Aggregation and Homogenization, Surface Charge and Structural

Dec 22, 2010 - The results also indicated that SS CO2 could enhance inactivation effect of the ... One of the frequently discussed areas is green chem...
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Aggregation and Homogenization, Surface Charge and Structural Change, and Inactivation of Mushroom Tyrosinase in an Aqueous System by Subcritical/Supercritical Carbon Dioxide Wanfeng Hu, Yan Zhang, Yuanyuan Wang, Linyan Zhou, Xiaojing Leng, Xiaojun Liao,* and Xiaosong Hu College of Food Science and Nutritional Engineering, China Agricultural University; Key Lab of Fruit and Vegetable Processing, Ministry of Agriculture; Engineering Research Centre for Fruit and Vegetable Processing, Ministry of Education, Beijing 100083, China Received September 1, 2010. Revised Manuscript Received November 22, 2010 The subcritical/supercritical carbon dioxide (SS CO2) has gained considerable attention in green chemistry industry for its advantage as nontoxic, nonflammable, and inexpensive. The effects of SS CO2 treatments on aggregation and homogenization, surface charge, secondary and tertiary structure, and activity of mushroom tyrosinase in an aqueous system were investigated using a number of methods including dynamic light scattering (DLS), zeta potential measurement, circular dichroism (CD) spectropolarimeter, and spectrofluorometer. With a treatment time of 20 min, three treatment temperatures (35, 45, and 55 °C) and four pressures (5, 8, 12, and 15 MPa) had been selected. The aggregation and homogenization of the globular protein particles was induced by SS CO2 as suggested by the particle size distribution (PSD) patterns that were closely related to the pressure and temperature. The surface charge of the tyrosinase decreased following the SS CO2 treatments, and its variation tendency shows a favorable consistency with that of its PSD patterns. The R-helix conformation in secondary structure and fluorescence intensity reflecting tertiary structure also decreased, together with the λmax red-shifted with the increasing pressure. The results also indicated that SS CO2 could enhance inactivation effect of the temperature on the tyrosinase with its lowest residual activity being about 60% under the condition of 8 MPa, 55 °C, and 20 min treatment time. The loss in the activity of the tyrosinase was correlated to its aggregation and homogenization effect induced by SS CO2, which led to the change of surface charge as well as secondary and tertiary structure.

Introduction Tyrosinase (EC 1.14.18.1), widely distributed in plants and microorganisms, belongs to a polyphenol oxidase (PPO) family with the catalytic center containing dinuclear copper, which catalyzes the ortho-hydroxylation of monophenol to diphenols and the subsequent oxidation of the diphenolic product to the resulting quinine.1 The quinone products are reactive precursors for the synthesis of melanin pigments.2 This process is called enzymatic browning, generally undesirable during the processing and storage of vegetables and fruits. CO2 has a low critical temperature and pressure (critical temperature=31.1 °C, critical pressure=7.4 MPa) and can be either in the subcritical (liquid or gaseous) or in supercritical state depending on the prevailing temperature and pressure. Subcritical/ supercritical CO2 (SS CO2) gains a special characteristic of gaslike viscosity and liquidlike density,3 which makes it an excellent solvent for various applications in a wide range of key and emerging industrial processes. One of the frequently discussed areas is green *Corresponding author: e-mail [email protected]; Ph þ861062737434-602; Fax þ8610-62737434-602.

(1) Mayer, A. M. Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 2006, 67(21), 2318–2331. (2) Yoruk, R.; Marshall, M. R. Physicochemical properties and function of plant polyphenol oxidase: A review. J. Food Biochem. 2003, 27(5), 361–422. (3) Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Enzymatic catalysis in a supercritical fluid. Biotechnol. Lett. 1985, 7(5), 325–328. (4) Leitner, W. Supercritical Carbon Dioxide as a Green Reaction Medium for Catalysis. Acc. Chem. Res. 2002, 35(9), 746–756. (5) Nalawade, S. P.; Picchioni, F.; Janssen, L. P. B. M. Supercritical carbon dioxide as a green solvent for processing polymer melts: Processing aspects and applications. Prog. Polym. Sci. 2006, 31(1), 19–43.

