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Biochemical characterization of highly organic solvent-tolerant cutinase from Fusarium oxysporum
Biocatalysis and Agricultural Biotechnology 2 (2013) 372–376
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Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Original Research Paper
Biochemical characterization of highly organic solvent-tolerant cutinase from Fusarium oxysporum Paula Speranza n, Gabriela Alves Macedo Department of Food Science, Faculty of Food Engineering—University of Campinas, Rua Monteiro Lobato, 80, Caixa Postal 6121, CEP 13083-970 Campinas, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 15 March 2013 Received in revised form 25 June 2013 Accepted 25 June 2013 Available online 5 July 2013
Biochemical characterization of cutinase from Fusarium oxysporum produced by submerged fermentation indicated that the enzyme showed a significant increase in activity, after exposure for 1 h in organic solvents, especially octanol, 2-nonanol, hexane, octane, isooctane and decane. After exposure to these solvents, the enzyme activity was higher by more than 40% when compared with the control without exposure to organic solvents. The highest activity observed in organic solvents, indicates the great potential of this enzyme in non-aqueous systems. The enzyme retained its activity almost completely in the temperature range between 28 and 50 1C, maintaining its activity more than 80% after 1 h at these temperatures. The enzyme retained its activity superior to 50% in the pH range between 5.6 and 7.0 after 24 h at 30 1C. The multivariate study showed that the activity is higher at temperature of 28 1C and pH 6.0. The enzyme has been activated in most metal ions tested, with the best result observed in the presence of Na+ (1 mM). The enzyme exhibited low activity in the ionic liquids tested (BMIM-PF4, BMIMPF6). The presence of sodium oxalate, sodium citrate, sodium bisulfite and sodium azide (1 mM), significantly increased the activity of the enzyme, indicating that these salts bind to certain metal ions, which interfere with the action of the enzyme. The biochemical properties observed, indicating the potential of this enzyme for industrial and biotechnological applications. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Enzyme Cutinase Fusarium oxysporum Characterization Organic solvent
1. Introduction Cutinase (3.1.1.74) secreted by phytopathogenic microorganisms are carboxylic ester hydrolases that are capable of degrading cutin polymers of plant cell walls (Purdy and Kolattukudy, 1975). The enzyme has hydrolytic activity on a wide variety of esters such as water-soluble simple esters and insoluble triglycerides (Maeda et al., 2005; Pio et al., 2008). Positive results were obtained in hydrolysis reaction of polyesters to obtain a material with higher hydrophilicity (Eberl et al., 2009). Cutinase has shown positive results for the removal of fat from cotton. The removal of this fat prevents the occurrence of the breakdown of triglycerides into smaller fragments during washing. These fragments have rancid smell and would be hardly removed from the interior of the fibers by normal detergents (Silva et al., 2012). Cutinase has shown a great potential for esterification and transesterification reactions in different reaction media, where it showed selectivity toward the production of short-chains carboxylic acid esters (Barros et al., 2012). It has been successfully
n
Corresponding author. Tel.: +55 19 3521 2175; fax: +55 19 3521 2153. E-mail addresses: [email protected], [email protected] (P. Speranza). 1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.06.005
applied in the production of aroma compounds (Dutta and Dasu, 2011; Barros et al., 2009). Recent works indicates that this enzyme is capable of catalyzing the separation of enantiomers, with possible applications in areas such as pharmaceuticals, chemical and foods (Speranza et al., 2011; Fraga et al., 2012). Such applications often involve the use of organic solvents; therefore enzymes that exhibit tolerance to these solvents are of great interest. The use of organic solvents in reaction media shifts the thermo dynamic equilibrium to favor synthesis over hydrolysis. Furthermore, in organic solvent the conformation of the enzyme appear to be more rigid. These characteristics enable controlling some of the enzyme's catalytic properties, such as the substrate specificity, the chemo-regio and enantioselectivity by variation of the solvent (Faber, 2011). One relevant limitation to industrial application of microbial enzymes is their production cost, which is determined by the production yield, experimental conditions of the process and enzyme stability. Therefore the search for new sources of microbial enzymes and their characterization can enable the application of them. Understanding the influence of parameters such as pH, temperature, metal ions, organic solvents and chelating agents is essential and should be evaluated and optimized. In the literature, a few studies indicate that cutinase shows an increase in its enzymatic activity after exposure to organic solvents. Thus, the
P. Speranza, G. Alves Macedo / Biocatalysis and Agricultural Biotechnology 2 (2013) 372–376
present study, investigated the detailed biochemical properties of an organic solvent-tolerant cutinase from Fusarium oxysporum.
2. Materials and Methods p-Nitrophenylbutyrate (pNPB) was purchased from SigmaAldrich Brazil Co. (São Paulo, SP, Brazil). All other reagents and solvents were purchased from Merck (São Paulo, SP, Brazil). 2.1. Microorganism preservation and preparation of the preinoculum A F. oxysporum strain (CBMAI 1274) isolated from soil and plants was selected in a previous study with 400 strains of fungi as the best cutinase producer. The culture medium used for this previous selection contained apple cutin as the sole carbon source (Macedo and Pio, 2005). The strain was maintained in potato dextrose agar slants and stored at 4 1C. The pre-inoculum was prepared by adding 2.5 mL of distilled water to remove the spores, obtaining a suspension containing 1.2 108 spores/mL. 2.2. Cutinase production The medium used for the development of the microorganism and production of the enzyme has been described in previously published work (Pio and Macedo, 2007). The fermented culture medium was filtered through a Whatman no. 1 paper and treated with solid ammonium sulfate (80%) overnight at 4 1C. The precipitate was collected by centrifugation (9050g for 15 min at 4 1C), dissolved in distilled water (enough to clear the pellet in the centrifuge tubes) and dialyzed against distilled water. The preparation was freeze-dried and used as crude cutinase preparations in all experiments. The initial activity of cutinase produced in this study was 3.4 U/mg. 2.3. Cutinase assay The activity against p-NPB (p- nitrophenyl butyrate) was determined as previously reported (Macedo and Pio, 2005). The hydrolysis of p-NPB was spectrophotometrically monitored for the formation of p-nitrophenol at 405 nm. One unit of cutinase activity was defined as the amount of enzymes required to convert 1 mmol of p-NPB to p-nitrophenol per minute, under the specified conditions (Calado et al., 2002). 2.4. Biochemical characteristics of the crude cutinase 2.4.1. pH stability The pH of the crude cutinase for cutinolytics activities was determined by using the following buffers (all at 50 mM): acetate buffer pH 3.6, 4.0, 5.0, 5.6; phosphate buffer pH 6.0, 6.5, 7.0; TrisHCl buffer pH 8.0, 8.5, 9.0 and borate–NaOH pH 9.5, 10.0. A mixture of 5 mg of crude cutinase and 1 mL of buffer given above was incubated for 24 h at 30 1C. After this period, the enzyme activity was determined with the cutinase assay described previously. The percentage residual activities were calculated by comparison with untreated control enzyme. 2.4.2. Thermal stability In order to determine the enzyme activity after being subjected to different temperatures, aliquots of 5 mg/mL of crude cutinase prepared in 50 mM phosphate buffer (pH 7.0) in Eppendorf tubes were incubated for 1 h at temperatures between 30 1C to the boiling temperature. After incubation, the tubes were rapidly cooled in an ice bath and then brought to room temperature.
