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Immobilization of enzymatic extract from Penicillium camemberti with lipoxygenase activity onto a hybrid layered double hydroxide
Biochemical Engineering Journal 48 (2009) 93–98
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Immobilization of enzymatic extract from Penicillium camemberti with lipoxygenase activity onto a hybrid layered double hydroxide Rogelio Morales Borges a,∗ , Gregorio Guadalupe Carbajal Arizaga b,∗ , Fernando Wypych c a Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías. Universidad de Guadalajara. Blvd. Marcelino García Barragán 1421, CP 44430. Guadalajara, Jalisco, Mexico b Centro de Nanociencias y Nanotecnología. Universidad Nacional Autónoma de México. Apdo. Postal 356, CP 22800. Ensenada, Baja California, Mexico c Departamento de Química, Universidade Federal do Paraná, CP 19081, Centro Politécnico, CEP 81531-990. Curitiba, PR, Brazil
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Article history: Received 7 May 2009 Received in revised form 26 August 2009 Accepted 27 August 2009
Keywords: Enzyme Microbial Immobilized Lipoxygenase Composite
a b s t r a c t A Zn/Al layered double hydroxide was synthesized by alkaline co-precipitation with azelate ions (− OOC(CH2 )7 COO− ). The interlayer space of the layered material is occupied by organic ions as confirmed by X-ray diffraction and FTIR spectroscopy. The resulting hybrid material was tested as support for Penicillium camemberti enzymatic extract, containing lipoxygenase (LOX) activity. The optimal condition for LOX immobilization is done with 0.6 mol L−1 potassium phosphate buffer and pH 6.0. The affinity for the substrate is the same after immobilization, however the specific activity slightly decreases. The immobilization enhanced the thermal stability, which was evident with incubation at 60 ◦ C where the immobilized enzyme retains 68% of specific activity while the free enzyme is inhibited. Recycling assays showed that after eight reaction cycles, the immobilized enzyme retains 60% of the activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The layered double hydroxides (LDH), also known as synthetic clays, are compounds whose structure is a modification of the layered magnesium hydroxide, where a fraction of the cationic sites are occupied by trivalent cations, which causes an excess of positive charge neutralized by the presence of additional anions in the interlayer space. These compounds are represented by the general formula: [M2+ 1−x M3+ x (OH)2 ]x+ Am− x/m ·nH2 O; where: M2+ and M3+ are the cations involved and A, an anion of charge m− [1,2]. The physico-chemical characteristics of the interlayer space depends on the anion, which can be organic and could form interesting new supports for the immobilization of catalytic species through intercalation or through adsorption on the external surfaces. Despite size restrictions regarding to intercalation of enzymes, small organic intercalated anions allow to interact with the enzymes [3]. Other proposed mechanism is the interaction of the enzyme with OH groups at the outer surface of the LDH crystals. Lipoxygenases (EC 1.13.11.12) are oxidoreductases that catalyze the specific addition of molecular oxygen to polyunsaturated fatty acids (containing the cis, cis-1,4-pentadiene moiety) yielding the
∗ Corresponding authors. Tel.: +52 646 174 46 02; fax: +52 646 174 46 03. E-mail addresses: [email protected] (R.M. Borges), [email protected] (G.G.C. Arizaga). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.08.011
1,3-cis-transdiene-5-hydroperoxides. Among the fatty acids with this functionality, linoleic and linolenic acids are the best-known substrates for LOX [4]. Penicillium camemberti is a fungus widely used in food industry as starter culture. Volatile compounds are produced in P. camemberti due to its lipolytic and proteolytic activity, which contribute to the formation of the aroma and flavor of Brie and Camembert cheeses [5]. The production of certain natural flavor compounds, including C5 to C9 aliphatic alcohols and carbonyl compounds, results from a biosynthetic pathway involving several enzymatic activities, like lipoxygenase (LOX) and hydroperoxide lyase (HPL) with polyunsaturated fatty acids as substrates. Although the increasing interest in the LOX production for aroma compounds [5,6], the limited stability of these enzymes has restricted their biotechnological applications [7] as well as the possibility of the reusability of the biocatalyst in continuous packed-bed reactors [7]. Additional benefits of immobilization, include the easy separation of the enzyme from its end products, which minimizes downstream processing costs [8–10]. LDHs have the capability to immobilized enzymes like lipases [11–13], however, it is known that the organic acids used as substrates can be easily intercalated into the LDHs structures [14] or react to form esters in the presence of alcohols and LDH [15,16]. The two facts can lead to misinterpretation of the results. The objective of this study is to synthesize a Zn/Al LDH hybridized with azelate ions in order to saturate the positive charge of the layers and avoid the exchange reaction with linoleic acid,
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which is the substrate in the peroxidation reaction. The immobilization of an enzymatic extract with LOX activity, in addition to its importance in food industry, is ideal because the reaction to quantify the yield of the reaction involves the peroxide group, unlike the reactions with lipase, where the carboxylic groups is quantified, avoiding the misinterpretation of enzymatic activity of the immobilized enzyme. We also aim to find the optimal conditions for immobilization and the highest LOX activity. 2. Experiments Linoleic acid (cis-9, cis-12-octadecadienoic acid) was purchased from Nu-Chek Prep (Elysian, MN). Trishydroxy methylaminomethane (TRIS) was obtained from Aldrich (Milwaukee, WI). Xylenol orange [(3,3-bis(N,N-di(carboxymethyl)aminomethyl)-ocresol)] was purchased from Sigma Chemical Co. (St. Louis, MO). Mono and dibasic potassium phosphate were purchased from Fisher Scientific (Fair Lawn, NJ). The inorganic support was synthesized with reagents of analytical grade: Zn(NO3 )2 ·6H2 O and Al(NO3 )3 ·9H2 O were purchased from Vetec and Synth, Brazil.