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chemistry industry, which applies supercritical CO2 as a sustainable alternative reaction medium for chemical synthesis4-6 and effective solvent for chemical extraction7-9 to totally eliminate or greatly reduce the side effect of numerous conventional organic solvents. In biocatalysis processing, enzyme stability and activity are of vital importance for the catalytic efficiency and may depend on the enzyme species, characteristics of compressed fluids, water content of the enzyme/support/reaction mixture, and process variables manipulated.10 Enzymes are more stable in nonaqueous systems and can effectively contact with substrates that are more soluble in hydrophobic solvent.3,11 Reports on enzyme activity in supercritical CO2 had shown contradictive results. Some reported that the enzyme activity increased several-fold in supercritical CO2;12,13 however, others detected an activity loss of the enzyme (6) Burgener, M.; Ferri, D.; Grunwaldt, J.; Mallat, T.; Baiker, A. Supercritical Carbon Dioxide: An Inert Solvent for Catalytic Hydrogenation? J. Phys. Chem. B 2005, 109(35), 16794–16800. (7) Grosso, C.; Ferraro, V.; Figueiredo, A. C.; Barroso, J. G.; Coelho, J. A.; Palavra, A. M. Supercritical carbon dioxide extraction of volatile oil from Italian coriander seeds. Food Chem. 2008, 111(1), 197–203. (8) Mekki, S.; Wai, C. M.; Billard, I.; Moutiers, G.; Burt, J.; Yoon, B.; Wang, J. S.; Gaillard, C.; Ouadi, A.; Hesemann, P. Extraction of lanthanides from aqueous solution by using room-temperature ionic liquid and supercritical carbon dioxide in conjunction. Chem.;Eur. J. 2006, 12(6), 1760–1766. (9) Mendes, R. L.; Nobre, B. P.; Cardoso, M. T.; Pereira, A. P.; Palavra, A. F. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorg. Chim. Acta 2003, 356, 328–334. (10) Oliveira, D.; Feihrmann, A. C.; Rubira, A. F.; Kunita, M. H.; Dariva, C.; Oliveira, J. V. Assessment of two immobilized lipases activity treated in compressed fluids. J. Supercrit. Fluids 2006, 38(3), 373–382. (11) Klibanov, A. M. Improving Enzymes by Using Them in Organic Solvents 2001, 409(6817), 241–246.

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and speculated that higher water content in the processing may led to the reduction of enzyme activity.14-20 In nonaqueous enzymology, water content is a key parameter in enzymatic catalysis when other conditions are the same because it affects both reaction rates and equilibrium conversions.15,16 Researchers had found that as the degree of hydration of the catalyst particles increased, the rate of uptake of the substrates decreased and particle aggregation occurred.17 The enzyme activity could also decrease with the increasing water activity (aw).15 Water inhibition has been considered as a predominantly physical effect.17 Therefore, in aqueous systems, enzymes treated with SS CO2 are definitely inactivated due to the high water content. A number of studies had shown an effective inactivation effect of enzymes in aqueous systems treated with SS CO2, including pectinesterase (PE) or pectin methylesterase (PME),18,19 lipoxygenase (LOX),20,21 and peroxidase (POD).22 They not only observed the activity loss of the enzymes but also detected the secondary and tertiary structural change of the enzymes treated with SS CO2. The structural changes were proposed to be responsible for the activity loss. Except for the structural changes caused by the treatments, some researchers stated a qualitative aggregation effect of SS CO2 on LOX particle in solution by transmission electron microscopy,21 but the aggregation effect was not quantitatively analyzed and the homogenization effect of SS CO2 was not yet observed. Moreover, the surface charge of enzymes treated with SS CO2 in solutions was also not analyzed. The surface charge that a particle acquires in a particular medium can be illustrated by zeta potential measurement for understanding electrostatic colloidal dispersion stability.23 In the literature, the relationship among the aggregation and homogenization, the variation of surface charge, structural changes, and inactivation of enzymes following SS CO2 treatments was not proposed until recently. Globular proteins disperse to form a stable gel solution; any perturbation in the solution may destroy the balance between (12) Giessauf, A.; Gamse, T. A simple process for increasing the specific activity of porcine pancreatic lipase by supercritical carbon dioxide treatment. J. Mol. Catal. B: Enzym. 2000, 9(1-3), 57–64. (13) Yan, H.; Noritomi, H.; Nagahama, K. A rise in the hydrolysis activity of Candida rugosa lipase caused by pressurized treatment with supercritical carbon dioxide. Kobunshi Ronbunshu 2001, 58(12), 674–678. (14) Habulin, M.; Knez, Z. Activity and stability of lipases from different sources in supercritical carbon dioxide and near-critical propane. J. Chem. Technol. Biotechnol. 2001, 76(12), 1260–1266. (15) Peres, C.; Gomes Da Silva, M. D. R.; Barreiros, S. Water activity effects on geranyl acetate synthesis catalyzed by novozym in supercritical ethane and in supercritical carbon dioxide. J. Agric. Food Chem. 2003, 51(7), 1884–1888. (16) Gamse, T.; Marr, R. Investigation of influence parameters on enzyme stability during treatment with supercritical carbon dioxide (SC-CO2). In Proc. 5th Int. Symp. Supercrit, Fluids Atlanta, 2000; pp 8-12. (17) Vazquez-Lima, F.; Pyle, D. L.; Asenjo, J. A. Factors affecting the esterification of lauric acid using an immobilized biocatalyst: Enzyme characterization and studies in a well-mixed reactor. Biotechnol. Bioeng. 1995, 46(1), 69–79. (18) Balaban, M. O.; Arreola, A.; Marshall, M.; Peplow, A.; Wei, C.; Cornell, J. Inactivation of Pectinesterase in Orange Juice by Supercritical Carbon Dioxide. J. Food Sci. 1991, 56(3), 743–746. (19) Zhi, X.; Zhang, Y.; Hu, X.; Wu, J.; Liao, X. Inactivation of apple pectin methylesterase induced by dense phase carbon dioxide. J. Agric. Food Chem. 2008, 56(13), 5394–5400. (20) Tedjo, W.; Eshtiaghi, M.; Knorr, D. Impact of Supercritical Carbon Dioxide and High Pressure on Lipoxygenase and Peroxidase Activity. J. Food Sci. 2000, 65(8), 1284–1287. (21) Liao, X.; Zhang, Y.; Bei, J.; Hu, X.; Wu, J. Alterations of molecular properties of lipoxygenase induced by dense phase carbon dioxide. Innovative Food Sci. Emerging Technol. 2009, 10(1), 47–53. (22) Gui, F.; Chen, F.; Wu, J.; Wang, Z.; Liao, X.; Hu, X. Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide. Food Chem. 2006, 97(3), 480–489. (23) Santiago, P. S.; Carvalho, F. A. O.; Domingues, M. M.; Carvalho, J. W. P.; Santos, N. C.; Tabak, M. Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions. Langmuir 2010, 26(12), 9794–9801. (24) Fennema, O. R. Major Food Components. In Food Chemistry (Food Science and Technology); CRC Press: Boca Raton, FL, 1996; p 460.

