIM][PF6] [C5MIM][PF6] [C6MIM][PF6] [C7MIM][PF6]
8.6 10.8 16.5 14.6 13.1 12.0
47.3 48.2 48.7 48.3 47.4 47.0
86.9 89.1 95.7 93.8 92.5 90.5
37 48 145 92 73 55
Figure 1. Residual activity of CLEAs of mbEHs after being incubated in biphasic systems containing various water-immiscible ILs or nhexane for 24 h. The relative residual activity of CLEAs before incubation (i.e., the original activity) was defined as 100%. Incubation conditions: [C4MIM][PF6] or n-hexane/phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/6), 40 °C, 220 rpm, 0.79 U/mL CLEAs of mbEHs.
significantly that of A. niger EHs immobilized onto Eupergit C (E = 56)38 but also was much higher than that of free EHs from other microorganisms (E value < 30).3,5,9,34 Therefore, it could be inferred that [C4MIM][PF6] displays a lower toxicity to and a higher biocompatibility with CLEAs of mbEHs. Clearly, [C4MIM][PF6] was considered as the most suitable second phase for the enzymatic reaction. 3.2. Optimization of Asymmetric Hydrolysis of SO with CLEAs of mbEHs in [C4MIM][PF6]-Containing Biphasic System. The above-described results clearly showed that the [C4MIM][PF6]/buffer biphasic system was the optimum reaction medium for the enzymatic hydrolysis of SO to (R)-PED catalyzed by CLEAs of mbEHs, and consequently the biocatalytic process was systematically optimized in the presence of [C4MIM][PF6] to further improve the reaction efficiency, in terms of several crucial variables such as volume ratio of [C4MIM][PF6] to buffer (VIL/ Vaq), reaction temperature, buffer pH, shaking rate, and SO concentration. As depicted in Figure 2, the VIL/Vaq value manifested significant influence on the reaction. The initial reaction rate became slower with increasing value of VIL/Vaq from 1:9 to 1:1 (especially from 1:5 to 1:1). Although the decrease of VIL/Vaq value from 1:5 to 1:9 did not result in an obvious alteration of the product yield, the product ee at a yield of around 49% went down drastically from 96.1% to 85.7%, mainly because of the increasing rate of nonenzymatic hydrolysis of SO with the decrease of VIL/Vaq. Increasing value of VIL/Vaq from 1:5 to 1:1 led to a remarkable drop in the product yield from 48.4% to 28.6%. When VIL/Vaq was 1:1, the yield showed no appreciable increase with prolonging reaction time, possibly owing to the inactivation of CLEAs of mbEHs at the interface of the two-phase system. Obviously, the optimum volume ratio of [C4MIM][PF6] to buffer for the CLEAsprompted hydrolysis of SO was 1:5, at which the observed initial reaction rate, yield, and product ee were 1.60 mmol/min, 48.4%, and 96.1%, respectively. The effects of various pHs (6.5−8.5) on the activities of CLEAs of mbEHs were examined for the biohydrolysis of SO to (R)-PED. As illustrated in Figure 3, lowering buffer pH from 7.5 to 6.0 gave rise to the diminished initial reaction rate (1.60
a
Reaction conditions: 8 mL of various ILs/phosphate buffer (100 mM, pH 7.5; volume ratio: 1/6), 40 °C, 220 rpm, 30 mM SO, 0.79 U/mL CLEAs of mbEHs.
