Highly Active Nanobiocatalyst from Lipase Noncovalently Immobilized

Research Article pubs.acs.org/journal/ascecg

Highly Active Nanobiocatalyst from Lipase Noncovalently Immobilized on Multiwalled Carbon Nanotubes for Baeyer−Villiger Synthesis of Lactones Magdalena Markiton,† Sławomir Boncel,‡ Dawid Janas,‡,§ and Anna Chrobok*,† †

Department of Chemical Organic Technology and Petrochemistry and ‡Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland § Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Rd, Cambridge CB3 0FS, United Kingdom S Supporting Information *

ABSTRACT: A new method for the chemo-enzymatic Baeyer−Villiger oxidation of cyclic ketones to lactones in the presence of a new heterogeneous nanobiocatalyst consisting of Candida antarctica lipase B immobilized on multiwalled carbon nanotubes (MWCNTs) has been developed. To ensure safety and meet the contemporary environmental criteria nanobiocatalyst was used for the in situ generation of peracid, thereby avoiding the direct handling of dangerous peroxy substance. The reaction was carried out under mild conditions at 20−40 °C using 30% aq H2O2 as the primary oxidant, with octanoic acid as the precursor of peracid. The influence of the reaction parameters and various carbon materials as supports were studied. The activities of the new biocatalysts were compared with the benchmark Novozyme-435. Recycling studies demonstrated the possibility of utilizing the most active MWCNTs-lipase biocatalyst five times without any significant loss of activity. The main advantage of this study is the superior activity of the new nanobiocatalyst, what caused a significant reduction of reaction times compared to those previously reported in the literature. KEYWORDS: Immobilization of lipase, Multiwalled carbon nanotubes, Chemo-enzymatic Baeyer−Villiger oxidation, Lactones

■

on acrylic resin (commercially available as Novozyme-435),4 CALB immobilized as cross-linked enzyme aggregates (CLEA),5 perhydrolase immobilized as CLEA, 6 CALB incorporated into mesoporous silica materials,7 or the native form of acyltransferase and CALB.8 Studies showed that the activities of CALB immobilized on silica or CLEA were 2−4 times higher than equal amounts of CALB immobilized on acrylic resin (Novozyme-435).5−7 The oxidation of ketones by the peracid generated in situ in the reaction system catalyzed by Novozyme-435 could be significantly accelerated by the presence of ionic liquids.9,10 To illustrate the practical application, we presented the possibility of recycling the biocatalyst based on mesoporous silica.7 However, the cost of preparing the biocatalyst based on mesoporous silica materials modified with organosilanes is far too high for industrial application. Recently, nanoscale materials have opened up opportunities in the field of nanobiocatalysis.11−14 Carbon nanomaterials have been described as versatile supports for enzyme immobilization due to their small size, large surface area,

INTRODUCTION Increasingly stringent environmental regulations of industrial processes with regard to safety and waste disposal have compelled the modification of a number of oxidation processes, including the Baeyer−Villiger oxidation of cyclic ketones to lactones. The synthesis of lactones is of significant interest in organic chemistry due to the wide range of possible applications, e.g., in the synthesis of antibiotics, steroids, pheromones, and monomers for polymerization.1 E factor increases dramatically on going downstream from bulk to fine chemicals and pharmaceuticals. Large E factors are mainly a consequence of multistep syntheses and the use of classical stoichiometric reagents, such as oxidants, acids, and bases, resulting in the generation of amounts of waste.2 Lactones are fine chemicals manufactured mainly by the Baeyer−Villiger oxidation of cyclic ketones with peracids, which are expensive and/or hazardous (shock sensitive).3 To ensure safety and meet all of the contemporary environmental criteria, enzymes can be used for the in situ generation of peracids. The chemo-enzymatic approach involves the oxidation of long- or medium-chain carboxylic acids with H2O2 to generate peracids, which are later used to oxidize ketones to lactones. To date, the former reaction can be effectively catalyzed by enzymes in various forms: Candida antarctica lipase B (CALB) immobilized © 2016 American Chemical Society

Received: October 8, 2016 Revised: December 20, 2016 Published: December 29, 2016 1685