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attractive and repulsive forces among protein gel to cause aggregation.24 This process is affected by a considerable number of parameters, including pH, ionic strength, protein concentration, and disturbing conditions such as thermal or high pressure treatments.25,26 Protein aggregation was mostly restricted to thermal treatment and high pressure homogeneous area, especially of whey proteins and soy proteins.27-29 The aggregation induced by the treatments may contribute to structural change of the enzymes and ultimately resulted in activity loss. To our knowledge, the aggregation and homogenization, surface charge, and structural changes on the tyrosinase in an aqueous system subject to SS CO2 have not yet been reported. Therefore, the present study is directed to perform a detailed investigation on this subject and to examine the activity of the tyrosinase, aiming at utilizing green solvent such as SS CO2 to inactivate enzyme and improving our understanding of possible mechanism for SS CO2induced inactivation of tyrosinase.

Materials and Methods Materials and Reagents. Mushroom tyrosinase (EC 1.14.18.1, product T-3824) was purchased from Sigma-Aldrich Co. (Beijing, China) as a lyophilized powder containing activity of 25 000 units/ mg. The molecular weight is 119.5 kDa. One activity unit causes, at pH 6.5 and 25 °C, an absorption increase of 0.001 per minute at 420 nm taking 0.1 M tyrosine as substrate. Mushroom tyrosinase was dissolved in phosphate buffer (pH 6.5, 0.05 M) to the concentration of 0.48 mg/mL before treatments. All the other chemicals were of analytical grade. The purity of CO2 during SS CO2 treatment is 99.9%, which was purchased from Beijing Analytical Apparatus Co. (Beijing, China) and was purified with a filter loading with active carbon. SS CO2 Processing System. The details of the SS CO2 system have been introduced in the work of Zhou et al.30 The samples were placed in an 850 mL stainless steel pressure vessel. The vessel temperature was maintained using a THYS-15 thermostatic bath (Ningbo Tianheng Instrument Factory, Zhejiang, China) equipped with a XMTA-7512 temperature controller (Yuyao Temperature Meter Factory, Zhejiang, China). The vessel pressure was controlled using a 2TD plunger pump (Huaan Supercritical Fluids Extraction Co. Ltd., Jiangsu, China) equipped with a DBY-300 pressure transducer (Shanxi Qingming Electronic Group Corporation, Shanxi, China), which can provide a maximum pressure of 50 MPa and a maximum flow rate of 50 L/h. A 2XZ-4 vacuum pump (Huangyan Qiujing Vacuum Pump Factory, Zhejiang, China) was connected to the vessel to control the vacuum state. Inactivation of Tyrosinase by SS CO2 Treatments. For each experiment, 1 mL of tyrosinase solution (enzyme concentration was 0.0313 mg/mL in sodium phosphate buffer, pH 6.5) in a 15 mL plastic tube without cap (Beijing Bomex Co., Beijing, China) was placed in the SS CO2 vessel at desired temperature. (25) Capes, J. S.; Kiley, P. J.; Windle, A. H. Investigating the Effect of pH on the Aggregation of Two Surfactant-Like Octapeptides. Langmuir 2010, 26(8), 5637– 5644. (26) Zhang, Y.; Tang, C.; Wen, Q.; Yang, X.; Li, L.; Deng, W. Thermal aggregation and gelation of kidney bean (Phaseolus vulgaris L.) protein isolate at pH 2.0: Influence of ionic strength. Food Hydrocolloids 2010, 24(4), 266–274. (27) Grcia-Juli, A.; Ren, M.; Corts-Muoz, M.; Picart, L.; Lpez-Pedemonte, T.; Chevalier, D.; Dumay, E. Effect of dynamic high pressure on whey protein aggregation: A comparison with the effect of continuous short-time thermal treatments. Food Hydrocolloids 2008, 22(6), 1014–1032. (28) Schmitt, C.; Bovay, C.; Vuilliomenet, A.; Rouvet, M.; Bovetto, L.; Barbar, R.; Sanchez, C. Multiscale Characterization of Individualized β-Lactoglobulin Microgels Formed upon Heat Treatment under Narrow pH Range Conditions. Langmuir 2009, 25(14), 7899–7909. (29) Ikeda, S.; Morris, V. J. Fine-Stranded and Particulate Aggregates of HeatDenatured Whey Proteins Visualized by Atomic Force Microscopy. Biomacromolecules 2002, 3(2), 382–389. (30) Zhou, L.; Wang, Y.; Hu, X.; Wu, J.; Liao, X. Effect of high pressure carbon dioxide on the quality of carrot juice. Innovative Food Sci. Emerging Technol. 2009, 10(3), 321–327.

