Immobilization of a Cutinase from Fusarium oxysporum and

Apr 13, 2017 - Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Heroon Polytechniou Street, Zograp...
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Immobilization of a Cutinase from Fusarium oxysporum and Application in Pineapple Flavor Synthesis Efstratios Nikolaivits, Georgios Makris, and Evangelos Topakas* Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Heroon Polytechniou Street, Zographou Campus, 15780 Athens, Greece S Supporting Information *

ABSTRACT: In the present study, the immobilization of a cutinase from Fusarium oxysporum was carried out as cross-linked enzyme aggregates. Under optimal immobilization conditions, acetonitrile was selected as precipitant, utilizing 9.4 mg protein/ mL and 10 mM glutaraldehyde as cross-linker. The immobilized cutinase (imFocut5a) was tested in isooctane for the synthesis of short-chain butyrate esters, displaying enhanced thermostability compared to the free enzyme. Pineapple flavor (butyl butyrate) synthesis was optimized, leading to a conversion yield of >99% after 6 h, with an initial reaction rate of 18.2 mmol/L/h. Optimal reaction conditions were found to be 50 °C, a vinyl butyrate/butanol molar ratio of 3:1, vinyl butyrate concentration of 100 mM, and enzyme loading of 11 U. Reusability studies of imFocut5a showed that after four consecutive runs, the reaction yield reaches 54% of the maximum. The efficient bioconversion offers a sustainable and environmentally friendly process for the production of “natural” aroma compounds essential for the food industry. KEYWORDS: cross-linked enzyme aggregates, immobilization, cutinase, transesterification, flavor esters, biocatalysis

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INTRODUCTION Short-chain esters (usually fewer than 10 carbon atoms)1 are used as flavor compounds by the food, cosmetic, and pharmaceutical industries.2 So far, the demand of these markets is filled by compounds either isolated from natural sources or chemically synthesized. Natural sources, such as higher plants, contain very low concentrations of the desired compounds, which also suffer from seasonal variations. Additionally, the extraction methods usually involve toxic solvents, whereas the isolation steps increase the cost of the whole process.2 On the other hand, chemical synthesis is cheaper but not environmentally friendly, because the reactions take place at high temperatures (150−240 °C) using strong acid or base catalysts. Furthermore, numerous byproducts need to be removed, as chemical synthesis is not selective.3 The answer to these issues is natural synthesis of flavor compounds taking place either by microorganisms (de novo synthesis) or by enzymatic bioconversion. For the production of flavor esters specifically, the enzymatic path seems to be more suitable and easier.2 According to Grand View Research Inc., the fatty acid ester market will be worth U.S. $2.44 billion by 2022, up from U.S. $1.83 billion in 2014. This market growth is supported by the high demand from the aforementioned industries (personal care, cosmetics, food processing) and the substitution of synthetic chemicals by biosynthesized esters.4 Nevertheless, enzymes present some drawbacks that usually exclude them from widespread industrial applications, such as low operational or storage stability and/or heat and organic solvent sensitivity. Immobilization of enzymes increases their stability properties and also allows them to be easily separated from the reaction and reused.5 There are three types of immobilization: binding to a support, encapsulation, and crosslinking. For the first two, a great disadvantage is that the carrier © 2017 American Chemical Society

makes up 90−99% of the biocatalyst mass, diluting the enzyme and leading to low productivities (product mass/biocatalyst mass).6 Cross-linking methods use glutaraldehyde as a cross-linker, which is inexpensive and has a low molecular weight, meaning that the resulting immobilized biocatalyst consists almost entirely of the enzyme itself. For the creation of cross-linked enzyme aggregates (CLEAs), the first step is the precipitation of the enzyme with the use of salts, organic solvents, or nonionic polymers. These physical aggregates are held together by noncovalent bonding without losing their tertiary architecture, and the cross-linking takes place between glutaraldehyde and the free amino groups on the surface of the enzyme.7,8 In this work, we report the study of CLEAs production from a crude Fusarium oxysporum cutinase preparation (namely, imFocut5a) produced heterologously in Escherichia coli BL21 cells.9 To optimize the immobilization process, various parameters were studied such as the precipitant, the concentration of cross-linker, and the concentration of the protein preparation. The immobilized cutinase following the CLEA methodology was subsequently characterized in terms of thermostability. The recombinant cutinase, in its free form, has been shown to possess synthetic potential in a water−oil biphasic system for the preparation of tyrosyl esters.10 In this case, the immobilized biocatalyst was tested for the production of short-chain butyrate esters through the transesterification of vinyl butyrate (VB) with various aliphatic alcohols in isooctane (Figure 1). The synthesis of butyl butyrate, a compound with Received: Revised: Accepted: Published: 3505