of the Lowry method [18]. Bovine serum albumin (Sigma Chemical Co., St. Louis, MO) was used as a standard for the calibration curve. 2.3. Substrate preparation Linoleic acid was used as substrate for LOX studies. The stock solution was prepared at a concentration of 4.0 mmol L−1 in the appropriate buffer solutions (0.6 mol L−1 ), according to the procedure outlined by Perraud et al. [19]. 2.4. Effect of ionic strength and pH on protein immobilization The effect of ionic strength on protein immobilization was studied by varying the buffer molarity from 0.1 mol L−1 to 0.8 mol L−1 KH2 PO4 –K2 HPO4 . The immobilization was carried out at 4 ◦ C for 18 h, using 0.15 g of support and 2.5 mL of protein solution (1.5 mg mL−1 ). In a similar way, the most effective pH to immobilize the enzyme was detected by using a wide range of buffer solutions (0.1 mol L−1 ), including citrate phosphate for the pH range 3.0–5.5; potassium phosphate for pH 6.0–8.0 and glycine–NaOH for pH values between 8.5 and 9.0.
2.1. Synthesis of the LDH and modification with azelate ions 2.5. Immobilization of LOX The Zn/Al LDH was synthesized by co-precipitation. The salts Zn(NO3 )2 ·6H2 O and Al(NO3 )3 ·9H2 O, in a molar ratio Zn/Al = 3, were dissolved in decarbonated water and then precipitated by addition of NH4 OH at a final pH of 8.5. An aliquot from this suspension was taken to confirm whether at this point the LDH was formed. In a separated vessel, 300 mL of 0.25 mol L−1 azelate solution was prepared by dissolving azelaic acid, HOOC(CH2 )7 COOH, with NH4 OH. Finally, this azelate solution was added to the LDH suspension and stirred for 24 h at room temperature. The solid was recovered by centrifugation at 4000 rpm, washed with water four times and dried at 65 ◦ C for 24 h. The white powder was identified as LDHA. The structures were analyzed by powder X-ray diffractometry (Shimadzu XDR-6000, with CuK␣ radiation = 1.5418 Å, operated at 30 kV and 30 mA) and Fourier transform infrared (FTIR) spectroscopy (Bio-Rad spectrometer, Model FTS 3500GX), using pellets prepared with KBr. 2.2. Culture growth and preparation of the enzymatic extract P. camemberti was induced to sporulation according to the procedure outlined by Perraud and Kermasha [17]. The medium contained glucose (10.0 g L−1 ), KH2 PO4 (1.0 g L−1 ), FeSO4 ·7H2 O (10.0 mg L−1 ), MgSO4 ·7H2 O (0.5 g L−1 ), NaNO3 (3.0 g L−1 ) and KCl (0.5 g L−1 ), whose pH was adjusted to 6.0 with NaOH (1 mol L−1 ) before sterilization at 120 ◦ C for 15 min. The spores, suspended in the medium, were counted using a Neubauer Counting Chamber (Hausser Scientific, Horsham, PA). The strains were inoculated in 2 L Erlenmeyer flasks containing 1 L of medium. After inoculation (107 spores mL−1 ), the cultures of P. camemberti were incubated on a rotary shaker (100 rpm) at 20 ◦ C for 10 days and then filtered through cheesecloth. The mycelia were washed (2 × 50 mL) with cold water (4 ◦ C) and potassium phosphate buffer solution (pH 6.5, 0.6 mol L−1 ). The recovered mycelia were blended (5 mL of the phosphate buffer per 1 g biomass) and homogenized, using 0.45–0.50 mm diameter glass beads in an MSK cell homogenizer (Braun, Melsungen, Germany) for 2 × 2 min. The LOX enzymatic extract was recovered by centrifugation (12,000 × g, 15 min) and the supernatants were lyophilized, whereas the pellets were discarded. The resulting extract from P. camemberti was considered to be the crude enzyme extract. The protein concentration of the enzymatic fractions was determined according to a modification
The potassium phosphate concentration and pH (0.6 mol L−1 and pH 6.0) of the buffer used from here and on were chosen from the previous experiments to determine the optimal ionic strength and pH. The immobilization was carried out with 0.15 g of support and different volumes of protein solution (1.5 mg mL−1 ) in order to have ratios from 10 to 60 mg protein per gram of support in conical 5 mL screw-cap tubes under mild agitation at 4 ◦ C for 18 h. The supernatants were recovered for protein determination. The supports containing the immobilized enzymatic extract were washed with deionized water (1 × 15 mL) and phosphate buffer solution (2 × 15 mL), where each wash solution was recovered for protein determination. The washed supports containing the immobilized enzymatic extract were re-suspended in the phosphate buffer solution (0.1 g wet support mL−1 ). The immobilization was followed by checking the activity of the immobilization suspensions and supernatants, and then compared to a reference solution where the volume of enzyme suspension was substituted by sodium phosphate buffer. The protein immobilization yield (%) was defined as the ratio of protein immobilized onto a support (mg), divided by the initial protein content (mg) multiplied by 100. The retention of enzyme activity (%) was defined as the specific activity of LOX of the immobilized enzyme extract, divided by the specific activity of LOX of the free extract multiplied by 100. 2.6. LOX activity assays LOX activity was measured according to the procedure outlined by Hall et al. [20]. The assay of the free enzymatic extract was initiated by the addition of 180 L of the enzyme suspension (1.5 mg protein mL−1 ) to 0.6 mL of substrate solution (4.0 mmol L−1 ), and the total volume was adjusted to 1.5 mL with buffer solution. The LOX assay for the immobilized enzymatic extract was initiated by the addition of 0.6 mL of immobilized enzyme suspension (0.1 g support mL−1 ) to 1 mL of substrate (4.0 mmol L−1 ), and the total volume was adjusted 2.6 mL with buffer solution. The amount of the immobilized enzyme and final volume are larger since the particles of support were uniformly distributed in larger volumes. The LOX assays for the free and immobilized enzyme extracts were carried out at 25 ◦ C, under mild stirring, for 12 min and 35 min, respectively. Aliquots of the homogenated solution
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Fig. 1. X-ray diffraction pattern of the (a) LDH and (b) the LDH-A. Fig. 2. FTIR spectra of the LDH-A.