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Figure 1. PSD of the tyrosinase following the SS CO2 or MT treatments. (a) SS CO2 treatments at 35 °C and at various pressures for 20 min. (b) MT treatments and SS CO2 treatments at 8 MPa and at various temperatures for 20 min.

The desired experimental pressure level of SS CO2 was built up for about 2-3 min. Upon finishing the treatment, the vessel was depressurized for about 3-4 min. The time for pressurization and depressurization was not counted in the SS CO2 treatment time. The SS CO2 treatment was performed at 5, 8, 12, and 15 MPa at 35 °C for 20 min and 35, 45, and 55 °C at 8 MPa for 20 min. The final tyrosinase solutions were analyzed after equilibrating at ambient temperature and pressure for 24 h.

Inactivation of Tyrosinase by Mild Thermal (MT) Treatments. For each experiment, 1 mL of tyrosinase solution was transferred into a 15 mL plastic tube without cap and then treated by MT in SS CO2 vessel at 35, 45, and 55 °C for 20 min. The vessel was preheated to the experimental temperatures before treatment, and the pressure was maintained at ambient pressure. The final tyrosinase solutions were analyzed after equilibrating at ambient temperature and pressure for 24 h. Dynamic Light Scattering (DLS). The size and zeta potential measurements were performed using a Zetasizer Nano-ZS device (Malvern Instruments, Malvern, Worcestershire, U.K.) equipped with a 532 nm excitation laser. The samples were prepared at 25 °C in 50 mM pH 6.5 phosphate buffers. The size and zeta potential measurements were reported as the mean and standard deviation of at least five readings. All measurements were carried out duplicate. Circular Dichroism (CD) Analysis. The far-UV CD spectra was recorded with a JASCO J-715 CD spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan), using a 1 mm path length quartz cell at ambient temperature (25 ( 1 °C), with protein concentration of 0.2936 mg/mL. CD spectra were scanned in the far-ultraviolet range from 200 to 260 at 50 nm/min. The photomultiplier absorbance in this work did not exceed 600 V in the spectral regions measured. Each spectrum was signal-averaged with four replicates and baseline-corrected by subtracting 50 mM sodium phosphate (pH 6.5) buffer spectrum. All the measurements mentioned before were taken under a nitrogen flow. A batch of mushroom tyrosinase treated by step temperature increases at the rate of 20 °C/5 min and then kept stable for data collecting for Langmuir 2011, 27(3), 909–916

5 min. The CD data were expressed in terms of mean residual ellipticity [θ] (deg cm2 dmol-1). The fractions in the secondary SS CO2- and MT-treated tyrosinase were estimated by CDSTTR algorithms; all the normalized root-mean-square deviation (NRMSD) values in this study were less than 0.1, which satisfied the requirements as suggested by CDSSTR algorithms.31 Tryptophan Fluorescence Spectroscopy Analysis. Tryptophan fluorescence spectra were measured with a Cary eclipse fluorescence spectrofluorometer (Varian Co. Ltd., Palo Alto, CA), using a 2 mm path length quartz cell at ambient temperature (25 ( 1 °C). The emission spectra (λem from 290 to 600 nm) were obtained at the excitation wavelength (λex) 280 nm with Ex 10 nm, Em 20 nm slits width. All the samples were determined immediately following treatments. Each spectrum was signal-averaged with three replicates and baseline-corrected by subtracting 50 mM pH 6.5 sodium phosphate buffer spectrum. The results were recorded as the mean value of three scans. Activity Assay of Tyrosinase. Mushroom tyrosinase activity was assayed by a spectrophotometric method32 with some modifications. Mushroom tyrosinase was diluted to a concentration of 0.0313 mg/mL. The assay was performed for all samples by incubating 0.1 mL of mushroom tyrosinase solution into 2.9 mL of 0.1 M phosphate buffer (pH 6.5) containing 0.1 M catechol as substrate. The increase in absorbance at 420 nm was monitored at intervals of 0.1 s-1 immediately after incubation in a Cary 50 spectrophotometer (Varian Co. Ltd., Palo Alto, CA), which equipped with a temperature control system: a Peltier device and a water pump (Varian Co. Ltd., Palo Alto, CA) to keep at 20 ( 0.1 °C. Prior to measurement, the substrate solution was pre-equilibrated to 20 °C with the temperature control system. The slope of the first (31) Whitmore, L.; Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 2008, 89(5), 392–400. (32) Rodrguez-Lpez, J.; Serna-Rodrguez, P.; Tudela, J.; Varn, R.; GarciaCnovas, F. A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid. Anal. Biochem. 1991, 195(2), 369–374.