[Tf2N] or [PF6]) as the anion) as the second phase, the anion [Tf2N]-based ILs with the imidazolium cations ([C2MIM][Tf2N], [C4MIM][Tf2N]) gave relatively lower initial reaction rate, product ee, and E value compared to the [PF6]-based ILs ([CnMIM][PF6], n = 4−7). Additionally, it was noticed that in the biphasic systems involving [CnMIM][PF6] (n = 4−7), the initial reaction rate, the yield, the product ee, and the E value all decreased with the with the elongation of the alkyl chain of the IL cation (i.e., increasing n value), which could be ascribed to the increased viscosity and toxicity of these ILs with the increased n value.37 The lowered partition coefficients of SO between IL phase and aqueous phase with increasing n value could also contribute to the unsatisfactory results.24 To further understand the influences of all the tested hydrophobic ILs on the hydrolysis with CLEAs, the biocompatibility of ILs with the CLEAs of mbEHs was determined by measuring the residual activity of the CLEAs before and after 24 h exposure to the biphasic systems containing various water-immiscible ILs or nhexane (Figure 1). Except for [C2MIM][Tf2N], other ILs tested, especially [C4MIM][PF6] and [C5MIM][PF6], afforded higher relative residual activity of CLEAs than n-hexane, showing better biocompatibility with CLEAs of mbEHs. Moreover, the relative residual activity of CLEAs with [CnMIM][PF6] (n = 4−7) clearly surpassed that with [C2MIM][Tf2N] or [C4MIM][Tf2N] and significantly decreased with increasing n value, which was in good accordance with the results listed in Table 2. Among all the seven hydrophobic ILs examined, [C4MIM][PF6] manifested the best biocompatibility with the CLEAs of mbEHs and gave the best results in terms of the initial reaction rate (1.65 mmol/min), yield (48.7%), product ee (95.7%), and E value (145), which were much higher than the corresponding values with the nhexane/buffer biphasic system (Table 1). In particular, the E value (E = 145) of CLEAs of mbEHs toward SO in the [C4MIM][PF6]-containing biphasic system not also surpassed 7926
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Figure 4. Effect of reaction temperature on CLEAs of mbEHscatalyzed asymmetric hydrolysis of SO: (Δ) initial reaction rate; (□) maximum yield; (○) product ee. Reaction conditions: [C4MIM][PF6]/phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/5), various temperatures, 220 rpm, 30 mM SO, 0.79 U/mL CLEAs of mbEHs.
Figure 2. Effect of volume ratio of IL to buffer on CLEAs of mbEHscatalyzed asymmetric hydrolysis of SO: (Δ) initial reaction rate; (□) maximum yield; (○) product ee. Reaction conditions: [C4MIM][PF6]/phosphate buffer (100 mM, pH 7.5) (various volume ratios), 40 °C, 220 rpm, 30 mM SO, 0.79 U/mL CLEAs.
SO at higher temperatures. So 40 °C was chosen as the optimum reaction temperature for the biohydrolysis. Owing to the high viscosity of ILs, the [C4MIM][PF6]containing biphasic system can limit the mass transfer of the substrate and the product to and from CLEAs of mbEHs. Therefore, it is also of significance to investigate the impact of shaking rate on the reaction. It was found that the reaction sped up remarkably with increasing shaking rate up to 240 rpm (from 1.15 to 1.89 mmol/min), indicating that the mass transfer was the rate-limiting step for the CLEAs-catalyzed process in [C4MIM][PF6]/buffer biphasic system (Figure 5). Both the product yield and the product ee were also increased to a certain extent, possibly due to the improved enzymatic rate relative to nonenzymatic rate. The optimal shaking rate was found to be 240 rpm, above which the enzymatic process proceeded slowly with further rise in shaking rate, in that the Figure 3. Effect of buffer pH on CLEAs of mbEHs-catalyzed asymmetric hydrolysis of SO: (Δ) initial reaction rate; (□) maximum yield; (○) product ee. Reaction conditions: [C4MIM][PF6]/ phosphate buffer (100 mM, various pHs) (volume ratio: 1/5), 40 °C, 220 rpm, 30 mM SO, 0.79 U/mL CLEAs of mbEHs.