February April 11, April 13, April 13,

16, 2017 2017 2017 2017 DOI: 10.1021/acs.jafc.7b00659 J. Agric. Food Chem. 2017, 65, 3505−3511

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Journal of Agricultural and Food Chemistry pineapple-like aroma,11 was further optimized, reaching a high yield of bioconversion (99%).

The concentration of VB was 100 mM, and the molar ratio of VB/ alcohol was 1:3. Optimization of Butyl Butyrate Synthesis by imFocut5a. Several parameters were studied to determine the optimal conditions for the production of butyl butyrate in 24 h reactions, such as reaction temperature (20−60 °C), VB/butanol molar ratio (5:1, 3:1, 1:1, 1:3, 1:5), VB concentration (50−300 mM), and enzyme loading (0−14.5 U) of imFocut5a. At the optimal conditions found, the time course of the reaction was also studied. Operational Stability of ImFocut5a. To test the stability of the immobilized biocatalyst under the reaction conditions, the same amount of imFocut5a was used for consequent reaction runs. After each run, the enzyme was separated from the reaction medium by centrifugation and washed four times with equal volumes of isooctane. The enzyme was then left to dry overnight at ambient temperature before being used again under the same reaction conditions. HPLC Analysis. Reaction samples (20 μL injections) were quantitatively analyzed using a Jasco PU-987 Intelligent Prep. pump operated at 0.8 mL min−1. Stationary phase was a C-18 reverse-phase Nucleosil 100-5 (Macherey-Nagel, Germany) eluted isocratically with 70% methanol in water at ambient temperature. Detection of VB and products was performed by a Jasco UV-975 detector at 210 nm after appropriate sample dilution in methanol. Conversion was calculated from the amount of VB reacted compared to its starting amount. All experiments were carried out in duplicate.

Figure 1. Schematic depiction of the transesterification reaction performed in isooctane with immobilized cutinase (imFocut5a) as catalyst.

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MATERIALS AND METHODS

Enzyme and Chemicals. The crude Focut5a cutinase preparation used for immobilization studies was the cell-free extract of E. coli BL21 cultures, as described previously.9 VB was purchased from SigmaAldrich (St. Louis, MO, USA), and all of the organic solvents used were of HPLC grade. Enzyme Assays. Cutinase activity was assayed using 0.96 mM pnitrophenyl butyrate (pNPB) or laurate (pNPL) as substrate in 0.1 M phosphate−citrate buffer, pH 6, at 40 °C for 10 min. The immobilized cutinase (imFocut5a) was assayed using 0.96 mM pNPL under the same conditions as previously for 15 min of incubation time. The release of p-nitrophenol was monitored at 410 nm, using a SpectraMax-250 microplate reader equipped with SoftMaxPro 1.1 software (Molecular Devices, Sunnyvale, CA, USA).12 One unit of enzyme is defined as the enzyme amount that releases 1 μmol of pNP per minute. Units assayed with pNPL were correlated to units measured with pNPB to have the same activity basis between free and immobilized enzyme. The enzyme concentration was calculated using the method of Bradford13 and a standard curve constructed by known solutions of bovine serum albumin. Preparation of ImFocut5a Cutinase. The procedure for the cross-linking of Focut5a cutinase was adapted from the work of Vafiadi et al.14 In brief, to optimize the immobilization method three parameters were studied: (a) precipitant, (b) cross-linker concentration, and (c) protein concentration. The standard cross-linking technique included precipitation of 1 mL of protein solution with 9 mL of precipitant, addition of 100 mM glutaraldehyde (cross-linking agent), and stirring for 3 h at 20 °C in an orbital shaker. Afterward, the mixture was diluted with 10 mL of 20 mM Tris-HCl, pH 8, buffer prior to centrifugation. The pellet was resuspended in 10 mL of the same buffer and centrifuged again (this step was repeated twice). The pellet was then freeze-dried, and imFocut5a activity was estimated. After the estimation of the optimum conditions for imFocut5a preparation, the same conditions were also employed for the intracellular fraction of the wild-type E. coli BL21 to determine the esterase activity that is not attributed to the recombinant cutinase. The percentage of the recombinant enzyme in the crude cell-free extract was determined by SDS-PAGE using an InGenius gel imaging and analysis system (Syngene, UK), incorporating the GeneTools software. Study of ImFocut5a Thermostability. The immobilized enzyme was checked in terms of thermal stability. ImFocut5a was incubated in 20 mM Tris-HCl, pH 8, buffer at various temperatures (20−40 °C) in an Eppendorf Thermomixer Comfort (Eppendorf, Germany) for up to 5 h, and its residual cutinase activity was measured in each case. The activity of imFocut5a incubated at room temperature was considered as 100% of relative activity. Synthesis of Aliphatic Butyl Esters. To test the synthetic ability of the imFocut5a produced under optimal immobilization conditions, the transesterification of VB with different short-chain aliphatic alcohols was performed. A standard transesterification reaction took place for 24 h at 40 °C in sealed serum glass 2 mL vials under stirring containing 0.5 mL of reactants in isooctane and 0.73 U of imFocut5a.