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Hu et al. Table 1. PSD of the Tyrosinase Following the SS CO2 or MT Treatments

treatment conditions

35 °C, 5 MPa

35 °C, 8 MPa

35 °C, 12 MPa

35 °C, 15 MPa

45 °C, 8 MPa

55 °C, 8 MPa

35 °C

35 °C

45 °C

55 °C

untreated

peak particle diameter (nm) peak value (%) span (nm)

564 16.7 180

627 8.9 317

580 14.7 172

550 16.1 190

508 12.1 180

380 22.2 53

400 34.5 11

521 4.8 153

422 16.1 110

433 16.0 121

119 23.6 13

linear part of the reaction curve was taken as the tyrosinase specific activity (Abs/min). The residual activity of mushroom tyrosinase was calculated according to the following formula: residual activity ¼

specific activity of PPO after treated specific activity of PPO before treated  100%

Statistical Analysis. Analysis of variance (ANOVA) was carried out by using the software Microcal Origin 7.5 (Microcal Software, Inc., Northampton, MA). ANOVA tests were performed to determine the significance at 95% confidence. All experiments were performed at least in duplicate.

Results and Discussion DLS Analysis of the Tyrosinase Treated with SS CO2. Figure 1 shows the particle size distribution (PSD) of the untreated and treated tyrosinases using DLS analysis. As shown in Figure 1a, the PSD pattern of the untreated tyrosinase was characterized with a narrow span of 13 nm in particle diameter and a steep peak value of 23.6% in volume fraction with the corresponding particle diameter of 119 nm, suggesting the untreated tyrosinase in the aqueous system to be very monodisperse. Moreover, the MT treatment at 35 °C changed the PSD pattern of the tyrosinase, which showed two size distributions with values of 400 and 521 nm, respectively, indicating this treatment resulted in noticeable aggregation of the enzyme and the system becoming polydisperse (Table 1). However, as the temperature increased to 45 and 55 °C in the MT treatments, the PSD of tyrosinase occurred at 420 and 440 nm (Figure 1b), with a wider span of 110 and 121 nm, respectively. The results suggested that higher temperatures in the MT treatments favored the aggregation of the enzyme. Compared with the MT treatments, the PSD of the tyrosinase was more severely destroyed and stronger aggregation was observed following SS CO2 treatments (Figure 1a,b). This SS CO2-induced aggregation enhancement was also detected in the particle size of LOX in an earlier study21 using transmission electron microscopy and turbidity analysis. Khorshid et al.33 utilized supercritical CO2 to precipitation soybean protein as a clean purification process. The aggregation was closely related to the pressure level during the SS CO2 treatment at 35 °C (Figure 1a). When the pressure of 5 MPa was applied, the size of the enzyme increased up to 564 nm with a span of 180 nm. If the pressure increased to 8 MPa, the size reached 627 nm, where the span was enlarged to 317 nm. The results indicated that more inhomogeneous and larger aggregates produced from 5 to 8 MPa during SS CO2 treatments. Nevertheless, when the pressure continue to increase to 12 and 15 MPa, respectively, the size was reduced to 580 and 550 nm with spans of 172 and 190 nm, respectively (Table 1). The phenomenon suggested that higher pressure caused a homogeneous effect on the tyrosinase solution. Thus, these observations showed that the PSD pattern of the tyrosinases exhibited a two-phase transition during SS CO2 treatments. The aggregation was also related to the temperature during the SS CO2 treatment at a fixed 8 MPa (Figure 1b). When (33) Khorshid, N.; Hossain, M. M.; Farid, M. M. Precipitation of food protein using high pressure carbon dioxide. J. Food Eng. 2007, 79(4), 1214–1220.

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Figure 2. Zeta potential of the tyrosinase following the SS CO2 or MT treatments. (a) SS CO2 treatments at 35 °C and at various pressures for 20 min. (b) MT treatments and SS CO2 treatments at 8 MPa and at various temperatures for 20 min.