vs 1.30 mmol/min), yield (around 49% vs 45%), and product ee (96.1% vs 83.8%), implying that the CLEAs of mbEHs markedly lost their activities at buffer pHs below 7.5. In addition, when buffer pH was raised from 7.5 to 8.5, similarly, the initial reaction rate and the product ee decreased significantly. Thus, the optimal buffer pH was shown to be 7.5. As can be seen in Figure 4, reaction temperature showed a tremendous influence on the CLEAs of mbEHs-catalyzed hydrolysis of SO. The initial reaction rate increased from 1.18 to 1.60 mmol/min with increasing reaction temperature from 30 to 40 °C, and the obtained product yield correspondingly increased from 41.5 to 49.0%. The product ee showed a slight decline. Further increase in temperature from 40 to 50 °C, however, led to a significant drop in initial reaction rate (1.08 mmol/min), product yield (36.9%), and product ee (86.4%), which might be attributable to the partial inactivation of CLEAs of mbEHs and the acceleration of nonenzymatic hydrolysis of
Figure 5. Effect of shaking rate on CLEAs of mbEHs-catalyzed asymmetric hydrolysis of SO: (Δ) initial reaction rate; (□) maximum yield; (○) product ee. Reaction conditions: [C4MIM][PF6]/ phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/5), 40 °C, various shaking rates, 30 mM SO, 0.79 U/mL CLEAs of mbEHs. 7927
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CLEAs of mbEHs were recovered by centrifugation, washed with aqueous buffer, and then reused in the next run. As shown in Figure 7, the CLEAs remained around 92% and 70% of their
adhering of a small part of mbEHs to the internal wall of the reactor was observed at a relatively shaking rate (260 rpm above) and resulted in partial loss of total activities of CLEAs. Thus, subsequent studies were conducted at a shaking rate of 240 rpm. Figure 6 depicted the significant influence of substrate concentration on the CLEAs of mbEHs-catalyzed hydrolysis of
Figure 7. Reusability of CLEAs in [C4MIM][PF6] or n-hexane containing biphasic system. The relative activity of CLEAs employed for the first batch was defined as 100%. Reaction conditions: [C4MIM][PF6] or n-hexane/phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/5), 40 °C, 240 rpm, 120 mM SO, 0.79 U/mL CLEAs.
Figure 6. Effect of substrate concentration on CLEAs of mbEHscatalyzed asymmetric hydrolysis of SO: (Δ) initial reaction rate; (□) maximum yield; (○) product ee. Reaction conditions: [C4MIM][PF6]/phosphate buffer (100 mM, pH 7.5) (volume ratio: 1/5), 40 °C, 240 rpm, 30−160 mM SO, 0.79 U/mL CLEAs of mbEHs.
original activity after being used repeatedly for three and five batches (24 h per batch), respectively, in the [C4MIM][PF6]/ buffer biphasic system. In contrast, the CLEAs retained only about 62% and 31%, respectively, of the relative activity after being reused for three and five batches (24 h per batch) in the n-hexane/buffer biphasic system. The above-described results clearly showed that the operational stability of the CLEAs of mbEHs was markedly enhanced in the presence of [C4MIM][PF6] compared to n-hexane. The relatively better biocompatibility of the IL [C4MIM][PF6] with mbEHs and its excellent solvent properties for the toxic substrate and product could partly account for the observations. The improved interactions between the IL and the CLEAs may also result in good operational stability of mbEHs in the [C4MIM][PF6]/buffer biphasic system. In addition, the coating and protection of the CLEAs by the IL may contribute to the good stability of the enzyme. 3.4. Preparative Scale Asymmetric Hydrolysis of SO with CLEAs of mbEHs. To further demonstrate the feasibility of applying ILs to practical use, the experiment of the asymmetric hydrolysis of SO to (R)-PED with the CLEAs of mbEHs was carried out on a 500 mL preparative scale under the optimized reaction conditions stated above. The yield of the isolated (R)-PED and the product ee obtained were 49.1% and 94.6%, respectively, after reaction for 24 h. Since the substrate concentration in the [C4MIM][PF6]/buffer biphasic system was much higher than that in the aqueous system (120 vs 5 mM) or the n-hexane/buffer biphasic system (120 vs 30 mM), the biocatalytic process with [C4MIM][PF6] and CLEAs of mbEHs became more efficient and facilitated the industrial production of valuable (R)-PED. Besides, the phases could be separated readily by centrifugation and the product (R)-PED can be easily recovered from the reaction system. So the CLEAs-mediated hydrolysis of SO to (R)-PED in the