DOI: 10.1021/acssuschemeng.6b02433 ACS Sustainable Chem. Eng. 2017, 5, 1685−1691

Research Article

ACS Sustainable Chemistry & Engineering

Transmission electron microscopy (TEM) images were acquired on a FEI Osiris system (200 kV acceleration voltage) equipped with a high-brightness field emission gun (X-FEG). The specimens were dispersed in ethanol and deposited onto Cu grids for imaging. Synthetic Procedures. Synthesis of Pristine MWCNTs, NMWCNTs, Fe-MWCNTs, and C10H21O-Nanocyl NC7000. Pristine MWCNTs were synthesized via catalytic chemical vapor deposition (cCVD) using a slightly modified protocol.24 N-MWCNTs were grown according to a previously described protocol.25 C10H21O-Nanocyl NC7000 was synthesized from prefunctionalized hydroxyl Nanocyl NC7000 MWCNTs obtained via a known procedure 26 and subsequently alkylated using n-decyl chloride (for details see the SI). Immobilization of CALB on the Surface of the Carbon Materials. An immobilization step was performed using a simple physical adsorption technique according to the procedure described in the literature27 (for details see the SI). General Procedures for the Baeyer−Villiger Oxidation of Cyclic Ketones in the Presence of the Synthesized (nano)Biocatalysts. A ketone (0.25 mmol) was introduced into a 25 mL round-bottom flask, to which the (nano)biocatalyst (0.005−0.030 g), octanoic acid (0.50− 6.25 mmol), toluene (0−0.5 mL), and n-decane (0.10 mmol, external standard) were subsequently added. Then, H2O2 (0.25−1.00 mmol) was added. The flask was sealed with a septum and mixed in a thermostatic electric shaker (±0.5 °C) with orbital stirring at 250 rpm at 20−40 °C for 1−20 h, depending on the reaction rate. Periodically, during the reaction, 10 μL of the samples diluted with methylene chloride were collected to monitor the progress of the reaction using GC. When the reaction was completed, the catalyst was filtered off and washed with toluene (5 mL). The filtrate was extracted with 10% NaHCO3 solution in water (3 × 5 mL). All collected water phases were then extracted with methylene chloride (3 × 10 mL). Combined organic phases were dried over anhydrous MgSO4 and, then, were concentrated and purified by column chromatography with a bed of Al2O3 (with methylene chloride as the eluent). The yields of the lactones were in the range of 86−95%. Recycling of the Nanobiocatalyst. In the experiments in which the nanobiocatalyst was recycled, the reactions were scaled up by a factor of 5. After completion of the reaction, the nanobiocatalyst was filtered off, washed with toluene (5 mL) and dried at 5 °C under vacuum for 24 h.

mechanical and thermal stability and other unique properties. The simple combination of a nanoscale support and enzyme has led to a much higher enzyme loading and, more importantly, increased enzyme stability.15 Among the various nanostructured materials, such as nanoparticles, nanofibers, or carbon nanotubes (CNTs), the latter are considered particularly promising by researchers. Two main types of CNTs, single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) have been used to immobilize enzymes. SWCNTs are attractive because of their higher surface area, but MWCNTs are desirable because of their easier dispersibility and environmental16 and health safety17 as well as lower cost. Particularly, MWCNTs are considered as less susceptible than SWCNTs to be airborne,18 are degradable by peroxidases19 and noncytotoxic when functionalized with hydrophilic groups20 or biomolecules.21 Noncovalent and covalent methods have been reported for the immobilization of various enzymes on CNTs.22 The economy of processes utilizing CNTs has become increasingly favorable as the prices of industrial-grade MWCNTs have dropped to 100$ per 1 kg (Nanocyl NC7000 MWCNTs). To the best of our knowledge, CNT/ lipase hybrids have not been applied in chemo-enzymatic Baeyer−Villiger oxidation until now. The fairly long reaction times required, modest yields of lactones, sometimes poor stability of the biocatalyst in the presence of H2O2, and availability at low cost remain major challenges to overcome in chemo-enzymatic Baeyer−Villiger reaction. The aim of this work was to design a high-performance and recyclable biocatalyst for the chemo-enzymatic Baeyer−Villiger reaction. We propose a generic approach to nanobiocatalysis as a replacement of hazardous peracids in industrially relevant oxidation processes in which the challenge is to generate both environmentally and economically sustainable processes.

■

■

EXPERIMENTAL SECTION

RESULTS AND DISCUSSION Selection and Characterization of the Support for Candida antarctica Lipase B (CALB). For the preparation of the heterogeneous (nano)biocatalysts, the following multiwalled carbon nanotubes (MWCNTs), pristine and surfacemodified (functionalization with n-decyloxy groups or nitrogen doping, N-MWCNTs) were used: commercially available Nanocyl NC7000 (MWCNTs) and C 10 H 21 O-Nanocyl NC7000 (Nanocyl NC7000 modified with n-decyloxy groups) and the unmodified and modified (nitrogen-doped and ironencapsulated) MWCNTs prepared in our laboratory according to the procedure described elsewhere (see Experimental Section and Materials and Methods). Functionalization of the MWCNTs was performed to examine whether lipophilic alkyl groups or N-doping of the nanotube walls (by carbon-to-nitrogen replacement) will affect the interactions between the enzyme and nanotube surface. Additionally, active carbon and graphite, as other carbon-based supports, were selected for comparison. Importantly for cCVD-derived CNTs, their integral feature is the presence of core-encapsulated ferromagnetic iron-based nanoparticles which are typically irremovable under lower technological and biotechnological regimes. This characteristic allows for easy removal of the nanobiocatalysts from the postreaction mixtures by means of a magnetic field, which is a significant advantage in light of the processing economy.