(0.1 mL) were taken at selected time intervals and were immediately added to 1 mL of xylenol orange reagent solution, which was prepared as a mixture of deionized/degassed water, ferrous sulfate (0.25 mmol L−1 ), perchloric acid (85.0 mmol L−1 ) and xylenol orange salt (0.1 mmol L−1 ) [21]. The absorbance of the reaction mixture was measured after 20 min of color development at 560 nm (10-HPOD; MEC 18,765 M−1 cm−1 ), using a Beckman DU-650 spectrophotometer (Beckman Instruments Inc.; San Raman, CA) [20]. LOX specific activity was defined as nmol of conjugated diene linoleic acid hydroperoxide per mg protein per min. All LOX assays were performed in duplicate in tandem with a blank trial, containing all components of the enzymatic assay with the exception of that LOX extract was thermally inactivated (95 ◦ C, 1 h). 2.7. Kinetic parameters of enzymatic activities The effect of linoleic acid concentration on the specific activity of the free and immobilized LOXs was investigated, using substrate concentrations ranging from 1.5 to 12 mmol L−1 and 1.5 to 77 mmol L−1 , respectively. 2.8. Thermostability and reusability
dimension gives the distance between the inorganic layers, 12.9 Å, which is in agreement with the presence of azelate ions between the inorganic layers [22].
3.2. FTIR spectroscopy The FTIR spectra of the LDH-A showed sharp lines at 2850 cm−1 and 2995 cm−1 due to methylene vibrations and at 1550 cm−1 and 1395 cm−1 caused by the asymmetric and symmetric stretching of the COO− group (Fig. 2). The lattice vibrations, i.e. the M–O (M = Zn or Al) stretching in the inorganic layers, are seen at 466 cm−1 and 431 cm−1 . This data confirms the intercalation of the azelate ions in the LDH and it is in agreement with the proposed arrange of the azelate ions in the LDH-A showed in Fig. 3. This structure represents only two contiguous inorganic layers. It must be considered that each LDH-A crystal is formed by the stacking of numerous layers. The partial hydrophobicity of this hybrid material is conferred by the azelate ions, which is responsible to fix the enzyme on the support.
The free and immobilized enzymatic extract were incubated at different temperatures and the specific activity was measured at 30 ◦ C and pH 6.0 after 1 h of incubation. The concentration of the free enzymatic extract was 1.50 mg protein mL−1 , whereas that of the immobilized one was 0.15–0.50 mg protein mL−1 . For the reusability studies, the immobilized enzyme was recovered by centrifugation at 2000 rpm after each kinetics assay and used again. 3. Results and discussion 3.1. X-ray diffraction The X-ray diffraction pattern of the solid formed by precipitation of the zinc and aluminum salts corresponds to a pure LDH crystalline phase with a basal distance of 8.9 Å Fig. 1a, which after the addition of the azelate solution (LDH-A) increased to 17.7 Å (Fig. 1a). The subtraction of the inorganic layer thickness (4.8 Å) to this basal
Fig. 3. Arrange of the azelate ions in the interlayer space of the LDH-A.
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Fig. 4. Effect of (a) the KH2 PO4 -K2 HPO4 buffer molarity and (b) the pH during the immobilization controlled with different buffer solutions.
3.3. Effect of ionic strength and pH on protein immobilization The optimum ionic strength required to force the hydrophobic adsorption of the LOX enzymatic extract was found by varying the buffer molarity (KH2 PO4 –K2 HPO4 ) during the immobilization and measuring the specific activity Fig. 4a. It is clear that the ionic strength significantly affected the efficiency of immobilization and the best specific activity was reached with KH2 PO4 –K2 HPO4 buffer 0.6 mol L−1 . Besides, the effect of pH was also followed and the highest amount of protein retained was reached at pH 6.0 (Fig. 4b). 3.4. Selection of the enzymatic extract/support ratio The crude enzymatic extract contained 90% of total protein. Although the quantification of LOX was not conducted, the protein fractions was separated in a preliminary test and the LOX specific activity revealed a close value to the activity found in the crude extract, thus the purification process was eliminated and the immobilization experiments were conducted with the crude extract. The specific activity (in nanomol of hydroperoxides produced per gram of protein per minute) of different amounts of enzymatic extract immobilized in 1 g of the LDH-A was measured to detect any interference of the support. According to Fig. 5, the percentage of specific activity has a linear relationship with the grams of enzymatic extract used in the immobilization experiment up to 60 mg of protein per gram of support. This means that the LDH-A support does not interfere in the peroxidation of the substrate or that the azelate ions are not exchanged with linoleic acid.