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RESULTS AND DISCUSSION Immobilization of Focut5a through CLEA Methodology. For the immobilization process following the CLEA methodology, crude E. coli cell-free extract preparation exhibiting recombinant cutinase activity was employed, aiming for a low-cost immobilization process that utilizes the native E. coli intracellular proteins as a carrier for cross-linking. According to SDS-PAGE of the crude cell-free extract, Focut5a corresponds to 40% of the total protein (Figure S1, Supporting Information). Because the intracellular E. coli fraction may contain enzymes exhibiting esterase activity able to hydrolyze pNPL, CLEAs were also prepared by utilizing the cell-free extract of the E. coli BL21 strain without expressing cutinase activity. The control CLEA preparation was measured against pNPL, and it was found to contain only 1.5% of relative activity compared to imFocut5a, which is attributed to the endogenous E. coli esterases. Three parameters were studied to determine the optimum conditions for the immobilization of the recombinant cutinase preparation. First, the effect of the precipitant on the residual activity of the precipitated enzyme was studied. Five organic solvents, such as methanol, ethanol, acetone, acetonitrile, and dimethyl sulfoxide, and a salt (saturated ammonium sulfate) were used for the precipitation of the enzyme. The recovery of the enzymatic activity of precipitated cutinase after resolubilization is shown in Table 1. Dimethyl sulfoxide almost totally abolished enzymatic activity, whereas acetonitrile and saturated Table 1. Focut5a Enzymatic Activity after Recovery from Precipitation Using Different Precipitants precipitant buffer Tris-HCl, pH 8 methanol ethanol acetone acetonitrile saturated (NH4)2SO4 dimethyl sulfoxide 3506

residual activity (%) 100 24.4 14.2 30.4 74.9 89.5 0.02

± ± ± ± ± ± ±

0.0 0.0 0.5 0.6 0.5 0.2 0.0

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Tris-HCl pH 8 buffer. At 30 and 35 °C, the catalyst retains almost all of its activity (>97%) after 1 h, whereas incubation at 40 °C resulted in a 50% decrease in residual activity (Figure 2).

ammonium sulfate allowed the precipitated enzyme to retain a large part of its activity (75 and 90%, respectively). Even though the most efficient precipitant proved to be ammonium sulfate, acetonitrile was also chosen for further investigation, as the aggregate activity may differ from the redissolved enzyme activity. According to Kartal et al.,15 a precipitant may cause the enzyme to take an unfavorable conformation, but after resolubilization, it may fold back to its native active form showing high activity. On the contrary, when the precipitated enzyme is cross-linked, it maintains this unfavorable conformation and presents very low activity. The next optimization step was the glutaraldehyde concentration, using both precipitation agents. The low glutaraldehyde concentration of 10 mM showed the highest activity for both precipitants, with acetonitrile to be the best precipitant in terms of relative activity (Table 2). Therefore, Table 2. Relative ImFocut5a Activity, Using Saturated (NH4)2SO4 and Acetonitrile as Precipitants, for Increasing Glutaraldehyde Concentration Focut5a CLEAs relative activity glutaraldehyde concentration (mM) 0 10 50 100 150

saturated (NH4)2SO4 0 98.3 37.2 19.1 15.1

± ± ± ±

Figure 2. Effect of temperature on the enzymatic activity of imFocut5a incubated in 20 mM Tris-HCl at 30, 35, and 40 °C for 1 h (black bars) and 5 h (white bars).