Materials and Methods. All substances (ketones, phosphate buffer, octanoic acid, toluene and aqueous solution of Candida antarctica lipase B) were purchased from Sigma-Aldrich. Nanocyl NC7000 MWCNTs was purchased from Nanocyl (Belgium), and active carbon and graphite were commercial materials obtained from Avantor Performance Materials (Poland) and the United Quantum Factory (Poland), respectively. Substituted cyclobutanones (3-phenylcyclobutanone and 3-butylcyclobutanone) were synthesized using a standard two-step procedure: a [2 + 2] cycloaddition of dichloroketene to the appropriate vinyl derivative, followed by reduction of the resulting dichloroketone with zinc in acetic acid.23 GC analyses were performed using a PerkinElmer Clarus 500 equipped with an SPB-5 column (30 m × 0.2 mm × 0.2 μm film; see Supporting Information (SI), Table S1). 1H NMR spectra were recorded at 300 or 600 MHz, and 13C NMR spectra were recorded at 75 or 150 MHz (Varian system; see SI). The lipase loadings on the surface of the carbon materials were determined by thermogravimetry (TGA) using a Mettler Toledo STAR851 thermobalance. Samples of approximately 20 mg were heated from 25 to 800 °C at a rate of 20 °C/min in standard 70 μL Pt crucibles under a dynamic nitrogen flow of 100 mL/min. The TG, DTG, and DTA curves were recorded (see SI, Figure S4−S11). Nitrogen adsorption/desorption isotherms for the carbon materials were obtained using a Micrometrics ASAP 2420 M instrument at −196 °C to calculate their specific surface area (SBET) and pore volume. The size of the pores was obtained using the Barrett−Joyner− Halenda (BJH) method with the Kruk−Jaroniec−Sayari correction. Prior to the experiments, the samples were outgassed at 200 °C and 1.33 × 10−3 Pa for 5 h. 1686


Research Article


Next, screening studies of the most active (nano)biocatalyst per mass unit of the final (nano)biocatalyst were performed using the model reaction of the chemo-enzymatic oxidation of 2-methylcyclohexanone (Scheme 1). The applied method

First, the surface properties of the above materials were determined. The highest surface area was measured for active carbon, and the second highest was found for unmodified Nanocyl NC7000 MWCNTs (outer diameter of 9.5 nm, a fewwall structure, length of 1.5 μm; Table 1). After functionaliza-

Scheme 1. Chemoenzymatic Baeyer−Villiger Oxidation of 2Methylcyclohexanone

Table 1. Characterization of (nano)Carbon-Based Supports and (nano)Biocatalysts support Nanocyl NC7000 C10H21O-Nanocyl NC7000 MWCNTs N-MWCNTs Fe-MWCNTs active carbon graphite

SBET, m2/g

pore volume, cm3/g

CALB loading, wt %

253 177

1.06 1.37

19.8 15.1

34 39 46 1158 3

0.11 0.15 0.14 0.43 0.01

6.0 9.5 9.6 16.9 2.0

involved the heterogeneous (nano)biocatalyst for peracid formation, 30% aq. H2O2 as the primary oxidant (in 2-fold molar excess in relation to the ketone) and carboxylic acid as the peracid precursor. In order to avoid excessive acidity in the reaction mixture, the easy available and relatively inexpensive octanoic acid was chosen.10 The total volume of octanoic acid and toluene as solvent was 1 mL per 0.25 mmol of ketone. The (nano)biocatalysts were used in amounts of 0.020 g per 0.25 mmol of ketone. The reaction was carried out at 20 °C. As shown in Figure 1, the CALB-MWCNTs was the most active nanobiocatalyst, surprisingly more active than CALB-

