Thermal Stability of Dehydrated α-amylase in Trehalose Matrices in Relation to its Phase Transitions

Transcription

1 Lebensm.-Wiss. u.-technol., 3, (1997) Thermal Stability of Dehydrated α-amylase in Trehalose Matrices in Relation to its Phase Transitions Mauricio R. Terebiznik, María P. Buera and Ana M.R. Pilosof M. P. Buera, A. M. R. Pilosof: Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (1428) Buenos Aires (Argentina) M. R. Terebiznik: Departamento de Industrias y de Ingeniería Química, Universidad de Buenos Aires, (1428) Buenos Aires (Argentina) (Received August 5, 1996; accepted October 16, 1996) Thermal stability of α-amylase in trehalose matrices of reduced moisture content was studied as affected by phase transitions occurring as a result of increasing temperature at a moisture content of g/kg. Removal of water greatly enhanced thermal stability of α-amylase but when trehalose was present an extraordinary stabilization was achieved. Even in an initially rubbery condition, the protective effect of trehalose could be assessed up to C. Deactivation kinetics in the range 8 C were related to crystallization of amorphous trehalose which would occur because the system was above the glass transition temperature. According to available water, at most % of amorphous trehalose would crystallize. The remaining amorphous trehalose phase would increase its glass transition temperature leading to enhanced enzyme stability. At temperatures close to 9 C, trehalose dihydrate crystals start melting, releasing water which could promote further trehalose crystallization and enzyme deactivation. Once trehalose crystallizes, the protective effect may be lost since crystalline trehalose forms a separated phase no longer associated with the enzyme. These phase transitions were reflected as breaks in the Arrhenius plots Academic Press Limited Keywords: α-amylase; stability; trehalose; phase transitions Introduction Fungal α-amylase is currently employed in the baking industry, brewing of beer and saccharification. The problem of the long-term stability of the enzymes places an important limitation on their use. In order to produce stable, storable enzymes, drying is currently applied. Removal of water reduces the freedom of movement of the protein molecules and thus inhibits conformational changes leading to activity losses (1). The effect of trehalose as a protectant of biomolecules during drying has been extensively investigated since it was found to be optimal in protecting membranes (liposomes, microorganisms), proteins and DNA restriction enzymes (2 6). Trehalose, like most carbohydrates, forms amorphous structures in low moisture biological materials (7 9). The amorphous matrix may exist as either a very viscous glass or as a more liquidlike rubber. A glass is characterized by a very low molecular mobility, translational motion, and restricted rotational motion. The change from the glassy to the rubbery state occurs as a second order phase transition at a critical temperature (which depends on the amount of water and other plasticizers) known as the glass transition temperature (Tg). The main consequence of glass transition is an increase in molecular mobility and free volume, which may result in physical and physicochemical deteriorative changes (1 12). Once the system is above the Tg, first order transitions, such as crystallization and melting, are allowed to occur. Crystallization of amorphous sugars in food systems may enhance both physical and chemical deterioration (1, 13 16). The protective effect of trehalose, for instance, was observed to be lost in systems in which a high proportion of trehalose crystallized (8, 17). The objective of the present work was to study the thermal stability of α-amylase in trehalose matrices as affected by phase transitions occurring as a result of increasing temperature at a given moisture content. Materials and Methods Enzyme preparation Solid state fermentation of Aspergillus oryzae NRRL 3485 was performed on wheat bran according to Terebiznik et al. (18). Water extract of enzyme was obtained by adding distilled water ( ml/kg of dry substrate) to the substrate fermented for 48 h at 37 C. The liquid phase was recovered by filtering through a gauze and centrifuging for 15 min at 1, g. The resulting supernatant was filtered through a.22 µm /97/ $.//fs Academic Press Limited 513