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The activity was determined with the cutinase assay described previously. The percentage residual activities were calculated by comparison with untreated control enzyme. 2.4.3. Multivariate study of the effect of temperature and pH on cutinase stability Initially, the parameters pH and temperature were evaluated independently, as indicated in the above items. The values to which the enzyme showed greater activity were used in a central composite rotatable design (CCRD) in order to check possible interactions between these parameters. A 22 factorial design with 4 central points and 4 axial points, totalizing 12 experiments is shown in Table 1. 2.4.4. Effect of metal ions, some chemicals and chelating agent on cutinolytic activity The effects of Ca2+, Hg2+, Mn2+, Co2+, Fe2+, Mg2+, Zn2+, Na+, K+, ethylenediamine tetraacetic acid (EDTA), urea, ρ-cloromercuriobenzoic, sodium oxalate, sodium citrate, sodium laurel sulfate, sodium bissulphite, iodoacetamide, 2-mercaptoethanol, sodium azide and n-bromosuccinamide on the cutinolytic activity were investigated. Final concentrations of each metal ion in the reaction mixture were 1 mM and 10 mM. The percentages of activities were determined by comparison with the standard assay mixture with no metal ion, chemicals or chelating added. 2.4.5. Activity in non-aqueous system The activity of the crude cutinase in methanol, ethanol, propanol, butanol, heptanol, octanol, 2-nonanol, hexane, octane, isooctane, decane, toluene, acetone, tetrahydrofuran, 1-butyl-3methyl imidazolium hexafluorophosphate (BMIM-PF4) and 1-butyl-3-methyl imidazolium tetrafluorophosphate (BMIM-PF6) were tested. A mixture of 5 mg of crude cutinase and 1 ml of each organic solvent or ionic liquid described above, was incubated for 1 h at 30 1C. After this period, the solvent/ionic liquid was dried with N2 and the activities were determined in an aqueous medium. Cutinolytic activity was compared with the mixture without an organic solvent/ionic liquid added. 2.4.6. Statistical analysis All values reported in biochemical characterization of the enzyme represent the mean from duplicates; error bars represent the range of values. Significant differences between enzymes biochemical properties were determined by analysis of variance. The software STATISTICA v.8.0 (StatSoft, Inc., USA) was used for the statistical analyses. Table 1 Coded levels and real values (in parentheses) for the factorial design (12 trials) and cutinase activity (U/mg). Run assay
1 2 3 4 5 6 7 8 9 10 11 12
Variables
Response
Temperature (1C)
pH
Cutinase activity (U/mg)
-1.00 1.00 -1.00 1.00 -1.41 1.41 0.00 0.00 0.00 0.00 0.00 0.00
-1.00 (5.3) -1.00 (5.3) 1.00 (6.7) 1.00 (6.7) 0.00 (6.0) 0.00 (6.0) -1.41 (5.0) 1.41 (7.0) 0.00 (6.0) 0.00 (6.0) 0.00 (6.0) 0.00 (6.0)
4.4 2.4 5.5 2.9 8.7 2.3 3.6 4.6 5.9 6.4 6.1 6.4
(30) (50) (30) (50) (28) (54) (40) (40) (40) (40) (40) (40)
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3. Results and discussion
Table 3 ANOVA for the response of the dependent variables.
The use of cutinase in biotechnology process is growing and the characterization of new enzymes is required. Below are presented the results of biochemical characterization of the enzyme.
3.1. pH stability
Source Regression Residues Total SS F2,9;
0.05 ¼ 4.26,
Sum of squares
Degree of freedom
31.7 9.1
2 9 11
40.8
Mean square
F-test
15.8 1.0
15.8
R2 ¼0.80, p-value ¼0.001
Cutinase retained its activity almost completely at pH ranging from 5.6 to 6.0, when incubated for 24 h at 30 1C. At pH 7.0 and 5.0, the enzyme retained about 50% of its activity; with a decrease in pH there is a fast decay in enzyme activity. At pH 4.5 the enzyme retains 11% of its activity. The activity of the enzyme decreased gradually at alkaline pH; retaining 7.5% of its activity at pH 10.0. Previous work conducted in our laboratory with cutinase from F. oxysporum produced by solid-state fermentation presented similar results (Speranza et al., 2011).
3.2. Thermal stability The cutinase retained its activity almost completely in the temperature range of 30 and 50 1C when incubated for 1 h. At 60 1C the enzyme retains 20% of its activity, decreasing slowly with increasing temperature. At 100 1C the enzyme retains 9.3% of its activity. Other studies indicate that cutinase shows the range of temperature activity near encountered in this study (Petersen et al., 2001). The application of cutinase in the synthesis of aroma compounds indicated that the reaction was more efficiently in the temperature range between 35 and 40 1C (Dutta and Dasu, 2011). The enzymatic transesterification for the production of biodiesel, using cutinase as catalyzer, was successfully operated at the temperature of 30 1C (Maeda et al., 2005). Therefore, this range of temperature activity, allows the use of this enzyme in different process industries, indicating the versatility of this enzyme.