the temperature shifted from 35 to 55 °C, the PSD span of the tyrosinase decreased from 317 to 53 nm in particle diameter, and a peak value increased from 8.9 to 22.2% in volume fraction with the corresponding particle size decreasing from 627 to 380 nm (Table 1), indicating that higher temperature enhanced the SS CO2-induced homogenization effect. The aggregation was possibly due to the pH lowering effect by SS CO2 treatments in the aqueous system. When CO2 combines with water, it forms carbonic acid, which temporarily lowers the local environmental pH.18 The intensive electrostatic attraction and repulsion between side chains of proteins would be weakened by the reduced surface charges around the isoelectric point (pI) of one enzyme, which ultimately result in folding and unfolding of peptide chains24,34 and thus lead to aggregation.35 Researchers have reported a SS CO2-induced pH lowering effect in orange juice,18 carrot juice,36 apple juice,37 and peach juice.38 The pH value of the tyrosinase solution following SS CO2 treatments in this study reduced from 6.5 to 6.1; this small drop was caused by the buffer capacity and CO2 separation following SS CO2 treatments when the applied pressure was reduced to atmospheric level. However, the pI of the tyrosinase is 4.7-5 as shown in Sigma (34) Fink, A. L.; Calciano, L. J.; Goto, Y.; Kurotsu, T.; Palleros, D. R. Classification of acid denaturation of proteins: Intermediates and unfolded states. Biochemistry 1994, 33(41), 12504–12511. (35) Jachimska, B.; Wasilewska, M.; Adamczyk, Z. Characterization of Globular Protein Solutions by Dynamic Light Scattering, Electrophoretic Mobility, and Viscosity Measurements. Langmuir 2008, 24(13), 6866–6872. (36) Park, S. J.; Lee, J. I.; Park, J. Effects of a combined process of high-pressure carbon dioxide and high hydrostatic pressure on the quality of carrot juice. J. Food Sci. 2002, 67(5), 1827–1834. (37) Gui, F.; Wu, J.; Chen, F.; Liao, X.; Hu, X.; Zhang, Z.; Wang, Z. Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide. Food Chem. 2007, 100(4), 1678–1685. (38) Zhou, L.; Zhang, Y.; Hu, X.; Liao, X.; He, J. Comparison of the inactivation kinetics of pectin methylesterases from carrot and peach by highpressure carbon dioxide. Food Chem. 2009, 115(2), 449–455.

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Figure 3. Far-UV CD spectra of the tyrosinase following the SS CO2 or MT treatments. (a) MT treatments. (b) SS CO2 treatments at 35 °C and at various pressures for 20 min. (c) SS CO2 treatments at 8 MPa and at various temperatures for 20 min.

Product Information.39 The pH value of the tyrosinase solution during the treatments was not reduced to a level of 4.7-5; the aggregation may not be totally due to the small pH drop. Therefore, other factors may play major roles in the aggregation mechanism. During the depressurization of the SS CO2 treatments, a gasliquid interface forms when CO2 converts from an aqua state to a gas form as the pressure releases. At the gas-liquid interface, protein chains would unfold due to orientation of nonpolar residues to the gas phase and polar residues to the liquid phase; the unfolded molecule would then undergo aggregation over its (39) Robb, D. A.; Gutteridge, S. Polypeptide composition of two fungal tyrosinases. Phytochemistry 1981, 20(7), 1481–1485. (40) MacRitchie, F.; Alexander, A. E. Kinetics of adsorption of proteins at interfaces. Part II. The role of pressure barriers in adsorption. J. Colloid Sci. 1963, 18(5), 458–463.

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hydrophobic regions.40,41 In the meantime, the depressurization is followed by a subsequent sudden release of the applied CO2 pressure from the solvent, which results in rapid gas expansion from the enzymes.42,43 This gas expansion produced an explosive effect and induced a homogenization effect of the aggregates. High pressure and temperature brought about more drastic explosion and thus disassociated the aggregated enzymes into smaller particles. The alteration of the PSD following the SS CO2 treatments was determined to be an interaction among the (41) Macritchie, F. Proteins at interfaces. Adv. Protein Chem. 1978, 32, 283–326. (42) Kasche, V.; Schlothauer, R.; Brunner, G. Enzyme denaturation in supercritical CO2: Stabilizing effect of S-S bonds during the depressurization step. Biotechnol. Lett. 1988, 10(8), 569–574. (43) Giessauf, A.; Gamse, T. A simple process for increasing the specific activity of porcine pancreatic lipase by supercritical carbon dioxide treatment. J. Mol. Catal. B: Enzym. 2000, 9(1-3), 57–64.

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Figure 4. Fluorescence emission spectra of the tyrosinase following the SS CO2 or MT treatments. (a) MT treatments and SS CO2 treatments at 8 MPa and at various temperatures for 20 min. (b) SS CO2 treatments at 35 °C and at various pressures for 20 min.

acid- and interface-induced protein aggregation and the explosioninduced homogenization effect. Zeta Potential Analysis of the Tyrosinase Treated with SS CO2. The value of zeta potential of the untreated tyrosinase was -11.25 mV as shown in Figure 2a. The negative value indicated that the untreated enzyme was negatively charged at pH 6.5. By MT treatments, the value of zeta potential slightly decreased from -14.50 to -11.20 mV when increasing the temperature from 35 to 55 °C (Figure 2b), indicating that the surface charge reduced at a higher temperature. Such reduction in surface charge weakened the electrostatic repulsion and thus favored the enzyme aggregation. This observation was substantially in conformity with that of its PSD pattern (Figure 1). For the SS CO2 treatments at 35 °C, the value of zeta potential varied between -11.71 and -9.11 mV when the pressure varied between 5 and 15 MPa (Figure 2a). In contrast, when the temperature increased from 35 to 55 °C with fixing the pressure at 8 MPa, a decrease of the zeta potential was observed (Figure 2b). The variation of the zeta potential probably resulted from the conformational changed of the enzyme caused by the SS CO2induced aggregation and homogenization as described above, and thus made some of the negatively/positively charged amino acid residues moving to the surface of the enzyme, which ultimately produced the alteration of zeta potential. Moreover, the zeta potential may also be affected by the pH of the medium. It should be noted that the pH had only minor effects on the zeta potential when the pH was far from the protein pI.28,35 Indeed, the pH value was reduced by 0.4 unit following the SS CO2 in this work as 914 DOI: 10.1021/la103482x