Beyond this point, the specific activity becomes constant (points not plotted), suggesting that the support is saturated. The forthcoming assays were done with 40 mg of protein per gram of LHD-A, where the yield of immobilized protein was 99.3% with 76.2% of specific activity. 3.5. Kinetic parameters of enzymatic activity KM and Vmax values of the free and immobilized LOX enzymatic extract were determined by measuring the initial rate of linoleic acid hydroperoxidation at various concentrations of substrate. The kinetic parameters for the free and immobilized enzyme in Table 1 were obtained from the Michaelis–Menten and Lineweaver–Burk plot (not shown). The same KM value was found for the free and the immobilized enzyme. It suggests that the immobilization did not affect the affinity between the substrate and the enzyme. Nonetheless, the lower Vmax detected should be a result of a more difficult access of the substrate to the active site due to a repression of flexibility in the LOX polypeptidic chain [23] or because some enzyme units are attached to the LDH support near to the active site. 3.6. Thermostability The assays to determine the effect of the incubation temperature were assessed considering the specific activity of the enzyme (either free or immobilized) incubated at 0 ◦ C as 100%. The first observation was that the activity of the immobilized LOX enzymatic extract after incubation at temperatures between 25 ◦ C and 60 ◦ C was higher than that of the free enzyme (Fig. 6). Incubation of the free enzyme at 25 ◦ C decreased the initial activity to 47%, whereas with the immobilization 92% of activity is retained. The treatment of the free enzyme at 60 ◦ C totally inhibits the activity of the free enzyme, but if immobilized, the initial activity only falls up to 68%. The protection against thermal denaturation can be explained by the ability of the LDHs compounds to dissipate the heat. LDHs are mixed in polymers as fire-retardant additives because they absorb the heat and, if the temperature is high, form and release water [2,24]. But in this work, only the heat dissipation along the micro-layered structure could explain the stability of the Table 1 Kinetic parameters of free and immobilized LOX.
Fig. 5. Specific activity of different amounts of LOX enzymatic extract from Penicillium camemberti immobilized per gram of support.
LOX type
Vmax. a (nmol HPDO mg−1 )
KM (mmol L−1 )
Free Immobilized
57.14 45.45
0.302 0.302
a The specific activity of Vmax is defined as nmol of conjugated diene hydroperoxides produced per min per mg of protein.
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directly proportional to the amount of protein immobilized and then the exchange of azelate ions with linoleic acid is discarded. An manageable amount for immobilization is 40 mg of protein per gram of support and the optimal condition for immobilization is with buffer 0.6 mol L−1 of KH2 PO4 –K2 HPO4 at pH 6.0. The percentage of immobilized enzyme was 99.35% and the specific activity after immobilization was 77.64% and this quantification was done without interference of the LDH-A support in the catalysis of the substrate. The immobilized enzyme when incubated at 60 ◦ C, retains 68% of the initial activity, while the free enzyme is totally inhibited. The hybrid support retains more than 60% of the initial enzyme activity after eight cycles. The kinetic parameters showed that the enzyme does not change its affinity. The immobilization of enzyme such as LOX in hybrid derivatives of LDH is a promissory strategy in the biogeneration of natural flavors due their ability to produce aroma and flavor with low cost. In addition no pollutants compounds are involved in the synthesis of the support. Fig. 6. Thermostability of the free and immobilized LOX enzymatic extract after 1 h of incubation. The specific activity was measured at 30 ◦ C and pH 6.0.
Acknowledgment Financial support was provided by the CNPq, Capes and FINEP brazilian agencies. R.M.B. is grateful for the financial support by CONACYT (project 82537). G.G.C.A. thanks PEC/PG of CAPES-IEL Nacional-Brasil. M. Sc. Angela Ma. Palacio Cortés is thanked for the revision of the manuscript. References
Fig. 7. Reusability of the immobilized enzyme.
LOX enzymatic extract. Other authors associate the thermostability to multipoint covalent links between the enzyme and the support [9,25]. It is also feasible that the loss of flexibility (considering the decreasing Vmax ) makes the enzyme more stable for a thermal treatment. 3.7. Reusability Finally, it was explored the reusability of the immobilized enzyme and it was found that the activity decreased between 3 and 8% of activity after each cycle, so that in the eighth cycle it remains 60% of the initial activity (Fig. 7). The successful immobilization achieved by the hybrid LDH-A besides enhanced the thermostability and a reasonable retention of specific activity in the recycling tests makes the support a candidate for industrial applications. 4. Conclusion The hybridized LDH contains azelate ions in the galleries, which adds a hydrophobic environment suitable for the immobilization of the enzyme. The amount of peroxidized linoleic acid is