3.3. Multivariate study of the effect of temperature and pH on enzyme activity The results obtained from the 22 central composite design runs and respective analysis conditions are shown in Table 1. The results indicate that the cutinase activity varied widely in the range of pH and temperature studied, with values between 2.3 and 8.7 U/mg. The highest activity was obtained at pH 6.0 and temperature of 28 1C. The central point has a small variance, indicating good repeatability of the analysis. Table 2 shows the regression coefficients for the coded secondorder polynomial equation. The parameters pH and temperature do not show interaction. The non-significant terms were eliminated during the regression and eliminated from the resulting model (Eq. (1)). The resulting model was tested for adequate fitness by ANOVA. The fitted model was suitable, showing significant regression, low residual values, no lack of fit, and Table 2 Coefficients estimates by the regression mode in CCRD. Factor
Effect
Std. Err.
Mean/intercept Temperature (1C) (L)a Temperature (1C) (Q) pH (L) pH (Q)a Temperature pH
6.20 1.71 0.62 0.37 1.28 0.16
0.44 0.31 0.35 0.31 0.35 0.44
a
Significant factors (po 0.05)
t-value
p-value
14.01 5.48 1,77 1.19 3.67 0.36
o 0.001 o 0.001 0.12 0.28 0.01 0.73
Fig. 1. Response surface plots showing the effect of temperature and pH on the cutinase activity.
satisfactory determination coefficient (Table 3). Y ¼ 5:7–1:7x1 –1:2x22
ð1Þ
Where: cutinase activity is (Y), temperature (x1), and pH (x2). Fig. 1 shows the surface response obtained for the model. The enzyme activity was affected by the variables independently. The reduction in temperature provides an increase in enzyme activity. The highest activity was obtained at a temperature of 28 1C, and the enzyme retains up to 42% of this activity at temperatures up to 40 1C. Since most industrial applications using cutinase occur at temperatures around or above 30 1C, analysis at temperatures below 28 1C were not performed. Regarding pH, it was observed that the enzyme activity was higher in the range between 6.0 and 6.5, and retains high activity over the pH range studied (Silva et al., 2012; Dutta and Dasu, 2011). 3.4. Effect of metal ions, chemicals and chelating agent on cutinolytic activity To determine whether the cutinase requires cation cofactor for activity, it was incubated with the metal ions and then assayed for cutinase activity against p-NPB. It is known that a cation may function as an essential component of the active site of an enzyme, may be part of an essential cofactor or may be necessary to maintain the conformation of the active site of an enzyme (Whitaker, 1994). Therefore, the evaluation of these metals may help to elucidate the composition of the active site of the enzyme, and predict the behavior of the enzyme in complex mixtures, where these metals may be present. The results in Table 4 indicate that the enzyme was activated in the presence of most metal ions in a concentration of 1 mM, particularly Ca2, Fe2+, Mn2+ and Na+. This result may be confirmed by the inhibitory effect of the enzyme in the presence of EDTA, a known chelating metal. The enzyme was quite inhibited in the presence of Hg2+ in both concentrations, indicating that sulfhydryl groups are involved in the active site of the enzyme (Kademi et al., 2000). At higher concentration of ρ-cloromercuriobenzoic, occurs inhibition of cutinase, this compound form mercaptides with
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Table 4 Effect of metal ions, chemicals and chelating agent on cutinase activity. Values are means 7 SD. Relative activity (%) Metal ions, chelator and chemicals
Control CaCl2 HgCl2 KCl MnCl2 CoCl2 K2HPO4 NaNO3 FeSO4 MgSO4 ZnSO4 MnSO4 K2SO4 Na2SO4 NaHSO3 EDTA Urea 4-Cloromercuriobenzoic Sodium oxalate Sodium citrate Sodium lauryl suphate Sodium bissulphite Iodoacetamide 2-mercaptoethanol Sodium azide n-bromosuccinamide
1 mM
10 mM
100.0 73.9 429.8 7 4.1 N.D. 285.4 7 2.1 272.9 7 3.9 411.0 72.8 327.2 7 3.7 360.0 73.9 429.8 73.9 292.6 7 5.1 359.1 73.8 413.6 7 4.9 589.5 74.7 522.6 75.2 293.3 73.8 89.6 7 5.2 118.5 7 4.9 119.2 7 3.2 624.37 3.8 512.7 7 2.9 134.4 7 3.8 293.3 74.0 153.2 7 3.9 117.0 7 2.1 454.9 74.2 154.6 7 5.1
100.0 73.2 218.4 7 6.1 N.D. 151.3 73.1 N.D. N.D. 350.67 5.3 205.2 74.8 199.2 7 2.7 120.2 7 3.1 15.2 72.1 60.9 7 4.0 145.6 7 3.2 128.3 7 4.2 90.2 7 5.1 42.6 7 4.4 60.2 7 4.8 N.D.3.3 160.4 7 2.2 130.2 7 3.9 N.D. 90.2 7 2.1 28.5 7 3.0 200.8 7 3.3 291.8 7 2.8 95.17 2.2
N.D.: no detectable activity.
essential sulfhydryl group of the enzyme. The presence of potentially reactive thiol group in cutinase is also suggested by the observation that iodoacetamide in a high concentration also has inhibited the action of cutinase; this compound binds to the thiol group of the enzyme irreversibly (Whitaker, 1994). β-mercaptoethanol as reducing agent of disulfide bridges stimulated enzyme activity. The inhibition of cutinase in the presence of Zn2+ and Co2 may be due to direct inhibition of the catalytic site and/or formation of complexes between metal ions and amino acid residues with negative charge in specific sites. The increase in enzyme activity in the presence of sodium azide, sodium oxalate, sodium citrate and sodium bisulfite can be attributed to the ability of these salts complex with metals such as copper, zinc and molybdenum, which as shown in Table 4, show inhibitory effect on enzymatic activity. Urea in high concentration had an inhibitory effect on the enzyme; this compound disrupts hydrophobic interactions, which maintain the conformation of the enzyme (Nelson and Cox, 2000). The application of enzymes in industrial scale often occurs in unfavorable conditions, such as in the presence of surfactants. The increased activity of cutinase in the presence of 1 mM of sodium lauryl sulfate, an anionic surfactant, indicates the potential application of this enzyme in detergent formulations. Chen et al. Chen et al. (2010) evaluated the effect of different metal ions on the activity of two cutinases from Thermobifida fusca and one cutinase from Fusarium solani pisi recombinant. The results show that when the enzymes were incubated with 1 mM of metal ions divalent, Mn2+, Co2+, Mg2+, Ba2+, Cu2+ or Ca2+, they do not interfere in enzyme activity. However the ions, Zn2+, Fe2+ and Pb2+, showed inhibitory effect on the activity of enzymes, especially the bacterial enzymes. The Cr2+ ion almost completely inhibited the bacterial enzymes, while the ion Hg2+ completely inhibited the three cutinases.