SO carried out in [C4MIM][PF6]-based biphasic system. The enzymatic reaction accelerated markedly with increasing SO concentration up to 120 mM, while the product yield and the product ee showed only marginal variations, keeping more than 49% and 95%, respectively. Further raising substrate concentration above 120 mM resulted in a significant drop in the initial reaction rate, the product yield, and the product ee, possibly owing to the increasing inhibitory effect of high substrate concentrations in the [C4MIM][PF6]/buffer biphasic system on the CLEAs of mbEHs. Consequently, the optimal substrate concentration in the [C4MIM][PF6]-based biphasic system was 120 mM, which was 4.0-fold higher than that in the n-hexane-based biphasic system (120 mM vs 30 mM). For the CLEAs of mbEHs-mediated asymmetric hydrolysis of SO conducted in the [C4MIM][PF6]-based biphasic system, the optimal volume ratio of [C4MIM][PF6] to buffer, reaction temperature, buffer pH, shaking rate, and substrate concentration were found to be 1/5, 40 °C, 7.5, 240 rpm, and 120 mM, respectively. Under the optimized conditions, the initial reaction rate, product yield, product ee, and E value were 3.35 mmol/min, 49.4%, 95.8%, and 151, respectively, which were higher than the corresponding values reported previously.5,9,38 Obviously, the use of the [C4MIM][PF6]-containing biphasic system as reaction medium was able to significantly improve the reaction efficiency of hydrolysis of SO with CLEAs of mbEHs. 3.3. Reusability of CLEAs of mbEHs in a [C4MIM][PF6]/ Buffer Biphasic System. In order to assess the operational stability of the CLEAs of mbEHs in the presence of ILs, the reuse of the CLEAs was comparatively investigated by using the hydrolysis of SO as a model reaction in the [C4MIM][PF6]/ buffer biphasic system and in the n-hexane/buffer biphasic system. Between each cycle of the enzymatic reaction, the 7928
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Sphingopyxis sp. exhibiting a novel epoxide hydrolase activity. Biotechnol. Lett. 2013, 36 (2), 1−6. (10) Xu, W.; Xu, J. H.; Pan, J.; Gu, Q.; Wu, X. Y. Enantioconvergent hydrolysis of styrene epoxides by newly discovered epoxide hydrolases in mung bean. Org. Lett. 2006, 8 (8), 1737−1740. (11) Devi, A. V.; Lahari, C.; Swarnalatha, L.; Fadnavis, N. W. Gelozymes in organic synthesis. Part IV: Resolution of glycidate esters with crude mung bean (Phaseolus radiatus) epoxide hydrolase immobilized in gelatin matrix. Tetrahedron: Asym. 2008, 19 (9), 1139−1144. (12) Bala, N.; Chimni, S. S. Recent developments in the asymmetric hydrolytic ring opening of epoxides catalysed by microbial epoxide hydrolase. Tetrahedron: Asym. 2010, 21 (24), 2879−2898. (13) Tran, D. N.; Balkus, K. J. Perspective of recent progress in immobilization of enzymes. ACS Catal. 2011, 1 (8), 956−968. (14) Hartmann, M.; Kostrov, X. Immobilization of enzymes on porous silicas: benefits and challenges. Chem. Soc. Rev. 2013, 42 (15), 6277−6289. (15) Sheldon, R. A.; van Pelt, S.; Kanbak-Aksu, S.; Rasmussen, J. A.; Janssen, M. H. A. Cross-linked enzyme aggregates (CLEAs) in organic synthesis. Aldrichimica Acta 2013, 46 (3), 81−93. (16) Sulek, F.; Fernandez, D. P.; Knez, Z.; Habulin, M.; Sheldon, R. A. Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs). Process Biochem. 