tion with alkyl groups, the Nanocyl NC7000 MWCNTs maintained the same geometry (aspect ratio) and possessed a highly developed porosity with 30% lower surface area. The assynthesized pristine MWCNTs (3.2 wt % Fe) and NMWCNTs (0.4 wt % N, 15.2 wt % Fe, outer diameter of 26 ± 15 nm, length of 40 μm) were significantly thicker (outer diameter of 60 ± 25 nm, inner diameter of 10 ± 2 nm) and longer (250 ± 120 μm) with a ca. 7-fold lower surface area than the Nanocyl NC7000 MWCNTs. Fe-MWCNTs (Fe-rich and long MWCNTs) were ca. 2.5 ± 0.5 mm long and similar diameter to pristine MWCNTs. CALB was immobilized using a physical adsorption technique according to the procedure described in the literature.27 The direct physical adsorption method is based on hydrophobic, electrostatic and hydrogen bonding interactions between the CNTs and enzymes.28 Lipases present their specific catalytic mechanisms of action in two structural forms. The first one called “close” features a polypeptide chain (lid or flat) which isolates an active center from the reaction medium and hinders its chemical activity. In the second one the “open” form of lipasesthe lid moves and an active center is exposed to reactants.29 Under favorable conditions, lipases can be immobilized via the hydrophobic surrounding of their active center, fixing them in an open conformation. Interfacial activation on hydrophobic supports at low ionic strength has been reported to be a simple and efficient method to immobilize lipases on various supports.29,30 The immobilization of lipase on solid supports may additionally improve enzyme rigidity, stability in the presence of hydrogen peroxide and can avoid dissociation.31 After immobilization, the protein loading was determined by TGA (Table 1). The high surface area and pore volume of the nanosupports corroborated the high enzyme loadings (Table 1). In the case of active carbon, the highest surface area at a lower volume of pores leads to geometrical restriction and hence lower loadings in the final biocatalysts compared to the Nanocyl-based system. The TEM image of the CALBMWCNTs (with the highest activity) selected for further studies demonstrated a dense and uniform coverage of the MWCNT surface with CALB (see SI, Figure S1). The enzyme areas were visible to the naked eye as distinguishable nonagglomerated islands with a thickness and diameter of a few nanometers adhering to the outer nanotube walls.

Figure 1. Influence of the carbon-based support on the (nano)biocatalyst activity. Reaction conditions: 2-methylcyclohexanone (0.25 mmol), 30% aq H2O2 (0.50 mmol), octanoic acid (0.5 mL, 3.125 mmol), toluene (0.5 mL), (nano)biocatalyst (0.020 g), 250 rpm, 20 °C; average of three independent experiments.

Nanocyl NC7000 with the highest loading of CALB. The introduction of alkyl groups caused a decrease in the activity of CALB-C10H21O-Nanocyl NC7000. Additionally, CALB-graphite was exceptionally active, although characterized by low lipase loading and low surface area. The N-MWCNTs, as carriers for lipase, turned out to be inactive and brought the reaction to a halt after 50% ketone conversion. Interestingly, the CALB-active carbon composed of the support with the highest surface area and high lipase loading did not result in high activity of the catalytic system. The reason for this phenomenon can be explained by lower accessibility of the octanoic acid or different enzymes conformations, including nonactive for this substrate.32 The crowded agglomerations of lipase could be the other reason for 1687


Research Article

ACS Sustainable Chemistry & Engineering the decline in its activity. In heavily loaded biocatalysts, the enzyme molecules often tend to aggregate. This behavior impedes the reactants in accessing their active sites, with a negative effect on their specific activity. This phenomenon has been observed previously.26 The CALB-MWCNTs, which most likely maintain the flexibility of lipase in their activated conformation,33,34 were chosen for further studies. The CALB-Fe-MWCNTs with a higher Fe loading (5.8 wt % Fe), specifically designed for magnetic separation (see SI, Figure.S2) were also very active. The activities of the new (nano)biocatalysts were also compared with the benchmark Novozyme-435 (Figure 1). The commercial biocatalyst emerged as slightly less active butas we previously verifiedwas not promising for recycling. This resin swelled upon contact with organic solvent, and the enzyme lost its activity. Nanobiocatalyst Loading. Next, we studied the key parameters affecting the reaction, including the influence of the nanobiocatalyst loading on the rate of 2-methylcyclohexanone oxidation. Direct evaluation of the biocatalysts’ activities from the rates of peracid formation can be somewhat difficult (Figure 1). Hence, we decided to assess their performance from the rates of lactone synthesis, discriminating the regions of specific kinetic control of the Baeyer−Villiger process. This determination was made by varying the amount of the CALBMWCNTs in the range of 1−30 mg, while keeping the amount of the model ketone constant (0.25 mmol). At a fixed molar ratio of ketone: H2O2 (1:2) and an excess of octanoic acid, the rate of lactone formation at 20 °C appeared to be dependent on the biocatalyst loading if it was ≤20 mg and remained constant with higher loadings (see SI, Figure S3). These experiments clearly showed that the synthesis of peracetic acid with over 20 mg of CALB-MWCNTs had no effect on the Baeyer−Villiger kinetics in the system under study, in strong contrast to the synthesis with less than 20 mg of CALB-MWCNTs. These findings delineated the region of effective control of the Baeyer−Villiger process in peracid synthesis and hence of the biocatalyst itself. Primary Oxidant. The form of the primary oxidant influences the activity and stability of the biocatalyst. In order to study this effect, 30 or 60% aq H2O2 and urea hydrogen peroxide (UHP), which is the anhydrous form of H2O2, were applied in the reaction. As shown in Figure 2, 30 or 60% aq H2O2 and UHP were equally active among the different forms of hydrogen peroxide after 10 h. A double molar excess of ketone to H2O2 was sufficient to produce peracid, and a further increase in the hydrogen peroxide amount did not influence the reaction rate. The exposure of lipase to highly concentrated (60%) aqueous hydrogen peroxide may result in its deactivation, which was observed in further studies concerning the recycling of the nanobiocatalyst. Peracid Precursor Loading. The amount of octanoic acid is crucial for this reaction. As shown in Figure 3, the use of octanoic acid in a molar ratio of 1:4 was not as efficient as its application as a cosolvent or even as the solvent alone. However, dilution with toluene (1:1 v/v) lowered the viscosity of the reaction mixture. Both octanoic acid and tolueneafter the process and nanobiocatalyst isolationcould be distilled off from the reaction mixture and recycled for industrial application.