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3.5. Activity in non-aqueous system The effect of organic solvents on the activity of cutinase is of great interest. Cutinase in the presence of such solvents have been reported to show esterification and transesterification activities Dutta and Dasu (2011; Speranza et al., 2011; Badenes et al., 2010). The results in Fig. 2 indicate that the enzyme after being maintained for 1 h with organic solvents, showed a significant increase in activity especially; octanol, 2-nonanol, hexane, octane, isooctane and decane. A possible explanation for this increase in activity may be attributable to changes in conformation of the enzyme during incubation caused by the interaction of solvent molecules with the enzyme. The results show that use of acetone, methanol, ethanol, propanol and butanol in reaction system leads greater inactivation of the cutinase. The explanation may be due to protein molecules in aqueous solution are surrounded by a hydration layer, which is composed of water molecules bound to the surface of the protein. Some organic solvents tend to displace the water molecules both in the hydration layer and in the interior of the protein, thereby distorting the interactions responsible for maintaining the native conformation of the enzymes (Akbari et al., 2009; Khemelntisky et al., 1991). The increase in polarity of watermiscible solvents leads to increase the enzyme inactivation because of essential water stripping by polar organic solvent. Thus, solvent polarity is one of factors determining the activity and stability of the enzymes (Kwon et al., 2009; Macedo and Pio, 2005). These results show the potential of this enzyme in organic synthesis and its application in areas such as productions of biodiesel (Badenes et al., 2010), flavorings (Dutta and Dasu, 2011) and the separation of racemic mixtures (Speranza et al., 2011). The enzyme has a low stability in ionic liquids tested. These compounds probably act as enzyme deactivating agent by stripping essential water molecules from the enzyme micro environmental in reaction media. Speranza et al. Speranza et al. (2011) evaluated the stability of cutinases from F. oxysporum produced in different solid-state fermentation. The enzyme produced in medium with soybean rind and rice bran was relatively stable in the presence of hexane keeping 71% and 66%, respectively of their activities compared with control. The enzyme produced in medium with Jatropha curcas seed cake was fairly stable in the presence of organic solvents, its activity remained high in all solvents evaluated, highlighting the hexane, butanol and propanol. The results indicate that the cutinase from F. oxysporum produced by submerged or solid-state fermentation is quite stable to the presence of organic solvents.
Fig. 2. Effects of organic solvents and ionic liquids on the activity of cutinase.
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Chen et al. Chen et al. (2010) evaluated the effect of organic solvent on the activity of two cutinases from Thermobifida fusca and one cutinase from F. solani pisi recombinant. The bacterial cutinases were more stable to organic solvents than the fungal cutinase, exhibited tolerance to methanol, ethanol, acetone, n-hexane and dimethyl sulfoxide, but were less stable in isopropanol and butanol. In contrast, the fungal cutinase was very unstable in these solvents except in n-hexane in which nearly 70% activity remained.
4. Conclusions The stability of enzymes is one of the most important factors that limit their industrial application, the results demonstrated that cutinase from F. oxysporum has an increase of its activity after being incubated in organic solvents, which is an essential feature in many organic synthesis. Furthermore, the results indicate that chelating agents and minerals can greatly enhance the activity of the enzyme, which may represent a major advance in order to enable the application of cutinases in complex mixtures, where these compounds can be present naturally. Future work of immobilized enzyme can be tested with the aim of increasing its stability and make the enzyme more attractive for biotechnological applications.
Acknowledgment The authors wish to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship. References Akbari, N., Khajeh, K., Rezaie, S., Mirdamadi, S., Shavandi, M., Ghaemi, N., 2009. High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding. Protein Expression Purif. 70, 75–80. Badenes, S.M., Lemos, F., Cabral, J.M.S., 2010. Assessing the use of cutinase reversed micellar catalytic system for the production of biodiesel from triglycerides. J. Chem. Technol. Biotechnol. 2010 (85), 993–998. Barros, D.P.C., Fonseca, L.P., Fernandes, P., Cabral, J.M.S., Mojovic, L., 2009. Biosynthesis of ethyl caproate and other short ethy esters catalyzed by cutinase in organic solvente. J. Mol. Catal. B: Enzym. 60, 178–185.