acetonitrile 0 100.0 80.6 89.6 81.2

1.7 1.2 1.1 1.0

± ± ± ±

0.0 0.1 0.2 0.2

After 5 h of incubation, imFocut5a suffers minor activity losses at 30 °C (10%), unlike 35 and 40 °C, at which the enzyme loses 40 and 66% of its initial activity, respectively. However, compared to the free Focut5a cutinase, the immobilized biocatalyst exhibited enhanced thermostability. As reported by Nikolaivits et al.,12 cutinase in its purified form retains half of its initial activity compared to 90% of imFocut5a after 3 h at 30 °C, but retains only 23% compared to 66% for imFocut5a at 35 °C. The increase of thermal stability is a very common outcome after immobilization of a biocatalyst. Synthetic Potential of imFocut5a for the Production of Butyrate Esters. Several short-chain alkyl butyrates were synthesized by a transesterification reaction of the corresponding alcohols with VB catalyzed by imFocut5a, using isooctane as a solvent (Figure 3). Transesterification reaction was preferred, because direct esterification presents several disadvantages, such

acetonitrile was chosen as a precipitant and 10 mM glutaraldehyde as a cross-linking agent for the next step. Typically, according to the literature,15,16 the effect of crosslinker concentration shows a maximum. At low glutaraldehyde concentrations, part of the enzyme is not cross-linked, resulting in substantial loss during the washing step. On the other hand, high cross-linker concentrations decrease the enzyme’s required flexibility, reducing its activity. In our case, probably due to the low initial enzyme concentration, the lowest glutaraldehyde concentration resulted in the highest activity. In the final optimization step the effect of protein concentration was studied. The highest crude enzyme concentration used was 9.4 mg mL−1, which corresponds to 730.4 U/g of imFocut5a activity (Table 3). Decreasing enzyme Table 3. ImFocut5a Activity Using Different Concentrations of Protein Solution protein concentration (mg mL−1) 0.6 1.2 2.5 4.7 9.4

CLEAs activity (U g−1) 454.5 473.3 597.6 617.4 730.4

± ± ± ± ±

25.9 15.2 15.5 16.4 19.7

concentrations resulted in decreasing imFocut5a activity reaching 50% of the maximum at 0.6 mg mL−1, so 9.4 mg protein mL−1 was used as the best concentration. To conclude, the cutinase preparation from F. oxysporum (9.4 mgprotein/mL) was introduced in the form of CLEAs using acetonitrile as precipitant and 10 mM glutaraldehyde as cross-linker. Thermal Stability of ImFocut5a. The imFocut5a biocatalyst prepared under optimum immobilization conditions was tested for its thermal tolerance when incubated in 20 mM

Figure 3. Effect of acyl acceptor (alcohol) chain length on the conversion of VB under standard reaction conditions. 3507

DOI: 10.1021/acs.jafc.7b00659 J. Agric. Food Chem. 2017, 65, 3505−3511

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Figure 4. Effect of (A) temperature ([VB] = 100 mM, [butanol] = 300 mM), (B) butanol concentration (40 °C with [VB] = 100 mM), (C) VB concentration (50 °C, [VB]:[butanol] = 3:1), and (D) enzyme loading (50 °C, [VB]:[butanol] = 3:1, [VB] = 100 mM) on the conversion of VB.