Figure 2. Influence of the primary oxidant on activity of the nanobiocatalyst. Reaction conditions: 2-methylcyclohexanone (0.25 mmol), octanoic acid (0.5 mL, 3.125 mmol), toluene (0.5 mL) CALBMWCNTs (0.020 g), 250 rpm, 20 °C, 10 h; average of three independent experiments.

Figure 3. Influence of amount of peroxyacid precursor on activity of the nanobiocatalyst. Reaction conditions: 2-methylcyclohexanone (0.25 mmol), 30% aq H2O2 (0.50 mmol), octanoic acid, toluene (up to 1.0 mL of solvents), CALB-MWCNTs (0.02 g), 250 rpm, 20 °C; average of three independent experiments.

An additional test confirmed the stability of 6-methyl-εcaprolactone under the described conditions. This process was carried out following the standard procedure, but a lactone was introduced into the reaction system instead of the ketone. GC analysis verified a constant lactone concentration after 8 h of mixing. Reaction Temperature. At this stage, we also wanted to determine whether the temperature could be increased to ensure optimum kinetics while maintaining the structure of the enzyme with 30% aq H2O2 as the selected oxidant. The nanobiocatalyst was stable for one cycle of the reaction, even at 40 °C, producing the lactone in high yield after 3 h. The same result was obtained at 20 °C after 10 h. To demonstrate the mechanism of the reaction, an additional test was performed. Fast catalyst filtration after 1 h of reaction at 20 °C demonstrated almost the same rate of the oxidation of 2-methylcyclohexanone in the filtrate after removal of the catalyst (Figure 4). This result was not an effect of proteins leaching from the nanobiocatalyst, which was confirmed by TGA of the CALB-MWCNTs after 6 cycles at 20 °C (see SI, Table S2). The loss of protein from the surface of the MWCNTs was minor. 1688


Research Article


should be noted that recycling of the nanobiocatalyst at 20 °C with 60% aq H2O2 was not possible. Substrate Scope. The most active reaction system was examined in the chemo-enzymatic Baeyer−Villiger synthesis of various lactones from the corresponding ketones to determine its practical potential. As shown in Table 2, the oxidation Table 2. Oxidation of Selected Ketones to Lactones

Figure 4. Influence of the reaction temperature on activity of the nanobiocatalyst. Reaction conditions: 2-methylcyclohexanone (0.25 mmol), 30% aq H2O2 (0.50 mmol), octanoic acid (0.5 mL, 3.125 mmol), toluene (0.5 mL), CALB-MWCNTs (0.020 g), 250 rpm; (a) nanobiocatalyst was filtered off after 1 h of reaction; (b) 60% aq H2O2 (0.50 mmol); average of three independent experiments.

This phenomenon may be a result of rapidly achieving the steady state concentration of peracid; therefore, the enzyme no longer necessary and can be deactivated at higher temperatures. This is caused by a high molar excess of octanoic acid relative to the ketone compared to the other chemo-enzymatic methods described in the literature.9,10 Recycling Studies. The results of the nanobiocatalyst activity and reusability studies performed for 2-methylcyclohexanone oxidation at 20 °C over five successive runs showed very high activity, which dropped down in the sixth cycle (Figure 5). In the same experiment carried out at 30 °C, the biocatalyst was active in three cycles but was active only in the first cycle at 40 °C.

Figure 5. Recycling studies. Reaction conditions: 2-methylcyclohexanone (1.25 mmol), 30% aq H2O2 (2.50 mmol), octanoic acid (2.5 mL, 15.625 mmol), toluene (2.5 mL), CALB-MWCNTs (0.100 g), 250 rpm, 20 °C.