described above and therefore was not the main factor to promote the change of the zeta potential. CD Analysis of the Tyrosinase Treated with SS CO2. The CD spectra of the MT- and SS CO2-treated tyrosinases are shown in Figure 3. Two double-negative slots at 208 and 222 nm of the untreated tyrosinase were characterized for R-helix conformation in the secondary structure. The value of the slots gradually decreased with increasing temperatures from 35 to 95 °C (Figure 4a), indicating that higher temperature may result in the loss of R-helix conformation. For the SS CO2-treated tyrosinase, the values of double-negative slots decreased as the pressure/temperature increased (Figure 4b,c), also suggesting a loss of R-helix conformation. As estimated by CDSTTR algorithms, R-helix conformation decreased respectively by 7% when increasing the temperature from 35 to 55 °C in the MT treatments, by 10% when increasing the pressure from 5 to 15 MPa during the SS CO2 treatments at 35 °C, and by 10% when increasing the temperature from 35 to 55 °C at 8 MPa. Similar observations had been reported by a number of previous studies. Ishikawa et al.44 presented that the microbubbled SS CO2 treatment could reduce the residual R-helix content of lipase, alkaline protease, acid protease, and glucoamylase to 62.9, 31.3, 37.6, and 12.4%, respectively. Gui et al.22 found that the residual R-helix relative content of SS CO2-treated horseradish POD decreased when the pressure level increased (44) Ishikawa, H.; Shimoda, M.; Yonekura, A.; Osajima, Y. Inactivation of Enzymes and Decomposition of R-Helix Structure by Supercritical Carbon Dioxide Microbubble Method. J. Agric. Food Chem. 1996, 44(9), 2646–2649.

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based on CD analysis. Liao et al.21 also observed that the R-helix content of LOX was reduced following SS CO2 treatment. Fluorescence Spectroscopy Analysis of the Tyrosinase Treated with SS CO2. Figure 4 shows alterations in the tryptophan fluorescence intensity and emission maximum wavelength (λmax) of the tyrosinases following the MT and SS CO2 treatments. The untreated tyrosinase exhibited a λmax of 337 nm (Figure 4a). Compared with the untreated tyrosinase, the fluorescence intensities of all the treated tyrosinases decreased, and λmax was redshifted. For the MT-treated tyrosinases, the fluorescence intensity decreased by 17.1% when increasing the temperature from 35 to 55 °C; the λmax shows almost no change for varying the temperature from 35 to 45 °C but exhibits a red-shifted variation from 338 to 341 nm for temperature increased from 45 to 55 °C (Figure 4a). Higher temperature can cause the unfolding of the protein peptide chains, which consequently led to the exposure of fluorescent groups to a more polar environment. Increases in the concentration of exposed fluorescent groups in the solvent could result in energy transfer and release and thus quenched the fluorescence intensity.45 Compared to the untreated and MT-treated tyrosinases, the fluorescence intensity of the tyrosinase decreased significantly

Figure 5. Residual activity of the tyrosinase following the SS CO2

or MT treatments. (a) SS CO2 treatments at 35 °C and at various pressures for 20 min. (b) MT treatment and SS CO2 treatments at 8 MPa and at various temperatures for 20 min.

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following the SS CO2 treatments at 8 MPa (Figure 4a). The fluorescence intensity of the tyrosinase changed slightly by increasing the temperature from 35 to 45 °C and then decreased by about 20% at 55 °C. Additionally, the λmax showed no change for the temperatures of 35-45 °C but a red-shift from 338 to 343 nm at 55 °C. These results suggested that the temperature affected the tryptophan fluorescence intensity and λmax of the tyrosinase in the same manner, no matter whether the SS CO2 treatments are applied and that the pressure at 55 °C enhanced the changes in the tryptophan fluorescence intensity and λmax. As shown in Figure 4b, the fluorescence intensity decreased as the pressure increased from 5 to 12 MPa but showed a slight increase from 12 to 15 MPa. The λmax was red-shifted from 338 to 341 nm as the pressure increased from 5 to 8 MPa and then blueshifted from 341 to 339 nm for the pressure increase from 12 to 15 MPa. The fluorescent groups were exposed to a more polar environment when the aggregation occurred over its hydrophobic regions at the gas-liquid surface following the SS CO2 treatments as described above, which ultimately quenched the fluorescence and caused the λmax to be red-shifted. Moreover, the sudden release of CO2 homogenized the tyrosinase particle solution and buried the exposed fluorescent groups inside the molecules, thereby slightly increasing the fluorescence intensity but decreasing the λmax as the pressure increased from 12 to 15 MPa. These results were almost in consistence with the SS CO2-induced aggregation and homogenization effect as described above. Liao et al.21 also observed that the fluorescence intensity of SS CO2treated LOX was reduced with increasing pressure. However, this finding was inconsistent with another observation that the fluorescence intensity of horseradish POD exposed to SS CO2 increased with increased the pressure.22 A reduction or increase in the fluorescence intensity of the model enzymes exposed to SS CO2 was closely related to the structure of the enzymes. Effect of SS CO2 on the Activity of the Tyrosinase. The inactivation effect of SS CO2 on the tyrosinase is shown in Figure 5. At 35 °C, there was no significant pressure-related enhancement in inactivation effect of the tyrosinase when the pressure was below 8 MPa. However, a significant reduction of about 25-30% in the activity was observed when the pressure was above 12 MPa (Figure 5a), suggesting that there was a pressure threshold to benefit the inactivation of the tyrosinase at this temperature. However, Weemaes et al.46 revealed that PPO is enzyme thermosensitive but rather pressure stable. The reduction in the activity of the tyrosinase indicated that a complex interaction rather than