Effect of Reaction Temperature. Temperature is a very important parameter for a biocatalytic reaction, because the rise of temperature increases the reaction rate but also could deactivate the catalyst. The optimum temperature of the free Focut5a is 40 °C; however, its stability at that temperature is very low.9 Because the immobilization increased the thermostability, as shown previously, the optimum temperature for the transesterification reaction was found to be 50 °C, resulting in 41.1% conversion (Figure 4A). The conversion of VB increases in an almost linear form for temperatures starting from 20 (20%) to 50 °C, but decreases sharply at 60 °C (2.5%), probably due to quick deactivation of the enzyme. The optimal temperature found for 50 °C is a significant step toward an efficient cutinase biocatalyst, because other cutinase-catalyzed esterification reactions found in the literature were performed in the temperature range between 30 and 40 °C.18−21 Effect of Substrates’ Molar Ratio. Generally, in reactions in which two or more substrates are involved, one of those can be used in excess to shift the equilibrium of the reaction toward the desired direction.1 On the other hand, a high concentration of one or both of the starting compounds can be proven to be deleterious for the enzymatic activity, leading to lower conversion. Under this view, the effect of different concentrations of the alcohol and VB in conversion was investigated. First, the effect of the molar ratio of VB and butanol on the reaction yield was studied by keeping the VB concentration constant (100 mM) at 40 °C and the butanol concentration ranging from 20 to 500 mM, corresponding to molar ratios of VB/alcohol from 0.2 to 5. As can be seen from Figure 4B, the lowest and highest concentrations of butanol (20 and 500 mM) resulted in the lowest conversions of the vinyl ester (27.2 and 32.5%, respectively). The highest yield (47.1%) was achieved for 33 mM butanol (3:1 molar ratio), whereas for higher

as the destabilizing effect of acids to the biocatalyst and the production of water that shifts the reaction equilibrium toward hydrolysis.17 The resulting esters can be used as flavor additives in foods or cosmeceuticals. As shown in Figure 3, the maximum conversion was achieved using butanol (C4) as acyl acceptor (37.3%). The transesterification yield was decreased for longer (C5, C7) or shorter (C1, C2, C3) carbon chains, highlighting the preference of Focut5a for C4 substrates.9 In addition, the polarity of the acyl acceptor seems to affect enzyme activity and should be also considered. For instance, when methanol is used, the conversion is much lower (13.3%) compared to that when heptanol is used (21%) even though both substrates differ by three carbon atoms compared to butanol, which was found to be the most suitable acyl acceptor. Therefore, it seems that lower molecular weight alcohols might also denature the immobilized biocatalyst, in addition to the influence of substrate specificity. Except from methanol, the rest of the acyl acceptors tested led to the synthesis of the corresponding butyrate esters with satisfactory yields (>20%). In the literature, cutinases are known to show preference for short-chain aliphatic substrates. For example, Burkholderia cepacia cutinase exhibited higher esterification yields when utilizing butyric (C4) and valeric (C5) acids with butanol,18 in accordance with the cutinase from Fusarium solani, in esterification reactions with ethanol.19 On the other hand, de Barros et al.19 observed a shift in the specificity of cutinase toward longer chain-length acids in a mini-emulsion system compared to an organic solvent system. Optimization of Butyl Butyrate Synthesis. Because imFocut5a showed highest activity on the transesterification of butanol with VB, this reaction was chosen for further optimization. The product of this reaction, butyl butyrate, is the prevalent ester giving the pineapple flavor.11 3508