To verify whether the enzyme slowly detached and was washed out or simply lost its activity under the influence of H2O2 over time, additional experiments were performed. The latter suppositions were confirmed by TGA analysis of the applied nanobiocatalysts, which indicated a 28% loss in protein loading after the third cycle at 30 °C and only 7% at 20 °C. The enzyme was still visible in the TEM image (20 °C), but the activity dropped in the sixth cycle, which can be explained by its deactivation after the prolonged influence of hydrogen peroxide, what has been reported before (see the SI).19 It

a

Reaction conditions: ketone (1.25 mmol), 30% aq H2O2 (2.5 mmol), octanoic acid (2.5 mL, 15.625 mmol), toluene (2.5 mL), CALB-MWCNTs (0.100 g), 250 rpm, 30 °C. bYields determined using GC, isolated yields given in parentheses. 1689


Research Article


Table 3. Comparison of the Activity of Various Biocatalysts in Chemoenzymatic Oxidation of 2-Methylcyclohexanone (2-MCH) and Cyclohexanone (CH) biocatalyst, g/mol ketone Novozyme-435, 50 g Novozyme-435, 50 g Perhydrolase-CLEA, 1% w/v CALB-CLEA, 50 g native CALB, 200 g CALB/SiO2 functionalized, 20 g CALB-MWCNTs, 80 g a

price and availability of biocatalyst 242 Euro/10 g (SigmaAldrich) 242 Euro/10 g (SigmaAldrich) commercially not available 424 Euro/500 mg (Sigma-Aldrich) 77 Euro/50 mL (SigmaAldrich) support commercially not available support: 100$/kg (Nanocyl)

ketone

oxidant

2UHP, 2 equiv MCH CH 50% H2O2 1.1 equiv CH 30% H2O2 4 equiv 250% H2O2 MCH 1.1 equiv 250% H2O2 MCH 2 equiv 2UHP, 2 equiv MCH 230% H2O2 MCH 2 equiv

temp, °C, time, h

yield of lactone, %

ethyl acetate

27, 72

95b

no data4

octanoic acid, [choline]NO3

50, 5

62a

no data9

ethylene glycol diacetate, phosphate buffer ethyl acetate

RT, 48

62a

no data6

40, 48

84a

no data5

octanoic acid, [bmim][NTf2]

45, 10

96b

ethyl acetate

RT, 29

97b

recycle of [bmim] [NTf2]10 practicable7

octanoic acid, toluene

20, 10

99a

peracid precursor, solvent

recycle of catalyst

practicable (this work)

Yields determined using GC. bIsolated yields.

processes proceeded very efficiently at 30 °C. Cyclic ketones were readily oxidized to their corresponding lactones in high yields (86−95%) under mild conditions in reasonable reaction times. As model reactants, we selected strained ketones such as cyclobutanones, which formed lactones in high yields, and nonstrained cyclohexanones which were much more difficult to oxidize. The most reactive among these ketones were the cyclobutanones which were oxidized to adequate γ-butyrolactones in 95% yield within 1−2 h. Substituted cyclohexanones yielded ε-caprolactones after 6−20 h in yields of 86−95%. The oxidation of 2-adamantanone and norcamphor gave their corresponding lactones in high yields. Extremely unreactive cycloheptanone did not undergo oxidation under these conditions. Future Outlook. The conclusive finding of this study is that this new method for the synthesis of lactones has great potential for industrial application. Generally, comparing all the chemo-enzymatic methods proposed in the literature (Table 3), evident improvements were achieved using MWCNTs as a support for lipase. First, the reaction times were drastically shortened, e.g., the time necessary to obtain 99% yield of 6methyl-ε-caprolactone was reduced to 3 h at 40 °C or to 10 h at 20 °C. However, the impact of several parameters must be carefully considered. The activity of the nanobiocatalyst can be inversely proportional to its stability, i.e., increasing temperature may accelerate the chemo-enzymatic oxidation; however, beyond a certain temperature, the enzyme becomes inactive. It is worth emphasizing that only a few studies have considered the recycling of the biocatalyst.7,10 The test of Novozyme-435 recycling highlighted the problems with filtering off the biocatalyst after the reaction, which was caused by the swelling of the resin. Finally, from an economical point of view, CNTs are less expensive than the specifically functionalized silica or ionic liquids.

were synthesized from the corresponding ketones consisting of Candida antarctica lipase B immobilized on the surface of MWCNTs. CALB-MWCNTs emerged as a stable and highly enzyme-loaded system. Cyclic ketones were readily oxidized to their corresponding lactones in high yields (86−95%) under mild conditions in reasonable reaction times. Finally, operational stability tests showed no significant activity loss during up to 5 consecutive cycles of use and recovery at 20 °C. The easy recovered (magnetically) and reused of the nanobiocatalysts is critical for their application in scaled-up processes.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02433. 1 H and 13C NMR spectra of lactones, thermograms of biocatalysts, conditions for GC analysis (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. +48 322372917. Fax: +48 322371032. ORCID

Anna Chrobok: 0000-0001-7176-7100 Funding

This work was financed by the National Science Centre, Poland (grant no. UMO-2015/17/B/ST8/01422). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financed by the National Science Centre, Poland (grant no. UMO-2015/17/B/ST8/01422). Grupa Azoty, Zakłady Azotowe Puławy SA, is gratefully acknowledged for supporting these studies and Artur Herman from Silesian University of Technology for the synthesis of C10H21ONanocyl NC7000.