Barros, D.P.C., Azevedo, A.M., Cabral, J.M.S., Fonseca, L.P., 2012. Optimization of flavor esters synthesis by Fusarium solani pisi cutinase. J. Food Biochem. 36, 275–284. Calado, C.R.C., Monteiro, S.M.S., Cabral, J.M.S., Fonseca, L.P., 2002. Effect of prefermentation on the production of cutinase by recombinant Saccharomyces cerevisiae. J. Biosci. Bioeng. 93, 354–359. Chen, S., Lingqia, S., Billig, S., Zimmermann, W., Chen, J., Wu, J., 2010. Biochemical characterization of cutinase from Thermobifida fusca. J. Mol. Catal. B: Enzym. 63, 121–127. Dutta, K., Dasu, V.V., 2011. Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320. J. Mol. Catal. B: Enzym. 72, 150–156. Eberl, A., Heumann, S., Bruckener, T., Araujo, R., Cavaco-Paulo, A., Kaufmann, F., Kroutil, W., Guebitz, G.M., 2009. Enzymatic surface hydrolysis of poly (ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules. J. Biotechnol. 143, 207–212. Faber, K., 2011. Biotransformation in organic chemistry. Springer, New York. Fraga, L.P., Carvalho, P.O., Macedo, G.A., 2012. Production of cutinase by Fusarium oxysporum on brazilian agricultural by-products and its enantioselective properties. Food Bioprocess Technol. 5, 138–146. Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., Barati, J., 2000. Purification and characterization of a thermostable esterase from the moderate thermophilic bacterium Bacillus circulans. Appl. Microbiol. Biotechnol. 54, 173–179. Khemelntisky, Y.L., Mozhaev, V., Belova, V., Sergeeva, M.V., Martinek, K., 1991. Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis. Eur. J. Biochem. 198, 31–41. Kwon, M.A., Kim, H.S., Yang, T.H., Song, B.K., Song, J.K., 2009. High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris. Protein Expression Purif. 68, 104–109. Macedo, G.A., Pio, T.F., 2005. A rapid screening method for cutinase producing microrganisms. Braz. J. Microbiol. 36, 388–394. Maeda, H., Yamagata, Y., Abe, K., Hasegawa, F., Machida, M., Ishioka, R., Gomi, K., Nakajima, T., 2005. Purification and characterization of a biodegradable plasticdegrading enzyme from Aspergillus oryzae. Appl. Microbiol. Biotecnol. 2005 (67), 778–788. Nelson, D.L., Cox, M.M., 2000. Lehninger Principles of Biochemistry, Third Edition Worth Publisher, New York. Petersen, S.B., Fojan, P., Petersen, E.I., 2001. Petersen MTN. The thermal stability of the Fusarium solani pisi cutinase as a function of pH. J. Biomed. Biotechnol., 62–69. Pio, T.F., Macedo, G.A., 2007. Optimizing the production of cutinase by Fusarium oxysporum using response surface methodology. Enz. Microbiol Technol. 41, 613–619. Pio, T.F., Macedo, G.A., Kamimura, E.S., 2008. Fungal cutinases: properties and industrial applications. Quím. Nova 2008 (31), 2118–2123. Purdy, RE, Kolattukudy, PE., 1975. Hydrolysis of plant cuticle by plant pathogens. Prop. Cutinase I, Cutinase II and a Nonspecific Esterase Isolated from Fusarium solani pisi. Biochem.-US 14, 2832–2840. Silva, C., Azoia, N.G., Martins, M., Matamá, T., Wu, J., Cavaco-Paulo, A., 2012. Molecular recognition of esterase plays a major role on the removal of fatty soils during detergency. J. Biotechnol. 161, 228–234. Speranza, P, Carvalho, PO, Macedo, GA., 2011. Effects of different solid state fermentation substrate on biochemical properties of cutinase from Fusarium sp. J. Mol. Catal. B: Enzym. 72, 181–186. Whitaker, J.R., 1994. Principles of enzymology for the food sciences. Marcel Dekker, Inc., New York.
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Original Research Paper
Biochemical characterization of highly organic solvent-tolerant cutinase from Fusarium oxysporum Paula Speranza n, Gabriela Alves Macedo Department of Food Science, Faculty of Food Engineering—University of Campinas, Rua Monteiro Lobato, 80, Caixa Postal 6121, CEP 13083-970 Campinas, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 15 March 2013 Received in revised form 25 June 2013 Accepted 25 June 2013 Available online 5 July 2013
Biochemical characterization of cutinase from Fusarium oxysporum produced by submerged fermentation indicated that the enzyme showed a significant increase in activity, after exposure for 1 h in organic solvents, especially octanol, 2-nonanol, hexane, octane, isooctane and decane. After exposure to these solvents, the enzyme activity was higher by more than 40% when compared with the control without exposure to organic solvents. The highest activity observed in organic solvents, indicates the great potential of this enzyme in non-aqueous systems. The enzyme retained its activity almost completely in the temperature range between 28 and 50 1C, maintaining its activity more than 80% after 1 h at these temperatures. The enzyme retained its activity superior to 50% in the pH range between 5.6 and 7.0 after 24 h at 30 1C. The multivariate study showed that the activity is higher at temperature of 28 1C and pH 6.0. The enzyme has been activated in most metal ions tested, with the best result observed in the presence of Na+ (1 mM). The enzyme exhibited low activity in the ionic liquids tested (BMIM-PF4, BMIMPF6). The presence of sodium oxalate, sodium citrate, sodium bisulfite and sodium azide (1 mM), significantly increased the activity of the enzyme, indicating that these salts bind to certain metal ions, which interfere with the action of the enzyme. The biochemical properties observed, indicating the potential of this enzyme for industrial and biotechnological applications. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Enzyme Cutinase Fusarium oxysporum Characterization Organic solvent
1. Introduction Cutinase (3.1.1.74) secreted by phytopathogenic microorganisms are carboxylic ester hydrolases that are capable of degrading cutin polymers of plant cell walls (Purdy and Kolattukudy, 1975). The enzyme has hydrolytic activity on a wide variety of esters such as water-soluble simple esters and insoluble triglycerides (Maeda et al., 2005; Pio et al., 2008). Positive results were obtained in hydrolysis reaction of polyesters to obtain a material with higher hydrophilicity (Eberl et al., 2009). Cutinase has shown positive results for the removal of fat from cotton. The removal of this fat prevents the occurrence of the breakdown of triglycerides into smaller fragments during washing. These fragments have rancid smell and would be hardly removed from the interior of the fibers by normal detergents (Silva et al., 2012). Cutinase has shown a great potential for esterification and transesterification reactions in different reaction media, where it showed selectivity toward the production of short-chains carboxylic acid esters (Barros et al., 2012). It has been successfully
n
Corresponding author. Tel.: +55 19 3521 2175; fax: +55 19 3521 2153. E-mail addresses: [email protected], [email protected] (P. Speranza). 1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.06.005
applied in the production of aroma compounds (Dutta and Dasu, 2011; Barros et al., 2009). Recent works indicates that this enzyme is capable of catalyzing the separation of enantiomers, with possible applications in areas such as pharmaceuticals, chemical and foods (Speranza et al., 2011; Fraga et al., 2012). Such applications often involve the use of organic solvents; therefore enzymes that exhibit tolerance to these solvents are of great interest. The use of organic solvents in reaction media shifts the thermo dynamic equilibrium to favor synthesis over hydrolysis. Furthermore, in organic solvent the conformation of the enzyme appear to be more rigid. These characteristics enable controlling some of the enzyme's catalytic properties, such as the substrate specificity, the chemo-regio and enantioselectivity by variation of the solvent (Faber, 2011). One relevant limitation to industrial application of microbial enzymes is their production cost, which is determined by the production yield, experimental conditions of the process and enzyme stability. Therefore the search for new sources of microbial enzymes and their characterization can enable the application of them. Understanding the influence of parameters such as pH, temperature, metal ions, organic solvents and chelating agents is essential and should be evaluated and optimized. In the literature, a few studies indicate that cutinase shows an increase in its enzymatic activity after exposure to organic solvents. Thus, the
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present study, investigated the detailed biochemical properties of an organic solvent-tolerant cutinase from Fusarium oxysporum.