2011, 46 (3), 765−769. (17) Roessl, U.; Nahalka, J.; Nidetzky, B. Carrier-free immobilized enzymes for biocatalysis. Biotechnol. Lett. 2010, 32 (3), 341−350. (18) Kartal, F.; Janssen, M. H. A.; Hollmann, F.; Sheldon, R. A.; KIlInc, A. Improved esterification activity of Candida rugosa lipase in organic solvent by immobilization as cross-linked enzyme aggregates (CLEAs). J. Mol. Catal. B: Enzym. 2011, 71 (3−4), 85−89. (19) Sheldon, R. A. Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs). Appl. Microbiol. Biotechnol. 2011, 92 (3), 467−477. (20) Yu, C. Y.; Li, X. F.; Lou, W. Y.; Zong, M. H. Cross-linked enzyme aggregates of mung bean epoxide hydrolases: a highly active, stable and recyclable biocatalyst for asymmetric hydrolysis of epoxides. J. Biotechnol. 2013, 166 (1−2), 12−19. (21) Kang, S. G.; Woo, J. H.; Hwang, Y. O.; Kang, J. H.; Lee, H. S.; Kim, S. J. Enantioselective hydrolysis of racemic epichlorohydrin using an epoxide hydrolase from Novosphingobium aromaticivorans. J. Biosci. Bioeng. 2010, 110 (3), 295−297. (22) Chen, W. J.; Lou, W. Y.; Wang, X. T.; Zong, M. H. Asymmetric hydrolysis of styrene oxide catalyzed by mung bean epoxide hydrolase in organic solvent/buffer biphasic system. Chin. J. Catal. 2011, 32 (9), 1557−1563. (23) Chen, W. J.; Lou, W. Y.; Yu, C. Y.; Wu, H.; Zong, M. H. Use of hydrophilic ionic liquids in a two-phase system to improve mung bean epoxide hydrolases-mediated asymmetric hydrolysis of styrene oxide. J. Biotechnol. 2012, 162 (2), 183−190. (24) Chen, W. J.; Lou, W. Y.; Zong, M. H. Efficient asymmetric hydrolysis of styrene oxide catalyzed by mung bean epoxide hydrolases in ionic liquid-based biphasic systems. Bioresour. Technol. 2012, 115, 58−62. (25) Chiappe, C.; Leandri, E.; Lucchesi, S.; Pieraccini, D.; Hammock, B. D.; Morisseau, C. Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-β-methylstyrene oxide catalyzed by soluble epoxide hydrolase. J. Mol. Catal. B: Enzym. 2004, 27 (4), 243−248. (26) Chiappe, C.; Leandri, E.; Hammock, B. D.; Morisseau, C. Effect of ionic liquids on epoxide hydrolase-catalyzed synthesis of chiral 1,2diols. Green Chem. 2007, 9 (2), 162−168. (27) Jia, X.; Xu, Y.; Li, Z. Regio- and stereoselective concurrent oxidations with whole cell biocatalyst: simple and green syntheses of enantiopure 1,2-diols via oxidative kinetic resolution. ACS Catal. 2011, 1 (6), 591−596. (28) Liu, Q. P.; Hou, X. D.; Li, N.; Zong, M. H. Ionic liquids from renewable biomaterials: synthesis, characterization and application in the pretreatment of biomass. Green Chem. 2012, 14 (2), 304−307. (29) Jia, R.; Hu, Y.; Liu, L.; Jiang, L.; Zou, B.; Huang, H. Enhancing catalytic performance of porcine pancreatic lipase by covalent
[C4MIM][PF6]/buffer biphasic system is believed to be promising and competitive.
4. CONCLUSIONS The asymmetric hydrolysis of SO to (R)-PED with CLEAs of mbEHs was successfully conducted in IL-containing biphasic systems. Various ILs exerted significant but different influences on the biohydrolysis reaction. The nature of the cation and especially the anion components of the ILs showed marked effect on the reaction. The good catalytic performance of the CLEAs and the effective inhibition of nonenzymatic hydrolysis were observed in the [C4MIM][PF6]/buffer biphasic system. The CLEAs of mbEHs-catalyzed process with the IL [C4MIM][PF6] is promising for industrial production of chiral vicinal diols.
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-20-22236669. Fax: +86-20-22236669. E-mail address:
[email protected] (W.Y.L.). *E-mail address:
[email protected] (M.H.Z.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We wish to thank the National Science Found for Excellent Young Scholars (21222606), the State Key Program of National Natural Science Foundation of China (21336002), the National Natural Science Foundation of China (21376096), the Key Program of Guangdong Natural Science Foundation (S2013020013049), and the Fundamental Research Funds for SCUT (2013ZG0003) for partially funding this work.