2 Millipore filter. α-amylase was around g/l corresponding to 3% of the total protein contained in the fermentation water extract (18). The effect of ph on the stability of α-amylase was first studied in order to select the adequate ph for performing the thermal inactivation studies. From these studies it was concluded that ph 8 greatly prevented enzyme deactivation when stored for up to 1 month at 28 C. Thus, enzyme extracts which had a ph between 6.5 and 7 were adjusted to ph 8 with 1 mol/l NaOH solution to minimize the effect of this variable on enzyme denaturation during the following studies. Enzyme deactivation assays Thermal deactivation of the enzyme in water extracts was assayed over aliquots of.5 ml contained in microcentrifuge tubes. Dried enzyme was prepared by freeze-drying 1 ml aliquots of water extract poured in 2 ml vials and frozen at 2 C. The frozen samples were soaked in liquid air just before the freeze-drying process. A Stokes freeze-dryer model 21 (F.J. Stokes Company Equipment Div., Pennsal Chem. Corp. Philadelphia, PA), which operated at 4 C and at a chamber pressure of less than 1 µm Hg, was used. Following freeze-drying the samples were transferred into an evacuated desiccator and kept over saturated potassium acetate solution (relative humidity, RH 22%) for 1 week at C. Amorphous α-amylase trehalose systems were prepared as indicated above for the dried enzyme but with the addition of 2 g/l trehalose to the water enzyme extract (Trehalose enzyme ratio of 4 to 1). After equilibration to RH 22%, vials containing the systems were hermetically sealed and placed in a glycerol thermostatic bath operated at the indicated temperatures in each case. At suitable intervals two samples were removed from the bath, brought to the original volume with distilled water and the remaining α-amylase activity determined as described below. absorbance measured at 64 nm. One unit of α-amylase activity was defined as the amount of enzyme that could hydrolyse 1 mg of starch in 3 min to a stage at which no colour was detected with iodine. Differential scanning calorimetry (DSC) DSC was performed with a Polymer Laboratories thermal analysis system (Rheometric Scientific, Thermal Science Division Polymer Laboratories LTD., Surrey Business Park, Kilm Lone, Epson, Surrey KT17 15F, U.K). The thermograms were analysed using Plus V Polymer Laboratories software. Temperature calibration was done by ASTM E 474/8 norm using the melting thermograms of indium and ice (2). An empty aluminium pan was used as reference. Measurements were done in duplicate and the average value reported. Results and Discussion The remaining activities of α-amylase in water extracts heated at different temperatures are shown in Fig. 1a as a function of heating time. The deactivation was modelled on a first order reaction: A(t) = A o exp ( K (T) t) Eqn [1] where K (T) is first order deactivation rate constant. The reaction rate constants obtained by linear regression of Eqn [1] were plotted in an Arrhenius graph (Fig. 1b) to obtain the activation energy (E a ) of thermal deactivation: K (T) = K o exp ( E a /RT) Eqn [2] The calculated activation energy (E a ) was with a standard deviation of 9.6 J/mol. This value is close to the value reported for neutral protease of Aspergillus oryzae (21). Figure 2 shows the thermogram corresponding to a sample of freeze-dried α-amylase dissolved in distilled Determination of moisture content Moisture content of the samples was determined by the difference in weight before and after drying in a vacuum oven at 7 C for 48 h over magnesium perchlorate. These conditions had been shown to result in a constant weight (8). α-amylase activity α-amylase was assayed by a modification of the method of Smith and Roe (19). Ten microlitres of a dilution of the enzyme preparation and.5 ml of.5 g/l Lintner soluble starch (Merck) in.2 mol/l acetate buffer ph 5 were mixed and incubated at 37 C for 7.5 min. The reaction was stopped by acidification and the remaining starch reacted with.5 ml of lugol reagent (1 mmol I 2 /8 mmol IK) in 2 mmol HCl. The mixture was diluted to 5 ml with distilled water and the ln K (1/h) /T ( K) Fig. 1 (a) Remaining activity of α-amylase in water extracts heated at different temperatures. ( ) = 45 C ( ) = 52 C; ( ) = 6 C; ( ) = 62 C; ( ) = 65 C; (*) = 7 C. (b) Arrhenius plot for deactivation rate constants of α-amylase in water extracts 2. (b) (a)