2. Materials and Methods p-Nitrophenylbutyrate (pNPB) was purchased from SigmaAldrich Brazil Co. (São Paulo, SP, Brazil). All other reagents and solvents were purchased from Merck (São Paulo, SP, Brazil). 2.1. Microorganism preservation and preparation of the preinoculum A F. oxysporum strain (CBMAI 1274) isolated from soil and plants was selected in a previous study with 400 strains of fungi as the best cutinase producer. The culture medium used for this previous selection contained apple cutin as the sole carbon source (Macedo and Pio, 2005). The strain was maintained in potato dextrose agar slants and stored at 4 1C. The pre-inoculum was prepared by adding 2.5 mL of distilled water to remove the spores, obtaining a suspension containing 1.2 108 spores/mL. 2.2. Cutinase production The medium used for the development of the microorganism and production of the enzyme has been described in previously published work (Pio and Macedo, 2007). The fermented culture medium was filtered through a Whatman no. 1 paper and treated with solid ammonium sulfate (80%) overnight at 4 1C. The precipitate was collected by centrifugation (9050g for 15 min at 4 1C), dissolved in distilled water (enough to clear the pellet in the centrifuge tubes) and dialyzed against distilled water. The preparation was freeze-dried and used as crude cutinase preparations in all experiments. The initial activity of cutinase produced in this study was 3.4 U/mg. 2.3. Cutinase assay The activity against p-NPB (p- nitrophenyl butyrate) was determined as previously reported (Macedo and Pio, 2005). The hydrolysis of p-NPB was spectrophotometrically monitored for the formation of p-nitrophenol at 405 nm. One unit of cutinase activity was defined as the amount of enzymes required to convert 1 mmol of p-NPB to p-nitrophenol per minute, under the specified conditions (Calado et al., 2002). 2.4. Biochemical characteristics of the crude cutinase 2.4.1. pH stability The pH of the crude cutinase for cutinolytics activities was determined by using the following buffers (all at 50 mM): acetate buffer pH 3.6, 4.0, 5.0, 5.6; phosphate buffer pH 6.0, 6.5, 7.0; TrisHCl buffer pH 8.0, 8.5, 9.0 and borate–NaOH pH 9.5, 10.0. A mixture of 5 mg of crude cutinase and 1 mL of buffer given above was incubated for 24 h at 30 1C. After this period, the enzyme activity was determined with the cutinase assay described previously. The percentage residual activities were calculated by comparison with untreated control enzyme. 2.4.2. Thermal stability In order to determine the enzyme activity after being subjected to different temperatures, aliquots of 5 mg/mL of crude cutinase prepared in 50 mM phosphate buffer (pH 7.0) in Eppendorf tubes were incubated for 1 h at temperatures between 30 1C to the boiling temperature. After incubation, the tubes were rapidly cooled in an ice bath and then brought to room temperature.
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The activity was determined with the cutinase assay described previously. The percentage residual activities were calculated by comparison with untreated control enzyme. 2.4.3. Multivariate study of the effect of temperature and pH on cutinase stability Initially, the parameters pH and temperature were evaluated independently, as indicated in the above items. The values to which the enzyme showed greater activity were used in a central composite rotatable design (CCRD) in order to check possible interactions between these parameters. A 22 factorial design with 4 central points and 4 axial points, totalizing 12 experiments is shown in Table 1. 2.4.4. Effect of metal ions, some chemicals and chelating agent on cutinolytic activity The effects of Ca2+, Hg2+, Mn2+, Co2+, Fe2+, Mg2+, Zn2+, Na+, K+, ethylenediamine tetraacetic acid (EDTA), urea, ρ-cloromercuriobenzoic, sodium oxalate, sodium citrate, sodium laurel sulfate, sodium bissulphite, iodoacetamide, 2-mercaptoethanol, sodium azide and n-bromosuccinamide on the cutinolytic activity were investigated. Final concentrations of each metal ion in the reaction mixture were 1 mM and 10 mM. The percentages of activities were determined by comparison with the standard assay mixture with no metal ion, chemicals or chelating added. 2.4.5. Activity in non-aqueous system The activity of the crude cutinase in methanol, ethanol, propanol, butanol, heptanol, octanol, 2-nonanol, hexane, octane, isooctane, decane, toluene, acetone, tetrahydrofuran, 1-butyl-3methyl imidazolium hexafluorophosphate (BMIM-PF4) and 1-butyl-3-methyl imidazolium tetrafluorophosphate (BMIM-PF6) were tested. A mixture of 5 mg of crude cutinase and 1 ml of each organic solvent or ionic liquid described above, was incubated for 1 h at 30 1C. After this period, the solvent/ionic liquid was dried with N2 and the activities were determined in an aqueous medium. Cutinolytic activity was compared with the mixture without an organic solvent/ionic liquid added. 2.4.6. Statistical analysis All values reported in biochemical characterization of the enzyme represent the mean from duplicates; error bars represent the range of values. Significant differences between enzymes biochemical properties were determined by analysis of variance. The software STATISTICA v.8.0 (StatSoft, Inc., USA) was used for the statistical analyses. Table 1 Coded levels and real values (in parentheses) for the factorial design (12 trials) and cutinase activity (U/mg). Run assay
1 2 3 4 5 6 7 8 9 10 11 12
Variables
Response
Temperature (1C)
pH
Cutinase activity (U/mg)
-1.00 1.00 -1.00 1.00 -1.41 1.41 0.00 0.00 0.00 0.00 0.00 0.00
-1.00 (5.3) -1.00 (5.3) 1.00 (6.7) 1.00 (6.7) 0.00 (6.0) 0.00 (6.0) -1.41 (5.0) 1.41 (7.0) 0.00 (6.0) 0.00 (6.0) 0.00 (6.0) 0.00 (6.0)
4.4 2.4 5.5 2.9 8.7 2.3 3.6 4.6 5.9 6.4 6.1 6.4
(30) (50) (30) (50) (28) (54) (40) (40) (40) (40) (40) (40)
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3. Results and discussion
Table 3 ANOVA for the response of the dependent variables.
The use of cutinase in biotechnology process is growing and the characterization of new enzymes is required. Below are presented the results of biochemical characterization of the enzyme.