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REFERENCES
(1) Solares, F. L.; Mateo, C. Improvement of the epoxide hydrolase properties for the enantioselective hydrolysis of epoxides. Cur. Org. Chem. 2013, 17 (7), 744−755. (2) Lin, H.; Liu, J. Y.; Wang, H. B.; Ahmed, A. A. Q.; Wu, Z. L. Biocatalysis as an alternative for the production of chiral epoxides: a comparative review. J. Mol. Catal. B: Enzym. 2011, 72 (4), 77−89. (3) Sareen, D.; Kumar, R. Prospecting for efficient enantioselective epoxide hydrolases. Indian J. Biotechnol. 2011, 10 (2), 161−177. (4) Karboune, S.; Archelas, A.; Baratti, J. C. Free and immobilized Aspergillus niger epoxide hydrolase-catalyzed hydrolytic kinetic resolution of racemic p-chlorostyrene oxide in a neat organic solvent medium. Process Biochem. 2010, 45 (2), 210−216. (5) Zhao, J.; Chu, Y. Y.; Li, A. T.; Ju, X.; Kong, X. D.; Pan, J.; Tang, Y.; Xu, J. H. An unusual (R)-selective epoxide hydrolase with high activity for facile preparation of enantiopure glycidyl ethers. Adv. Synth. Catal. 2011, 353 (9), 1510−1518. (6) Carlsson, A. J.; Bauer, P.; Ma, H.; Widersten, M. Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity. Biochemistry 2012, 51 (38), 7627−7637. (7) Beloti, L. L.; Costa, B. Z.; Toledo, M. A.; Santos, C. A.; Crucello, A.; Fávaro, M. T. A novel and enantioselective epoxide hydrolase from Aspergillu sbrasiliensis CCT 1435: purification and characterization. Protein Expres. Purif. 2013, 91 (2), 175−183. (8) Jin, H. X.; Liu, Z. Q.; Hu, Z. C.; Zheng, Y. G. Biosynthesis of (R)epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase. Eng. Life Sci. 2013, 4 (33), 385−392. (9) Woo, J. H.; Lee, E. Y. Enantioselective hydrolysis of racemic styrene oxide and its substituted derivatives using newly-isolated 7929
dx.doi.org/10.1021/ie4037559 | Ind. Eng. Chem. Res. 2014, 53, 7923−7930
Industrial & Engineering Chemistry Research
Article
modification using functional ionic liquids. ACS Catal. 2013, 9 (3), 976−1983. (30) Jaeger, V. W.; Pfaendtner, J. Structure, dynamics, and activity of xylanase solvated in binary mixtures of ionic liquid and water. ACS Chem. Biol. 2013, 6 (8), 1179−1186. (31) Xiao, Z. J.; Du, P. X.; Lou, W. Y.; Wu, H.; Zong, M. H. Using water-miscible ionic liquids to improve the biocatalytic anti-Prelog asymmetric reduction of prochiral ketones with whole cells of Acetobacter sp. CCTCC M209061. Chem. Eng. Sci. 2012, 84, 695−705. (32) De Winter, K.; Verlinden, K.; Křen, V.; Weignerová, L.; Soetaert, W.; Desmet, T. Ionic liquids as cosolvents for glycosylation by sucrose phosphorylase: balancing acceptor solubility and enzyme stability. Green Chem. 2013, 15 (7), 1949−1955. (33) Lai, J. Q.; Li, Z.; Lv, Y. H.; Yang, Z. Specific ion effects of ionic liquids on enzyme activity and stability. Green Chem. 2011, 13 (7), 1860−1868. (34) Woo, J. H.; Lee, E. Y. Enantioselective hydrolysis of racemic styrene oxide and its substituted derivatives using newly-isolated Sphingopyxis sp. exhibiting a novel epoxide hydrolase activity. Biotechnol. Lett. 2014, 36 (2), 357−362. (35) Latala, A.; Nedzi, M.; Stepnowski, P. Toxicity of imidazolium and pyridinium based ionic liquids towards algae: Chlorella vulgaris, Oocystis submarina (green algae) and Cyclotella meneghiniana, Skeletonema marinoi (diatoms). Green Chem. 2009, 11 (4), 580−588. (36) Nockemann, P.; Thijs, B.; Driesen, K.; Janssen, C. R.; Van Hecke, K.; Van Meervelt, L.; Kossmann, S.; Kirchner, B.; Binnemans, K. Choline saccharinate and choline acesulfamate: ionic liquids with low toxicities. J. Phys. Chem. B 2007, 111 (19), 5254−5263. (37) Wang, W.; Zong, M. H.; Lou, W. Y. Use of an ionic liquid to improve asymmetric reduction of 4 ′-methoxyacetophenone catalyzed by immobilized Rhodotorula sp AS2.2241 cells. J. Mol. Catal. B: Enzym. 2009, 56 (1), 70−76. (38) Yildirim, D.; Tukel, S. S.; Alptekin, O.; Alagoz, D. Immobilized Aspergillus niger epoxide hydrolases: cost-effective biocatalysts for the preparation of enantiopure styrene oxide, propylene oxide and epichlorohydrin. J. Mol. Catal. B: Enzym. 2013, 88, 84−90.
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