Figure 6. Assumed inactivation mechanism of tyrosinase treated by SS CO2. Langmuir 2011, 27(3), 909–916

DOI: 10.1021/la103482x

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pressure may play a more important role in inactivating the tyrosinase during the SS CO2 treatments. As shown in Figure 5b, there was no significant difference in the activity of the tyrosinase when increasing the temperature during the MT treatments. However, the activity of the tyrosinase was significantly reduced during the SS CO2 treatments at 8 MPa with the increasing temperature. SS CO2 treatment at 8 MPa and 55 °C for 20 min led to a 40% reduction of the activity while the MT treatment at 55 °C for 20 min caused about 20% reduction of the activity (Figure 5b). The higher the temperature during SS CO2 treatments was, the greater the reduction of the activity of the tyrosinase was, indicating that the temperature may enhance the inactivation effect. No activation of the tyrosinase was detected in this study, but Liu et al.47 observed that high-pressure microfluidization treatment can induce the activation of the tyrosinase. Inactivation Mechanism of Tyrosinase Treated with SS CO2. As shown in Figure 6, an inactivation mechanism of the tyrosinase by the SS CO2 treatments could be assumed as follows: In an aqueous system, SS CO2 during pressurization could be dissolved into the hydration layer to lower pH value of local environmental and resulted in partial aggregation of the enzyme. On the other hand, the formation of a gas-aqueous interface and the quick release of CO2 during depressurization could exert an aggregation and homogenization effect on the enzyme particles. In addition, pressurized CO2 was rather hydrophilic. It could strip (45) L, C. A. S.; Sommer, F. B.; Teresa, N. M. Thermal denaturation of HRPA2:pH-dependent conformational changes. Enzyme Microb. Technol. 2007, 40(4), 696–703. (46) Weemaes, C.; Rubens, P.; De Cordt, S.; Ludikhuyze, L.; Van Den Broeck, I.; Hendrickx, M.; Heremans, K.; Tobback, P. Temperature sensitivity and pressure resistance of mushroom polyphenoloxidase. J. Food Sci. 1997, 62(2), 261–266. (47) Liu, W.; Liu, J.; Liu, C.; Zhong, Y.; Liu, W.; Wan, J. Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment. Innovative Food Sci. Emerging Technol. 2009, 10(2), 142– 147. (48) Yoshimura, T.; Shimoda, M.; Ishikawa, H.; Miyake, M.; Hayakawa, I.; Matsumoto, K.; Osajima, Y. Inactivation kinetics of enzymes by using continuous treatment with microbubbles of supercritical carbon dioxide. J. Food Sci. 2001, 66(5), 694–697.

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the essential water from the microenvironment around the tyrosinase, causing enzyme inactivation with the unfavorable water partitioning between the enzymatic support and the solvent,10 or alter the balance of the water-protein interactions, causing the hydrophobic groups of the protein to be exposed to the hydrophilic environment.48,49 As a consequence, the acid-induced and interface-induced protein aggregation, the explosion-induced homogenization, and the interaction between pressurized CO2 and the enzyme may may affect in combination the conformation of the enzyme and cause changes in the spacial, tertiary, secondary structures of the protein, which were observed through DLS determination, zeta potential measurement, CD spectra, and fluorescence spectra. These structural changes were responsible for the activity loss of the tyrosinase, demonstrating that the tyrosinase was inactivated by the SS CO2 treatments.

Conclusions SS CO2 treatments resulted in tyrosinase particle aggregation, and the aggregates were homogenized by its explosion during depressurization. The surface charge of the tyrosinase decreased following SS CO2 treatment. Its secondary and tertiary structure changed dependent on the pressures and the temperatures. These changes were substantially in conformity. SS CO2 in an aqueous system may effectively inactivate mushroom tyrosinase. The technique SS CO2 opened up a green way in inactivating enzymes and could apply in preservation of agricultural products. Acknowledgment. This research work is supported by of the National Natural Science Foundation of China (No. 30571297), Beijing National Natural Science Foundation (No. 6062015), the Science and Technology Support in the 11th Five-year Plan of China (No. 2006BAD05A02), and the Program for New Century Excellent Talents in University (No. NCET-06-0109). (49) Tsou, C. L. Location of the active sites of some enzymes in limited and flexible molecular regions. Trends Biochem. Sci. 1986, 11(10), 427–429.

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