3.1. pH stability
Source Regression Residues Total SS F2,9;
0.05 ¼ 4.26,
Sum of squares
Degree of freedom
31.7 9.1
2 9 11
40.8
Mean square
F-test
15.8 1.0
15.8
R2 ¼0.80, p-value ¼0.001
Cutinase retained its activity almost completely at pH ranging from 5.6 to 6.0, when incubated for 24 h at 30 1C. At pH 7.0 and 5.0, the enzyme retained about 50% of its activity; with a decrease in pH there is a fast decay in enzyme activity. At pH 4.5 the enzyme retains 11% of its activity. The activity of the enzyme decreased gradually at alkaline pH; retaining 7.5% of its activity at pH 10.0. Previous work conducted in our laboratory with cutinase from F. oxysporum produced by solid-state fermentation presented similar results (Speranza et al., 2011).
3.2. Thermal stability The cutinase retained its activity almost completely in the temperature range of 30 and 50 1C when incubated for 1 h. At 60 1C the enzyme retains 20% of its activity, decreasing slowly with increasing temperature. At 100 1C the enzyme retains 9.3% of its activity. Other studies indicate that cutinase shows the range of temperature activity near encountered in this study (Petersen et al., 2001). The application of cutinase in the synthesis of aroma compounds indicated that the reaction was more efficiently in the temperature range between 35 and 40 1C (Dutta and Dasu, 2011). The enzymatic transesterification for the production of biodiesel, using cutinase as catalyzer, was successfully operated at the temperature of 30 1C (Maeda et al., 2005). Therefore, this range of temperature activity, allows the use of this enzyme in different process industries, indicating the versatility of this enzyme.
3.3. Multivariate study of the effect of temperature and pH on enzyme activity The results obtained from the 22 central composite design runs and respective analysis conditions are shown in Table 1. The results indicate that the cutinase activity varied widely in the range of pH and temperature studied, with values between 2.3 and 8.7 U/mg. The highest activity was obtained at pH 6.0 and temperature of 28 1C. The central point has a small variance, indicating good repeatability of the analysis. Table 2 shows the regression coefficients for the coded secondorder polynomial equation. The parameters pH and temperature do not show interaction. The non-significant terms were eliminated during the regression and eliminated from the resulting model (Eq. (1)). The resulting model was tested for adequate fitness by ANOVA. The fitted model was suitable, showing significant regression, low residual values, no lack of fit, and Table 2 Coefficients estimates by the regression mode in CCRD. Factor
Effect
Std. Err.
Mean/intercept Temperature (1C) (L)a Temperature (1C) (Q) pH (L) pH (Q)a Temperature pH
6.20 1.71 0.62 0.37 1.28 0.16
0.44 0.31 0.35 0.31 0.35 0.44
a
Significant factors (po 0.05)
t-value
p-value
14.01 5.48 1,77 1.19 3.67 0.36
o 0.001 o 0.001 0.12 0.28 0.01 0.73
Fig. 1. Response surface plots showing the effect of temperature and pH on the cutinase activity.
satisfactory determination coefficient (Table 3). Y ¼ 5:7–1:7x1 –1:2x22
ð1Þ
Where: cutinase activity is (Y), temperature (x1), and pH (x2). Fig. 1 shows the surface response obtained for the model. The enzyme activity was affected by the variables independently. The reduction in temperature provides an increase in enzyme activity. The highest activity was obtained at a temperature of 28 1C, and the enzyme retains up to 42% of this activity at temperatures up to 40 1C. Since most industrial applications using cutinase occur at temperatures around or above 30 1C, analysis at temperatures below 28 1C were not performed. Regarding pH, it was observed that the enzyme activity was higher in the range between 6.0 and 6.5, and retains high activity over the pH range studied (Silva et al., 2012; Dutta and Dasu, 2011). 3.4. Effect of metal ions, chemicals and chelating agent on cutinolytic activity To determine whether the cutinase requires cation cofactor for activity, it was incubated with the metal ions and then assayed for cutinase activity against p-NPB. It is known that a cation may function as an essential component of the active site of an enzyme, may be part of an essential cofactor or may be necessary to maintain the conformation of the active site of an enzyme (Whitaker, 1994). Therefore, the evaluation of these metals may help to elucidate the composition of the active site of the enzyme, and predict the behavior of the enzyme in complex mixtures, where these metals may be present. The results in Table 4 indicate that the enzyme was activated in the presence of most metal ions in a concentration of 1 mM, particularly Ca2, Fe2+, Mn2+ and Na+. This result may be confirmed by the inhibitory effect of the enzyme in the presence of EDTA, a known chelating metal. The enzyme was quite inhibited in the presence of Hg2+ in both concentrations, indicating that sulfhydryl groups are involved in the active site of the enzyme (Kademi et al., 2000). At higher concentration of ρ-cloromercuriobenzoic, occurs inhibition of cutinase, this compound form mercaptides with
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Table 4 Effect of metal ions, chemicals and chelating agent on cutinase activity. Values are means 7 SD. Relative activity (%) Metal ions, chelator and chemicals
Control CaCl2 HgCl2 KCl MnCl2 CoCl2 K2HPO4 NaNO3 FeSO4 MgSO4 ZnSO4 MnSO4 K2SO4 Na2SO4 NaHSO3 EDTA Urea 4-Cloromercuriobenzoic Sodium oxalate Sodium citrate Sodium lauryl suphate Sodium bissulphite Iodoacetamide 2-mercaptoethanol Sodium azide n-bromosuccinamide
1 mM
10 mM
100.0 73.9 429.8 7 4.1 N.D. 285.4 7 2.1 272.9 7 3.9 411.0 72.8 327.2 7 3.7 360.0 73.9 429.8 73.9 292.6 7 5.1 359.1 73.8 413.6 7 4.9 589.5 74.7 522.6 75.2 293.3 73.8 89.6 7 5.2 118.5 7 4.9 119.2 7 3.2 624.37 3.8 512.7 7 2.9 134.4 7 3.8 293.3 74.0 153.2 7 3.9 117.0 7 2.1 454.9 74.2 154.6 7 5.1
100.0 73.2 218.4 7 6.1 N.D. 151.3 73.1 N.D. N.D. 350.67 5.3 205.2 74.8 199.2 7 2.7 120.2 7 3.1 15.2 72.1 60.9 7 4.0 145.6 7 3.2 128.3 7 4.2 90.2 7 5.1 42.6 7 4.4 60.2 7 4.8 N.D.3.3 160.4 7 2.2 130.2 7 3.9 N.D. 90.2 7 2.1 28.5 7 3.0 200.8 7 3.3 291.8 7 2.8 95.17 2.2
N.D.: no detectable activity.