■

CONCLUSIONS The environmentally benign chemo-enzymatic Baeyer−Villiger process proposed herein avoids the use of a relatively unstable peracid, which is in situ generated during the enzymatic stage in the proposed method. This heterogeneous process is based on the utilization of a biocatalyst To demonstrate the robustness of the nanobiocatalysts in the chemo-enzymatic Baeyer−Villiger reaction various lactones

■

REFERENCES

(1) Renz, M.; Meunier, B. 100 years of Baeyer−Villiger oxidations. Eur. J. Org. Chem. 1999, 1999, 737−750. 1690


Research Article

ACS Sustainable Chemistry & Engineering (2) Sheldon, R. A. Catalysis and pollution prevention. Chem. Ind. 1997, 12−15. (3) ten Brink, G.; Arends, I.; Sheldon, R. The Baeyer−Villiger reaction: New developments toward greener procedures. Chem. Rev. 2004, 104, 4105−4124. (4) Rios, M. Y.; Salazar, E.; Olivo, H. F. Baeyer-Villiger oxidation of substituted cyclohexanones via lipase-mediated perhydrolysis utilizing urea-hydrogen peroxide in ethyl acetate. Green Chem. 2007, 9, 459− 462. (5) Chavez, G.; Hatti-Kaul, R.; Sheldon, R. A.; Mamo, G. BaeyerVilliger oxidation with peracid generated in situ by CalB-CLEA catalyzed perhydrolysis. J. Mol. Catal. B: Enzym. 2013, 89, 67−72. (6) Chavez, G.; Rasmussen, J.; Janssen, M.; Mamo, G.; Hatti-Kaul, R.; Sheldon, R. A. Baeyer−Villiger oxidation of cyclohexanone in aqueous medium with in situ generation of peracid catalyzed by perhydrolase CLEA. Top. Catal. 2014, 57, 349−355. (7) Drożdż, A.; Chrobok, A.; Baj, S.; Szymańska, K.; Mrowiec-Białoń, J.; Jarzębski, A. The chemo-enzymatic Baeyer−Villiger oxidation of cyclic ketones with an efficient silica-supported lipase as a biocatalyst. Appl. Catal., A 2013, 467, 163−170. (8) Drożdż, A.; Hanefeld, U.; Szymańska, K.; Jarzębski, A.; Chrobok, A. A robust chemo-enzymatic lactone synthesis using acyltransferase from Mycobacterium smegmatis. Catal. Commun. 2016, 81, 37−40. (9) Kotlewska, J.; van Rantwijk, F.; Sheldon, R. A.; Arends, I. W. C. E. Epoxidation and Baeyer−Villiger oxidation using hydrogen peroxide and a lipase dissolved in ionic liquids. Green Chem. 2011, 13, 2154− 2160. (10) Drożdż, A.; Erfurt, K.; Bielas, R.; Chrobok, A. Chemo-enzymatic Baeyer−Villiger oxidation in the presence of Candida antarctica lipase B and ionic liquids. New J. Chem. 2015, 39, 1315−1321. (11) Feng, W.; Ji, P. Enzymes immobilized on carbon nanotubes. Biotechnol. Adv. 2011, 29, 889−95. (12) Garcia-Galan, C.; Berenguer-Murcia, A.; Fernandez-Lafuente, R.; Rodrigues, R. C. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 2011, 353, 2885−2904. (13) Rastian, Z.; Khodadadi, A. A.; Guo, Z.; Vahabzadeh, F.; Mortazavi, Y. Plasma functionalized multiwalled carbon nanotubes for immobilization of Candida antarctica Lipase B: production of biodiesel from methanolysis of rapeseed oil. Appl. Biochem. Biotechnol. 2016, 178, 974−989. (14) Jamie, A.; Alshami, A. S.; Maliabari, Z. O.; Ali Ateih, M.; Al Hamouz, O. C. S. Immobilization and enhanced catalytic activity of lipase on modified MWCNT for oily wastewater treatment. Environ. Prog. Sustainable Energy 2016, 35, 1441−1449. (15) Kim, J.; Grate, J. W. Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett. 2003, 3, 1219−1222. (16) Boncel, S.; Kyzioł-Komosińska, J.; Krzyżewska, I.; Czupioł, J. Interactions of carbon nanotubes with aqueous/aquatic media containing organic/inorganic contaminants and selected organisms of aquatic ecosystems. Chemosphere 2015, 136, 211−221. (17) Liu, Y.; Zhao, Y.; Sun, B.; Chen, C. Understanding the toxicity of carbon nanotubes. Acc. Chem. Res. 2013, 46, 702−713. (18) Luanpitpong, S.; Wang, L.; Manke, A.; Martin, K. H.; Ammer, A. G.; Castranova, V.; Yang, Y.; Rojansakul, Y. Induction of stemlike cells with fibrogenic properties by carbon nanotubes and its role in fibrogenesis. Nano Lett. 2014, 14, 3110−3116. (19) Zhao, Y.; Allen, B. L.; Star, A. Enzymatic degradation of multiwalled carbon nanotubes. J. Phys. Chem. A 2011, 115, 9536− 9544. (20) Boncel, S.; Müller, K. H.; Skepper, J. N.; Walczak, K. Z.; Koziol, K. K. Tunable chemistry and morphology of multi-wall carbon nanotubes as a route to non-toxic, theranostic systems. Biomaterials 2011, 32, 7677−7686. (21) Singh, S.; Ali, S. Z.; Tiwari, P. M.; Eroğlu, E.; Vig, K.; Dennis, V. A.; Vardharajula, S. Functionalized carbon nanotubes: biomedical applications. Int. J. Nanomed. 2012, 7, 5361−5374.