DOI: 10.1021/acs.jafc.7b00659 J. Agric. Food Chem. 2017, 65, 3505−3511

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Journal of Agricultural and Food Chemistry alcohol concentrations of 100 and 300 mM, the yield was close to 40%. Escandell et al.22 also found that the optimum molar ratio for the production of butyl acetate was 3:1 (vinyl acetate excess) using lipase B from Candida antarctica. In the case of cutinase from Thermobifida fusca, ethanol concentrations >0.1 M also sharply decreased the reaction yield.1 The low conversion seen at high alcohol concentrations is a common phenomenon for cutinases and can be attributed either to denaturation of the biocatalyst through dehydration18 or to the formation of dead-end enzyme−alcohol intermediates.19 On the other hand, the effect of VB concentration was also investigated using a concentration range between 50 and 300 mM with a constant substrate molar ratio of 3. The maximum conversion was found for 0.1 M VB (54.2%), whereas above that value, conversion decreased, leading to 23.7% for the highest concentration (Figure 4C). For the lowest concentration tested, the yield was low (34%), probably due to the low concentration of both substrates; therefore, the thermodynamic equilibrium is not directed toward product formation. In several esterification reactions studied in the literature, it has been proven that high acid concentrations affected enzyme activity, leading to low reaction yields due to the acidic microenvironment surrounding the enzyme surface. On the contrary, biocatalysts are usually more tolerant to the respective fatty acid esters, so higher substrate concentrations can be used.23 In our case, this hypothesis stands as the enzyme seems to perform better in the excess of vinyl ester compared to the alcohol. Effect of Enzyme Loading. The amount of biocatalyst used at a certain reaction is of paramount interest in a technoeconomical study to investigate the potential of this reaction to scale up to an industrial level. In most cases, the cost of the enzyme is one of the limiting steps of this kind of application. In this particular study, the amount of immobilized biocatalyst employed ranged from 1.5 to 29 U mLreaction−1. As depicted in Figure 4D, when 99%). Optimal enzyme loading was found to be 22 U mL−1. It appears that for small enzyme loading, the reaction cannot reach its equilibrium in 24 h, whereas for higher enzyme amounts (>22 U mL−1) the reaction in completed in 95% for the synthesis of all short-chain (C4−C6) esters tested.24 Time Course of Butyl Butyrate Synthesis under Optimal Conditions. The time the enzymatic system needs to reach the thermodynamic equilibrium is an important factor, especially when the biocatalyst is immobilized, and therefore mass transfer phenomena are present. As seen in Figure 5, conversion reaches 90% at 4 h, whereas in 6 h the reaction is completed, reaching a conversion of >99%. The reaction system optimized attains very fast its equilibrium compared to the literature, as for butyl butyrate synthesis reaction times of 24 h11 or even 48 h25 have been stated. The initial reaction rate calculated for the first 2 h of the reaction was 18.2 ± 1.1 mmol L−1 h−1 (R2 = 0.9943). This

Figure 5. Time course of the conversion of VB under optimal conditions.

initial reaction rate given by imFocut5a is lower compared to the one achieved from Thermomyces lanuginosus lipase for the synthesis of the same ester in isooctane, exhibiting an initial rate of 33 mmol L−1 h−1.26 Following all of the optimization steps, butyl butyrate synthesis catalyzed by imFocut5a was improved, reaching a 99% conversion under the reaction conditions of 50 °C, substrate ratio of VB/butanol 3:1, VB concentration of 100 mM, and enzyme loading of 11 U for 6 h of incubation. Reusability of the Immobilized Biocatalyst. One of the most important properties of an immobilized biocatalyst is its ability to be separated from the reaction mixture and be used again in a following reaction batch. This property can decrease the cost of the process even further and make it feasible on an industrial scale. Even though the cutinase from F. oxysporum has been shown to be relatively thermolabile, its immobilization following the CLEAs technique made it more stable and able to operate efficiently at temperatures >40 °C for several hours. To gain a better understanding of the immobilized biocatalyst and its biotechnological evaluation, the potential reuseability of imFocut5a in consecutive reactions under optimal conditions was tested. It was observed that after each of the first three runs, imFocut5a loses almost 20% of its activity (Figure 6). However, after the fourth run the activity loss was decreased to 10% compared to the first three rounds of biocatalyst reuse. Candida rugosa lipase immobilized on graphene oxide showed superior reusability, losing just 10% of its activity after 10 cycles of reaction.27 Similar behavior (20% loss after 10 runs) was shown by the commercial lipase Novozym 435.28 Other commercial lipases (Lipozyme RM-IM and Lipozyme TL-IM) proved to be much less stable, maintaining 30 and 0% (respectively) activity after just four consecutive reaction batches.28 The efficient enzymatic synthesis of a pineapple flavor compound that diminishes any byproduct formation together with the use of an immobilized biocatalyst allows the effective purification of the final product. In addition, the enzymatic reaction is considered as an alternative bioprocess that retains the natural characteristics of the starting materials, particularly when biobased compounds are being used, for example, from acetone-butanol-ethanol fermentation. The immobilized cutinase offers a sustainable and environmentally friendly 3509