[1] F. Wypych, K.G. Satyanarayana, Clay Surfaces: Fundamentals and Applications, Elsevier, Amsterdam, 2004, pp. 553. [2] C.M.C. Pereira, M. Herrero, F.M. Labajos, A.T. Marques, V. Rives, Preparation and properties of new flame retardant unsaturated polyester nanocomposites based on layered double hydroxides, Polym. Degrad. Stab. 94 (2009) 939–946. [3] H. Zhang, K. Zou, H. Sun, X. Duan, A magnetic organic–inorganic composite: Synthesis and characterization of magnetic 5-aminosalicylic acid intercalated layered double hydroxides, J. Solid State Chem. 178 (2005) 3485–3493. [4] H.W. Gardner, Lipoxygenase as a versatile biocatalyst, J. Am. Oil Chem. Soc. 73 (1996) 1347–1357. [5] C.C. Chen, S.D. Chen, J.J. Chen, C.M. Wu, Effects of pH value on the formation of volatiles of Shiitake (Lentinus edodes), an edible mushroom, J. Agric. Food Chem. 32 (1984) 999–1001. [6] S. Kermasha, N. Dioum, B. Bisakowski, M. Vega, Biocatalysis by immobilized lipoxygenase in a ternary micellar system, J. Mol. Catal. B: Enzyme 19–20 (2002) 305–317. [7] A.-F. Hsu, E. Wu, S. Shen, T.A. Foglia, K. Jones, Immobilized lipoxygenase in a packed-bed column bioreactor: continuous oxygenation of linoleic acid, Biotechnol. Appl. Biochem. 30 (1999) 245–250. [8] J. Kim, J.W. Grate, P. Wang, Nanostructures for enzyme stabilization, Chem. Eng. Sci. 61 (2006) 1017–1026. [9] C.E. Hall, S. Karboune, H. Florence, S. Kermasha, Immobilization of an enzymatic extract from Penicillium camemberti containing lipoxygenase and hydroperoxide lyase activities, J. Mol. Catal. B: Enzyme 52–53 (2008) 88–95. [10] A. Karout, C. Chopard, A.C. Pierre, Nanostructures for enzyme stabilization, J. Mol. Catal. B: Enzyme 44 (2007) 117–126. [11] M.B.A. Rahman, M. Basri, M.Z. Hussein, M.N.H. Idris, R.N.Z.R.A. Rahman, A.B. Salleh, Immobilisation of lipase from Candida rugosa on layered double hydroxides of Mg/Al and its nanocomposite as biocatalyst for the synthesis of ester, Catal. Today 93–95 (2004) 405–410. [12] M.B.A. Rahman, N.M.M. Yunus, M.Z. Hussein, R.N.Z.R.A. Rahman, A.B. Salleh, M. Basri, Application of advanced materials as support for immobilisation of lipase from Candida rugosa, Biocatal. Biotransform. 23 (2005) 233–239. [13] M.B.A. Rahman, U.H. Zaidan, M. Basri, M.Z. Hussein, R.N.Z.R.A. Rahman, A.B. Salleh, Enzymatic synthesis of methyl adipate ester using lipase from Candida rugosa immobilised on Mg, Zn and Ni of layered double hydroxides (LDHs), J. Mol. Catal. B: Enzyme 50 (2008) 33–39. [14] S. Carlino, The intercalation of carboxylic acids into layered double hydroxides: a critical evaluation and review of the different methods, Solid State Ionics 98 (1997) 73–84. [15] M.B.J. Roeffaers, B.F. Sels, H. Uji-I, F.C. de Schryver, P.A. Jacobs, D.E. de Vos, J. Hofkens, Spatially resolved observation of crystal-face dependent catalysis by single turnover counting, Nature 439 (2006) 572–575. [16] Y. Liu, E. Lotero, J.G. Goodwin, X. Mo, Transesterification of poultry fat with methanol using Mg–Al hydrotalcite derived catalysts, Appl. Catal., A. 331 (2007) 138–148. [17] X. Perraud, S. Kermasha, Characterization of lipoxygenase extracts from Penicillum sp, J. Am. Oil Chem. Soc. 77 (2000) 335–342.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Immobilization of enzymatic extract from Penicillium camemberti with lipoxygenase activity onto a hybrid layered double hydroxide Rogelio Morales Borges a,∗ , Gregorio Guadalupe Carbajal Arizaga b,∗ , Fernando Wypych c a Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías. Universidad de Guadalajara. Blvd. Marcelino García Barragán 1421, CP 44430. Guadalajara, Jalisco, Mexico b Centro de Nanociencias y Nanotecnología. Universidad Nacional Autónoma de México. Apdo. Postal 356, CP 22800. Ensenada, Baja California, Mexico c Departamento de Química, Universidade Federal do Paraná, CP 19081, Centro Politécnico, CEP 81531-990. Curitiba, PR, Brazil
a r t i c l e
i n f o
Article history: Received 7 May 2009 Received in revised form 26 August 2009 Accepted 27 August 2009
Keywords: Enzyme Microbial Immobilized Lipoxygenase Composite
a b s t r a c t A Zn/Al layered double hydroxide was synthesized by alkaline co-precipitation with azelate ions (− OOC(CH2 )7 COO− ). The interlayer space of the layered material is occupied by organic ions as confirmed by X-ray diffraction and FTIR spectroscopy. The resulting hybrid material was tested as support for Penicillium camemberti enzymatic extract, containing lipoxygenase (LOX) activity. The optimal condition for LOX immobilization is done with 0.6 mol L−1 potassium phosphate buffer and pH 6.0. The affinity for the substrate is the same after immobilization, however the specific activity slightly decreases. The immobilization enhanced the thermal stability, which was evident with incubation at 60 ◦ C where the immobilized enzyme retains 68% of specific activity while the free enzyme is inhibited. Recycling assays showed that after eight reaction cycles, the immobilized enzyme retains 60% of the activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The layered double hydroxides (LDH), also known as synthetic clays, are compounds whose structure is a modification of the layered magnesium hydroxide, where a fraction of the cationic sites are occupied by trivalent cations, which causes an excess of positive charge neutralized by the presence of additional anions in the interlayer space. These compounds are represented by the general formula: [M2+ 1−x M3+ x (OH)2 ]x+ Am− x/m ·nH2 O; where: M2+ and M3+ are the cations involved and A, an anion of charge m− [1,2]. The physico-chemical characteristics of the interlayer space depends on the anion, which can be organic and could form interesting new supports for the immobilization of catalytic species through intercalation or through adsorption on the external surfaces. Despite size restrictions regarding to intercalation of enzymes, small organic intercalated anions allow to interact with the enzymes [3]. Other proposed mechanism is the interaction of the enzyme with OH groups at the outer surface of the LDH crystals. Lipoxygenases (EC 1.13.11.12) are oxidoreductases that catalyze the specific addition of molecular oxygen to polyunsaturated fatty acids (containing the cis, cis-1,4-pentadiene moiety) yielding the
∗ Corresponding authors. Tel.: +52 646 174 46 02; fax: +52 646 174 46 03. E-mail addresses: [email protected] (R.M. Borges), [email protected] (G.G.C. Arizaga). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.08.011