(22) Gao, Y.; Kyratzis, I. Covalent immobilization of proteins on carbon nanotubes using the cross-linker 1-ethyl-3-(3dimethylaminopropyl)carbodiimide-a critical assessment. Bioconjugate Chem. 2008, 19, 1945−1950. (23) Bolm, O.; Beckmann, O.; Kuhn, T.; Palazzi, C.; Adam, W.; Rao, P. B.; Saha-Möller, C. R. Influence of hydroperoxides on the enantioselectivity of metal-catalyzed asymmetric Baeyer−Villiger oxidation and epoxidation with chiral ligands. Tetrahedron: Asymmetry 2001, 12, 2441−2446. (24) Boncel, S.; Koziol, K. K. K.; Walczak, K. Z.; Windle, A. H.; Shaffer, M. S. P. Infiltration of highly aligned carbon nanotube arrays with molten polystyrene. Mater. Lett. 2011, 65, 2299−2303. (25) Boncel, S.; Pattinson, S. W.; Geiser, V.; Shaffer, M. S. P.; Koziol, K. K. K. En route to controlled catalytic CVD synthesis of densely packed and vertically aligned nitrogen-doped carbon nanotube arrays. Beilstein J. Nanotechnol. 2014, 5, 219−233. (26) Blanco, R. M.; Terreros, P.; Fernández-Pérez, M.; Otero, C.; Diaz-González, G. Functionalization of mesoporous silica for lipase immobilization: characterization of the support and the catalysts. J. Mol. Catal. B: Enzym. 2004, 30, 83−93. (27) Boncel, S.; Zniszczoł, A.; Szymańska, K.; Mrowiec-Białoń, J.; Jarzębski, A.; Walczak, K. Z. Alkaline lipase from Pseudomonas fluorescens non-covalently immobilised on pristine versus oxidised multi-wall carbon nanotubes as efficient and recyclable catalytic systems in the synthesis of Solketal esters. Enzyme Microb. Technol. 2013, 53, 263−270. (28) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir 2004, 20, 11594−11599. (29) Fernández-Lorente, G.; Cabrera, Z.; Godoy, C.; FernandezLafuente, R.; Palomo, J. M.; Guisan, J. M. Interfacially activated lipases against hydrophobic supports: Effect of the support nature on the biocatalytic properties. Process Biochem. 2008, 43, 1061−1067. (30) Fernandez-Lorente, G.; Palomo, J. M.; Cabrera, Z.; Guisan, J. M.; Fernández-Lafuente, R. Specificity enhancement towards hydrophobic substrates by immobilization of lipases by interfacial activation on hydrophobic supports. Enzyme Microb. Technol. 2007, 41, 565− 569. (31) Hernandez, K.; Berenguer-Murcia, A.; Rodrigues, R. C.; Fernández-Lafuente, R. Hydrogen peroxide in biocatalysis. A dangerous liaison. Curr. Org. Chem. 2012, 16, 2652−2672. (32) Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernández-Lafuente, R. Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 2013, 42, 6290−6307. (33) Bordes, F.; Barbe, S.; Escalier, P.; Mourey, L.; Andre, I.; Marty, A.; Tranier, S. Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase. Biophys. J. 2010, 99, 2225−2234. (34) Feng, W.; Sun, X.; Ji, P. Activation mechanism of Yarrowia lipolytica lipase immobilized on carbon nanotubes. Soft Matter 2012, 8, 7143−7150.

1691


Highly Active Nanobiocatalyst from Lipase Noncovalently Immobilized

Recommend Documents