essential sulfhydryl group of the enzyme. The presence of potentially reactive thiol group in cutinase is also suggested by the observation that iodoacetamide in a high concentration also has inhibited the action of cutinase; this compound binds to the thiol group of the enzyme irreversibly (Whitaker, 1994). β-mercaptoethanol as reducing agent of disulfide bridges stimulated enzyme activity. The inhibition of cutinase in the presence of Zn2+ and Co2 may be due to direct inhibition of the catalytic site and/or formation of complexes between metal ions and amino acid residues with negative charge in specific sites. The increase in enzyme activity in the presence of sodium azide, sodium oxalate, sodium citrate and sodium bisulfite can be attributed to the ability of these salts complex with metals such as copper, zinc and molybdenum, which as shown in Table 4, show inhibitory effect on enzymatic activity. Urea in high concentration had an inhibitory effect on the enzyme; this compound disrupts hydrophobic interactions, which maintain the conformation of the enzyme (Nelson and Cox, 2000). The application of enzymes in industrial scale often occurs in unfavorable conditions, such as in the presence of surfactants. The increased activity of cutinase in the presence of 1 mM of sodium lauryl sulfate, an anionic surfactant, indicates the potential application of this enzyme in detergent formulations. Chen et al. Chen et al. (2010) evaluated the effect of different metal ions on the activity of two cutinases from Thermobifida fusca and one cutinase from Fusarium solani pisi recombinant. The results show that when the enzymes were incubated with 1 mM of metal ions divalent, Mn2+, Co2+, Mg2+, Ba2+, Cu2+ or Ca2+, they do not interfere in enzyme activity. However the ions, Zn2+, Fe2+ and Pb2+, showed inhibitory effect on the activity of enzymes, especially the bacterial enzymes. The Cr2+ ion almost completely inhibited the bacterial enzymes, while the ion Hg2+ completely inhibited the three cutinases.
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3.5. Activity in non-aqueous system The effect of organic solvents on the activity of cutinase is of great interest. Cutinase in the presence of such solvents have been reported to show esterification and transesterification activities Dutta and Dasu (2011; Speranza et al., 2011; Badenes et al., 2010). The results in Fig. 2 indicate that the enzyme after being maintained for 1 h with organic solvents, showed a significant increase in activity especially; octanol, 2-nonanol, hexane, octane, isooctane and decane. A possible explanation for this increase in activity may be attributable to changes in conformation of the enzyme during incubation caused by the interaction of solvent molecules with the enzyme. The results show that use of acetone, methanol, ethanol, propanol and butanol in reaction system leads greater inactivation of the cutinase. The explanation may be due to protein molecules in aqueous solution are surrounded by a hydration layer, which is composed of water molecules bound to the surface of the protein. Some organic solvents tend to displace the water molecules both in the hydration layer and in the interior of the protein, thereby distorting the interactions responsible for maintaining the native conformation of the enzymes (Akbari et al., 2009; Khemelntisky et al., 1991). The increase in polarity of watermiscible solvents leads to increase the enzyme inactivation because of essential water stripping by polar organic solvent. Thus, solvent polarity is one of factors determining the activity and stability of the enzymes (Kwon et al., 2009; Macedo and Pio, 2005). These results show the potential of this enzyme in organic synthesis and its application in areas such as productions of biodiesel (Badenes et al., 2010), flavorings (Dutta and Dasu, 2011) and the separation of racemic mixtures (Speranza et al., 2011). The enzyme has a low stability in ionic liquids tested. These compounds probably act as enzyme deactivating agent by stripping essential water molecules from the enzyme micro environmental in reaction media. Speranza et al. Speranza et al. (2011) evaluated the stability of cutinases from F. oxysporum produced in different solid-state fermentation. The enzyme produced in medium with soybean rind and rice bran was relatively stable in the presence of hexane keeping 71% and 66%, respectively of their activities compared with control. The enzyme produced in medium with Jatropha curcas seed cake was fairly stable in the presence of organic solvents, its activity remained high in all solvents evaluated, highlighting the hexane, butanol and propanol. The results indicate that the cutinase from F. oxysporum produced by submerged or solid-state fermentation is quite stable to the presence of organic solvents.
Fig. 2. Effects of organic solvents and ionic liquids on the activity of cutinase.
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Chen et al. Chen et al. (2010) evaluated the effect of organic solvent on the activity of two cutinases from Thermobifida fusca and one cutinase from F. solani pisi recombinant. The bacterial cutinases were more stable to organic solvents than the fungal cutinase, exhibited tolerance to methanol, ethanol, acetone, n-hexane and dimethyl sulfoxide, but were less stable in isopropanol and butanol. In contrast, the fungal cutinase was very unstable in these solvents except in n-hexane in which nearly 70% activity remained.
4. Conclusions The stability of enzymes is one of the most important factors that limit their industrial application, the results demonstrated that cutinase from F. oxysporum has an increase of its activity after being incubated in organic solvents, which is an essential feature in many organic synthesis. Furthermore, the results indicate that chelating agents and minerals can greatly enhance the activity of the enzyme, which may represent a major advance in order to enable the application of cutinases in complex mixtures, where these compounds can be present naturally. Future work of immobilized enzyme can be tested with the aim of increasing its stability and make the enzyme more attractive for biotechnological applications.
Acknowledgment The authors wish to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship. References Akbari, N., Khajeh, K., Rezaie, S., Mirdamadi, S., Shavandi, M., Ghaemi, N., 2009. High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding. Protein Expression Purif. 70, 75–80. Badenes, S.M., Lemos, F., Cabral, J.M.S., 2010. Assessing the use of cutinase reversed micellar catalytic system for the production of biodiesel from triglycerides. J. Chem. Technol. Biotechnol. 2010 (85), 993–998. Barros, D.P.C., Fonseca, L.P., Fernandes, P., Cabral, J.M.S., Mojovic, L., 2009. Biosynthesis of ethyl caproate and other short ethy esters catalyzed by cutinase in organic solvente. J. Mol. Catal. B: Enzym. 60, 178–185.
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