1,3-cis-transdiene-5-hydroperoxides. Among the fatty acids with this functionality, linoleic and linolenic acids are the best-known substrates for LOX [4]. Penicillium camemberti is a fungus widely used in food industry as starter culture. Volatile compounds are produced in P. camemberti due to its lipolytic and proteolytic activity, which contribute to the formation of the aroma and flavor of Brie and Camembert cheeses [5]. The production of certain natural flavor compounds, including C5 to C9 aliphatic alcohols and carbonyl compounds, results from a biosynthetic pathway involving several enzymatic activities, like lipoxygenase (LOX) and hydroperoxide lyase (HPL) with polyunsaturated fatty acids as substrates. Although the increasing interest in the LOX production for aroma compounds [5,6], the limited stability of these enzymes has restricted their biotechnological applications [7] as well as the possibility of the reusability of the biocatalyst in continuous packed-bed reactors [7]. Additional benefits of immobilization, include the easy separation of the enzyme from its end products, which minimizes downstream processing costs [8–10]. LDHs have the capability to immobilized enzymes like lipases [11–13], however, it is known that the organic acids used as substrates can be easily intercalated into the LDHs structures [14] or react to form esters in the presence of alcohols and LDH [15,16]. The two facts can lead to misinterpretation of the results. The objective of this study is to synthesize a Zn/Al LDH hybridized with azelate ions in order to saturate the positive charge of the layers and avoid the exchange reaction with linoleic acid,
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which is the substrate in the peroxidation reaction. The immobilization of an enzymatic extract with LOX activity, in addition to its importance in food industry, is ideal because the reaction to quantify the yield of the reaction involves the peroxide group, unlike the reactions with lipase, where the carboxylic groups is quantified, avoiding the misinterpretation of enzymatic activity of the immobilized enzyme. We also aim to find the optimal conditions for immobilization and the highest LOX activity. 2. Experiments Linoleic acid (cis-9, cis-12-octadecadienoic acid) was purchased from Nu-Chek Prep (Elysian, MN). Trishydroxy methylaminomethane (TRIS) was obtained from Aldrich (Milwaukee, WI). Xylenol orange [(3,3-bis(N,N-di(carboxymethyl)aminomethyl)-ocresol)] was purchased from Sigma Chemical Co. (St. Louis, MO). Mono and dibasic potassium phosphate were purchased from Fisher Scientific (Fair Lawn, NJ). The inorganic support was synthesized with reagents of analytical grade: Zn(NO3 )2 ·6H2 O and Al(NO3 )3 ·9H2 O were purchased from Vetec and Synth, Brazil.
of the Lowry method [18]. Bovine serum albumin (Sigma Chemical Co., St. Louis, MO) was used as a standard for the calibration curve. 2.3. Substrate preparation Linoleic acid was used as substrate for LOX studies. The stock solution was prepared at a concentration of 4.0 mmol L−1 in the appropriate buffer solutions (0.6 mol L−1 ), according to the procedure outlined by Perraud et al. [19]. 2.4. Effect of ionic strength and pH on protein immobilization The effect of ionic strength on protein immobilization was studied by varying the buffer molarity from 0.1 mol L−1 to 0.8 mol L−1 KH2 PO4 –K2 HPO4 . The immobilization was carried out at 4 ◦ C for 18 h, using 0.15 g of support and 2.5 mL of protein solution (1.5 mg mL−1 ). In a similar way, the most effective pH to immobilize the enzyme was detected by using a wide range of buffer solutions (0.1 mol L−1 ), including citrate phosphate for the pH range 3.0–5.5; potassium phosphate for pH 6.0–8.0 and glycine–NaOH for pH values between 8.5 and 9.0.
2.1. Synthesis of the LDH and modification with azelate ions 2.5. Immobilization of LOX The Zn/Al LDH was synthesized by co-precipitation. The salts Zn(NO3 )2 ·6H2 O and Al(NO3 )3 ·9H2 O, in a molar ratio Zn/Al = 3, were dissolved in decarbonated water and then precipitated by addition of NH4 OH at a final pH of 8.5. An aliquot from this suspension was taken to confirm whether at this point the LDH was formed. In a separated vessel, 300 mL of 0.25 mol L−1 azelate solution was prepared by dissolving azelaic acid, HOOC(CH2 )7 COOH, with NH4 OH. Finally, this azelate solution was added to the LDH suspension and stirred for 24 h at room temperature. The solid was recovered by centrifugation at 4000 rpm, washed with water four times and dried at 65 ◦ C for 24 h. The white powder was identified as LDHA. The structures were analyzed by powder X-ray diffractometry (Shimadzu XDR-6000, with CuK␣ radiation = 1.5418 Å, operated at 30 kV and 30 mA) and Fourier transform infrared (FTIR) spectroscopy (Bio-Rad spectrometer, Model FTS 3500GX), using pellets prepared with KBr. 2.2. Culture growth and preparation of the enzymatic extract P. camemberti was induced to sporulation according to the procedure outlined by Perraud and Kermasha [17]. The medium contained glucose (10.0 g L−1 ), KH2 PO4 (1.0 g L−1 ), FeSO4 ·7H2 O (10.0 mg L−1 ), MgSO4 ·7H2 O (0.5 g L−1 ), NaNO3 (3.0 g L−1 ) and KCl (0.5 g L−1 ), whose pH was adjusted to 6.0 with NaOH (1 mol L−1 ) before sterilization at 120 ◦ C for 15 min. The spores, suspended in the medium, were counted using a Neubauer Counting Chamber (Hausser Scientific, Horsham, PA). The strains were inoculated in 2 L Erlenmeyer flasks containing 1 L of medium. After inoculation (107 spores mL−1 ), the cultures of P. camemberti were incubated on a rotary shaker (100 rpm) at 20 ◦ C for 10 days and then filtered through cheesecloth. The mycelia were washed (2 × 50 mL) with cold water (4 ◦ C) and potassium phosphate buffer solution (pH 6.5, 0.6 mol L−1 ). The recovered mycelia were blended (5 mL of the phosphate buffer per 1 g biomass) and homogenized, using 0.45–0.50 mm diameter glass beads in an MSK cell homogenizer (Braun, Melsungen, Germany) for 2 × 2 min. The LOX enzymatic extract was recovered by centrifugation (12,000 × g, 15 min) and the supernatants were lyophilized, whereas the pellets were discarded. The resulting extract from P. camemberti was considered to be the crude enzyme extract. The protein concentration of the enzymatic fractions was determined according to a modification