3 Heat flow (mj/s) Temperature ( C) 1. ln K (1/h) Fig. 2 DSC thermogram for α-amylase in distilled water (3 g/l) (b) /T ( K) Fig. 3 (a) Remaining activity of freeze-dried α-amylase humidified to 22% RH and heated at different temperatures. ( ) = 6 C; ( ) = 66 C; ( ) = 7 C; ( ) = 76 C. (b) Arrhenius plot for deactivation rate constant of freeze-dried α-amylase (a) 3. water (3 g/l). The endotherm was characterized by a peak temperature of 69.9 ±.4 C and an apparent enthalpy of denaturation ( H) of 1.8 ± 1.7 J/g. The denaturation temperature of the present enzyme was higher than that reported by Fukada and Takahashi (22) (61 64 C) from differential scanning calorimetric studies of Taka-amylase from Aspergillus oryzae. Denaturational enthalpy value agrees with reported values for globular proteins (21). The observed denaturation temperature supports the results from deactivation studies which showed that α-amylase was fully deactivated by heating for 2 min at 7 C. Remaining activity of freeze-dried enzyme, previously equilibrated at 22% RH, is shown in Fig. 3a for different temperature treatments. Removal of water greatly enhanced thermal stability of α-amylase as the T value (temperature for which the half-life of the enzyme equals that obtained for the enzyme water extract heated at 6 C) increased by approximately 12 C. The activation energy calculated from the Arrhenius plot (Fig. 3b) was 212 ± 23 J/mol and was not significantly different from that obtained for the enzyme in solution suggesting that despite the fact that removal of water had profound effects on the rate of denaturation it might not affect the mechanism of deactivation. When the enzyme was freeze-dried in a trehalose matrix (2 g/l in the enzyme water extract) and equilibrated at 22% RH the half-life at 6 C (528 h) was about 43 times greater than that showed for the freeze-dried enzyme (1.22 h). Among the sugars, trehalose has been shown to induce a high protection of enzymes during freeze-drying (23) and air-drying (24). Colaco et al. (6) reported that DNA restriction and modifying enzymes can be dried in the presence of trehalose with no loss of activity even after prolonged storage and exposure to temperatures as high as 7 C. This stability was unique to trehalose and has not been found with other sugars. Recently thermal stability of enzyme invertase was studied in reduced moisture matrices of trehalose, maltodextrin and polyvynilpyrrolidone (8, ). Among these matrices, trehalose was found to be superior at preventing thermal deactivation of invertase both in the glassy or rubbery state for given temperature/moisture conditions, even when partial crystallization occurred. The ability of the freeze-dried enzyme/trehalose system to withstand exposure to high temperatures was assayed. Deactivation kinetics at 8, 85, 9 and C are shown in Fig. 4a d. The T value increased approximately 35 C relative to the dried enzyme without trehalose indicating that trehalose offers a high thermoprotection. While the shapes of curves in Fig. 4a and d were similar to those in Figs 2 and 3, the shapes of curves in Fig. 4b and c were quite different as they showed three phases. A first phase of rapid deactivation until 6% of retained enzyme activity was followed by a second phase in which activity changed little over time (85 C) or reached a defined plateau (9 C). Following this, a last fast deactivation phase was observed. The observed changes on deactivation kinetics were related to phase transitions of amorphous trehalose. Trehalose is a nonreducing disaccharide known to form dihydrated crystals (26, 27). The amorphous trehalose α-amylase system obtained by freeze-drying and equilibration at 22% RH (equilibrium moisture content was g/kg) had a Tg of 43 C (Fig. 5). This value agrees fairly well with a Tg of 45 C reported for amorphous trehalose equilibrated at 22% RH (8), indicating that the Tg of the system is mainly determined by the sugar matrix. This value is far below the Tg reported for dry trehalose (8 C) reflecting the plasticizing effect of water that decreases Tg. During thermal treatment, as temperatures assayed were above Tg, the trehalose matrix was initially in a rubbery condition. Above the glass transition temperature molecular mobility is greatly increased and many amorphous compounds crystallize. Crystallization of amorphous sugars occurs above Tg and below Tcr (crystallization temperature) as a function of time (28). Reported Tcr for trehalose is 12 C 515

4 (a) (b) 2 (c) (d) Fig. 4 Remaining activity of α-amylase freeze-dried in trehalose matrices humidified at 22% RH and heated at (a) 8 C, (b) 85 C, (c) 9 C and (d) C (8) at a heating rate of 5 C/min, and instantaneous crystallization will occur at (Tcr Tg) = 57 C. Trehalose amylase samples equilibrated at 22% RH had (T Tg) values between 35 and 55 C for heating temperatures between 8 and C, respectively. Thus approaching C, crystallization would occur instantaneously. Figure 6 shows the (T Tg) plots of deactivation rate constants. Above a (T Tg) = 35 C, deactivation rate was greatly enhanced according to increasing trehalose crystallization rates. Once trehalose crystallizes, it essentially forms a separated phase (27) and the protective effect may be lost since crystalline trehalose is no longer associated with the enzyme and thermal inactivation is free to occur. Karmas (27) reported that nonenzymatic browning of xylose and lysine in trehalose matrix was highly increased in samples humidified at RHs where crystal hydrate formation of trehalose occurred. In our experiments, systems were sealed so that when crystallization to the dihydrate crystals (the common type of crystal form) occurred water was absorbed by the crystal (27). The amount of water needed to form the dihydrate is 1.5% on a dry trehalose basis; this moisture value is two times the moisture content of equilibrated trehalose at 22% RH ( g/kg moisture on dry basis). Thus, according to available water, at most % of amorphous trehalose would crystallize. A competition between the amorphous phase and crystallizing trehalose for water leads to the removal of water from the amorphous phase (M. Karel, pers. comm.). The decrease of moisture content of the amorphous 3 2 Heat flow (mj/s) 43 C K (1/h) Temperature ( C) T Tg ( C) 6 Fig. 5 DSC thermogram showing Tg for α-amylase freezedried in trehalose matrix humidified at 22% RH 516 Fig. 6 Deactivation rates of α-amylase freeze-dried in trehalose matrices as a function of (T Tg)