DOI: 10.1021/acs.jafc.7b00659 J. Agric. Food Chem. 2017, 65, 3505−3511

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(7) Sheldon, R. A.; Schoevaart, R.; van Langen, L. M. Cross-linked enzyme aggregates. In Immobilization of Enzymes and Cells; Guisan, J. M., Ed.; Springer Science & Business Media, 2006; pp 31−45. (8) Sheldon, R. A. Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts. Biochem. Soc. Trans. 2007, 35 (6), 1583− 1587. (9) Dimarogona, M.; Nikolaivits, E.; Kanelli, M.; Christakopoulos, P.; Sandgren, M.; Topakas, E. Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential. Biochim. Biophys. Acta, Gen. Subj. 2015, 1850 (11), 2308−2317. (10) Nikolaivits, E.; Norra, G.-F.; Voutsas, E.; Topakas, E. Cutinase from Fusarium oxysporum catalyzes the acylation of tyrosol in an aqueous medium: optimization and thermodynamic study of the reaction. J. Mol. Catal. B: Enzym. 2016, 129, 29−36. (11) Lorenzoni, A. S. G.; Graebin, N. G.; Martins, A. B.; FernandezLafuente, R.; Záchia Ayub, M. A.; Rodrigues, R. C. Optimization of pineapple flavour synthesis by esterification catalysed by immobilized lipase from Rhizomucor miehei. Flavour Fragrance J. 2012, 27 (2), 196− 200. (12) Nikolaivits, E.; Kokkinou, A.; Karpusas, M.; Topakas, E. Microbial host selection and periplasmic folding in Escherichia coli affect the biochemical characteristics of a cutinase from Fusarium oxysporum. Protein Expression Purif. 2016, 127, 1−7. (13) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1−2), 248−254. (14) Vafiadi, C.; Topakas, E.; Alissandratos, A.; Faulds, C. B.; Christakopoulos, P. Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation. J. Biotechnol. 2008, 133 (4), 497−504. (15) Kartal, F.; Janssen, M. H. A.; Hollmann, F.; Sheldon, R. A.; Kılınc, 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. (16) Tandjaoui, N.; Tassist, A.; Abouseoud, M.; Couvert, A.; Amrane, A. Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of Brassica rapa peroxidase. Biocatal. Agric. Biotechnol. 2015, 4 (2), 208−213. (17) Pirozzi, D.; Greco, G. Lipase-catalyzed transformations for the synthesis of butyl lactate: a comparison between esterification and transesterification. Biotechnol. Prog. 2006, 22 (2), 444−448. (18) Dutta, K.; Dasu, V. V. Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320. J. Mol. Catal. B: Enzym. 2011, 72 (3−4), 150−156. (19) de Barros, D. P. C.; Fonseca, L. P.; Fernandes, P.; Cabral, J. M. S.; Mojovic, L. Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent. J. Mol. Catal. B: Enzym. 2009, 60 (3−4), 178−185. (20) de Barros, D. P. C.; Fernandes, P.; Cabral, J. M. S.; Fonseca, L. P. Synthetic application and activity of cutinase in an aqueous, miniemulsion model system: hexyl octanoate synthesis. Catal. Today 2011, 173 (1), 95−102. (21) de Barros, D. P. C.; Fonseca, L. P.; Cabral, J. M. S.; Weiss, C. K.; Landfester, K. Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media. Biotechnol. J. 2009, 4 (5), 674−683. (22) Escandell, J.; Wurm, D. J.; Belleville, M. P.; Sanchez, J.; Harasek, M.; Paolucci-Jeanjean, D. Enzymatic synthesis of butyl acetate in a packed bed reactor under liquid and supercritical conditions. Catal. Today 2015, 255, 3−9. (23) Cunnah, P. J.; Aires-Barros, M. R.; Cabral, J. M. S. Esterification and transesterification catalysed by cutinase in reverse micelles of CTAB for the synthesis of short chain esters. Biocatal. Biotransform. 1996, 14 (2), 125−146. (24) de Barros, D. P. C.; Azevedo, A. M.; Cabral, J. M. S.; Fonseca, L. P. Optimization of flavor etsers synthesis by Fusarium solani pisi cutinase. J. Food Biochem. 2012, 36 (3), 275−284.

Figure 6. Conversion of VB for each run of reactions under the optimal conditions for 6 h reusing the same biocatalyst.

production of a flavor compound, underpinning its industrial potential for use in food bioprocesses.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00659. Semiquantitative protein estimation of Focut5a cutinase in the cell-free extract of E. coli BL21 cells; percentage of recombinant protein was determined by SDS-PAGE using a gel imaging and analysis system (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(E.T.) Phone: +30-210-7723264. Fax: +30-210-7723163. Email: [email protected]. ORCID

Evangelos Topakas: 0000-0003-0078-5904 Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.7b00659 J. Agric. Food Chem. 2017, 65, 3505−3511