The potassium phosphate concentration and pH (0.6 mol L−1 and pH 6.0) of the buffer used from here and on were chosen from the previous experiments to determine the optimal ionic strength and pH. The immobilization was carried out with 0.15 g of support and different volumes of protein solution (1.5 mg mL−1 ) in order to have ratios from 10 to 60 mg protein per gram of support in conical 5 mL screw-cap tubes under mild agitation at 4 ◦ C for 18 h. The supernatants were recovered for protein determination. The supports containing the immobilized enzymatic extract were washed with deionized water (1 × 15 mL) and phosphate buffer solution (2 × 15 mL), where each wash solution was recovered for protein determination. The washed supports containing the immobilized enzymatic extract were re-suspended in the phosphate buffer solution (0.1 g wet support mL−1 ). The immobilization was followed by checking the activity of the immobilization suspensions and supernatants, and then compared to a reference solution where the volume of enzyme suspension was substituted by sodium phosphate buffer. The protein immobilization yield (%) was defined as the ratio of protein immobilized onto a support (mg), divided by the initial protein content (mg) multiplied by 100. The retention of enzyme activity (%) was defined as the specific activity of LOX of the immobilized enzyme extract, divided by the specific activity of LOX of the free extract multiplied by 100. 2.6. LOX activity assays LOX activity was measured according to the procedure outlined by Hall et al. [20]. The assay of the free enzymatic extract was initiated by the addition of 180 L of the enzyme suspension (1.5 mg protein mL−1 ) to 0.6 mL of substrate solution (4.0 mmol L−1 ), and the total volume was adjusted to 1.5 mL with buffer solution. The LOX assay for the immobilized enzymatic extract was initiated by the addition of 0.6 mL of immobilized enzyme suspension (0.1 g support mL−1 ) to 1 mL of substrate (4.0 mmol L−1 ), and the total volume was adjusted 2.6 mL with buffer solution. The amount of the immobilized enzyme and final volume are larger since the particles of support were uniformly distributed in larger volumes. The LOX assays for the free and immobilized enzyme extracts were carried out at 25 ◦ C, under mild stirring, for 12 min and 35 min, respectively. Aliquots of the homogenated solution
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Fig. 1. X-ray diffraction pattern of the (a) LDH and (b) the LDH-A. Fig. 2. FTIR spectra of the LDH-A.
(0.1 mL) were taken at selected time intervals and were immediately added to 1 mL of xylenol orange reagent solution, which was prepared as a mixture of deionized/degassed water, ferrous sulfate (0.25 mmol L−1 ), perchloric acid (85.0 mmol L−1 ) and xylenol orange salt (0.1 mmol L−1 ) [21]. The absorbance of the reaction mixture was measured after 20 min of color development at 560 nm (10-HPOD; MEC 18,765 M−1 cm−1 ), using a Beckman DU-650 spectrophotometer (Beckman Instruments Inc.; San Raman, CA) [20]. LOX specific activity was defined as nmol of conjugated diene linoleic acid hydroperoxide per mg protein per min. All LOX assays were performed in duplicate in tandem with a blank trial, containing all components of the enzymatic assay with the exception of that LOX extract was thermally inactivated (95 ◦ C, 1 h). 2.7. Kinetic parameters of enzymatic activities The effect of linoleic acid concentration on the specific activity of the free and immobilized LOXs was investigated, using substrate concentrations ranging from 1.5 to 12 mmol L−1 and 1.5 to 77 mmol L−1 , respectively. 2.8. Thermostability and reusability
dimension gives the distance between the inorganic layers, 12.9 Å, which is in agreement with the presence of azelate ions between the inorganic layers [22].
3.2. FTIR spectroscopy The FTIR spectra of the LDH-A showed sharp lines at 2850 cm−1 and 2995 cm−1 due to methylene vibrations and at 1550 cm−1 and 1395 cm−1 caused by the asymmetric and symmetric stretching of the COO− group (Fig. 2). The lattice vibrations, i.e. the M–O (M = Zn or Al) stretching in the inorganic layers, are seen at 466 cm−1 and 431 cm−1 . This data confirms the intercalation of the azelate ions in the LDH and it is in agreement with the proposed arrange of the azelate ions in the LDH-A showed in Fig. 3. This structure represents only two contiguous inorganic layers. It must be considered that each LDH-A crystal is formed by the stacking of numerous layers. The partial hydrophobicity of this hybrid material is conferred by the azelate ions, which is responsible to fix the enzyme on the support.
The free and immobilized enzymatic extract were incubated at different temperatures and the specific activity was measured at 30 ◦ C and pH 6.0 after 1 h of incubation. The concentration of the free enzymatic extract was 1.50 mg protein mL−1 , whereas that of the immobilized one was 0.15–0.50 mg protein mL−1 . For the reusability studies, the immobilized enzyme was recovered by centrifugation at 2000 rpm after each kinetics assay and used again. 3. Results and discussion 3.1. X-ray diffraction The X-ray diffraction pattern of the solid formed by precipitation of the zinc and aluminum salts corresponds to a pure LDH crystalline phase with a basal distance of 8.9 Å Fig. 1a, which after the addition of the azelate solution (LDH-A) increased to 17.7 Å (Fig. 1a). The subtraction of the inorganic layer thickness (4.8 Å) to this basal
Fig. 3. Arrange of the azelate ions in the interlayer space of the LDH-A.
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Fig. 4. Effect of (a) the KH2 PO4 -K2 HPO4 buffer molarity and (b) the pH during the immobilization controlled with different buffer solutions.