5 trehalose phase would increase its glass transition temperature and decrease the mobility of enzyme in the remaining amorphous trehalose matrix. However, from Fig. 4a it can be seen that when treated at 8 C enzyme activity reached values that were lower than % after 7 d storage indicating that besides the increasing of the Tg of the remaining amorphous trehalose matrix, α-amylase continued to be lost. Karmas (27), on studying nonenzymatic browning reactions, and Mazzobre et al. (17) studying invertase deactivation demonstrated that there are separate effects of temperature and moisture content which are not accounted for by the physical condition of the matrix. The extraordinary increase in deactivation rates that took place at heating temperatures above 8 C reflects a specific effect of temperature on the rate of trehalose crystallization and subsequent phase transition of dihydrate crystals. In fact, at temperatures close to 9 C trehalose dihydrate crystals start to melt (8). Jeffrey and Nanni (29) reported a melting point of C followed by crystallization of the anhydrous form above 12 C. Thus, temperatures above 85 C, while increasing the rate of trehalose dihydrate crystallization, would also promote melting of the crystals. The moisture released by melted dihydrate crystals would eventually diffuse into the remaining amorphous trehalose promoting its crystallization. Thus a cycle of crystallization, melting and further crystallization would be able to take place. The plateau phase in Fig. 4b and c would occur as a result of decreased deactivation rates of α-amylase included in the remaining trehalose amorphous phase due to the increased Tg of this phase as it is depleted of water by crystallizing trehalose. Simultaneously, melting of trehalose dihydrate would occur and released water would accelerate crystallization of the remaining amorphous trehalose phase leading to the fast last deactivation phase (third phase in Fig. 4b and c). When heating at C, denaturation and phase transitions of trehalose would take place at accelerated rates so that the plateau is not so apparent. At C almost all the activity was lost within 1 h. The Arrhenius plot of deactivation rate constants (for 9 C the first kinetic phase was considered) for the trehalose α-amylase system is shown in Fig. 7. According to the Arrhenius equation a linear relationship exists between ln k and 1/T. If a change in reaction mechanism or physical state of the system occurs at a particular temperature, a break in the curve may appear (3). Undoubtedly, the two breaks in the Arrhenius curve were related to first order phase transitions of trehalose indicating that crystallization is the rate determining step for α-amylase deactivation. The low activation energy obtained between 6 and 8 C (4 J/mol) reflects a molecular mobility-limiting environment. The activation energy of reactions in a diffusion limited environment is expected to be 33 J/mol (31). Between 8 and 85 C an extraordinary increase in α-amylase deactivation rate constants reflects the rapid crystallization of the trehalose matrix ln K (1/h) /T ( K).31 Fig. 7 Arrhenius plot for deactivation rates constant of α-amylase freeze-dried in trehalose matrices in this temperature range. A high activation energy (4 J/mol) was associated with the crystallization phenomenon. The activation energy above 85 C was 128 J/mol reflecting an increased molecular mobility as a result of partial trehalose crystallization and melting. Conclusions Removal of water greatly enhanced thermal stability of α-amylase. However, when dehydration was carried out on a trehalose matrix an extraordinary stabilization was achieved. Even in an initially rubbery condition, the protective effect of trehalose could be assessed up to C. Partial crystallization of the matrix could remove water from the amorphous phase thus increasing its glass transition temperature and leading to enhanced enzyme stability. At temperatures close to 9 C, trehalose dihydrate crystals start melting, releasing water which could promote further trehalose crystallization. The breaks in the Arrhenius plots could be attributed to first order transitions (crystallization/melting) which may occur cyclically as a consequence of heating the system above its initial Tg, close to the melting temperature of trehalose dihydrate crystals. Present results show the impact of first order phase transitions (which are on time related to second order phase transitions) on the kinetics of deteriorative reactions in low moisture systems. Acknowledgements The authors acknowledge financial support from the University of Buenos Aires (Secretaría de Ciencia y Técnica), Consejo Nacional de Investigaciones Cientificas y Técnicas de la República Argentina, and International Foundation for Science (Sweden). We thank Dr Marcus Karel for his helpful comments and Dr Jorge Wagner for his technical assistance in DSC measurements.

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