3.3. Effect of ionic strength and pH on protein immobilization The optimum ionic strength required to force the hydrophobic adsorption of the LOX enzymatic extract was found by varying the buffer molarity (KH2 PO4 –K2 HPO4 ) during the immobilization and measuring the specific activity Fig. 4a. It is clear that the ionic strength significantly affected the efficiency of immobilization and the best specific activity was reached with KH2 PO4 –K2 HPO4 buffer 0.6 mol L−1 . Besides, the effect of pH was also followed and the highest amount of protein retained was reached at pH 6.0 (Fig. 4b). 3.4. Selection of the enzymatic extract/support ratio The crude enzymatic extract contained 90% of total protein. Although the quantification of LOX was not conducted, the protein fractions was separated in a preliminary test and the LOX specific activity revealed a close value to the activity found in the crude extract, thus the purification process was eliminated and the immobilization experiments were conducted with the crude extract. The specific activity (in nanomol of hydroperoxides produced per gram of protein per minute) of different amounts of enzymatic extract immobilized in 1 g of the LDH-A was measured to detect any interference of the support. According to Fig. 5, the percentage of specific activity has a linear relationship with the grams of enzymatic extract used in the immobilization experiment up to 60 mg of protein per gram of support. This means that the LDH-A support does not interfere in the peroxidation of the substrate or that the azelate ions are not exchanged with linoleic acid.
Beyond this point, the specific activity becomes constant (points not plotted), suggesting that the support is saturated. The forthcoming assays were done with 40 mg of protein per gram of LHD-A, where the yield of immobilized protein was 99.3% with 76.2% of specific activity. 3.5. Kinetic parameters of enzymatic activity KM and Vmax values of the free and immobilized LOX enzymatic extract were determined by measuring the initial rate of linoleic acid hydroperoxidation at various concentrations of substrate. The kinetic parameters for the free and immobilized enzyme in Table 1 were obtained from the Michaelis–Menten and Lineweaver–Burk plot (not shown). The same KM value was found for the free and the immobilized enzyme. It suggests that the immobilization did not affect the affinity between the substrate and the enzyme. Nonetheless, the lower Vmax detected should be a result of a more difficult access of the substrate to the active site due to a repression of flexibility in the LOX polypeptidic chain [23] or because some enzyme units are attached to the LDH support near to the active site. 3.6. Thermostability The assays to determine the effect of the incubation temperature were assessed considering the specific activity of the enzyme (either free or immobilized) incubated at 0 ◦ C as 100%. The first observation was that the activity of the immobilized LOX enzymatic extract after incubation at temperatures between 25 ◦ C and 60 ◦ C was higher than that of the free enzyme (Fig. 6). Incubation of the free enzyme at 25 ◦ C decreased the initial activity to 47%, whereas with the immobilization 92% of activity is retained. The treatment of the free enzyme at 60 ◦ C totally inhibits the activity of the free enzyme, but if immobilized, the initial activity only falls up to 68%. The protection against thermal denaturation can be explained by the ability of the LDHs compounds to dissipate the heat. LDHs are mixed in polymers as fire-retardant additives because they absorb the heat and, if the temperature is high, form and release water [2,24]. But in this work, only the heat dissipation along the micro-layered structure could explain the stability of the Table 1 Kinetic parameters of free and immobilized LOX.
Fig. 5. Specific activity of different amounts of LOX enzymatic extract from Penicillium camemberti immobilized per gram of support.
LOX type
Vmax. a (nmol HPDO mg−1 )
KM (mmol L−1 )
Free Immobilized
57.14 45.45
0.302 0.302
a The specific activity of Vmax is defined as nmol of conjugated diene hydroperoxides produced per min per mg of protein.
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directly proportional to the amount of protein immobilized and then the exchange of azelate ions with linoleic acid is discarded. An manageable amount for immobilization is 40 mg of protein per gram of support and the optimal condition for immobilization is with buffer 0.6 mol L−1 of KH2 PO4 –K2 HPO4 at pH 6.0. The percentage of immobilized enzyme was 99.35% and the specific activity after immobilization was 77.64% and this quantification was done without interference of the LDH-A support in the catalysis of the substrate. The immobilized enzyme when incubated at 60 ◦ C, retains 68% of the initial activity, while the free enzyme is totally inhibited. The hybrid support retains more than 60% of the initial enzyme activity after eight cycles. The kinetic parameters showed that the enzyme does not change its affinity. The immobilization of enzyme such as LOX in hybrid derivatives of LDH is a promissory strategy in the biogeneration of natural flavors due their ability to produce aroma and flavor with low cost. In addition no pollutants compounds are involved in the synthesis of the support. Fig. 6. Thermostability of the free and immobilized LOX enzymatic extract after 1 h of incubation. The specific activity was measured at 30 ◦ C and pH 6.0.
Acknowledgment Financial support was provided by the CNPq, Capes and FINEP brazilian agencies. R.M.B. is grateful for the financial support by CONACYT (project 82537). G.G.C.A. thanks PEC/PG of CAPES-IEL Nacional-Brasil. M. Sc. Angela Ma. Palacio Cortés is thanked for the revision of the manuscript. References
Fig. 7. Reusability of the immobilized enzyme.
LOX enzymatic extract. Other authors associate the thermostability to multipoint covalent links between the enzyme and the support [9,25]. It is also feasible that the loss of flexibility (considering the decreasing Vmax ) makes the enzyme more stable for a thermal treatment. 3.7. Reusability Finally, it was explored the reusability of the immobilized enzyme and it was found that the activity decreased between 3 and 8% of activity after each cycle, so that in the eighth cycle it remains 60% of the initial activity (Fig. 7). The successful immobilization achieved by the hybrid LDH-A besides enhanced the thermostability and a reasonable retention of specific activity in the recycling tests makes the support a candidate for industrial applications. 4. Conclusion The hybridized LDH contains azelate ions in the galleries, which adds a hydrophobic environment suitable for the immobilization of the enzyme. The amount of peroxidized linoleic acid is
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