- Email: [email protected]
Kinetics of thermal inactivation of free Aureobasidium pullulans fructosyltransferase
Enzyme and Microbial Technology 47 (2010) 134–139
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Kinetics of thermal inactivation of free Aureobasidium pullulans fructosyltransferase a ˇ Zdenka Onderková a , Jolanta Bryjak b , Katarína Vanková , Milan Polakoviˇc a,∗ a Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic b Department of Bioorganic Chemistry Faculty of Chemistry Wroclaw University of Technology, Norwida 4/6, 50-373 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 15 February 2010 Received in revised form 21 June 2010 Accepted 23 June 2010 Keywords: Fructosyltransferase Thermal stability Biocatalyst operational lifetime Inactivation Kinetics Modelling
a b s t r a c t A batch catalytic process of fructooligosaccharide production with the initial sucrose concentration of 700 g dm−3 was carried out in the temperature interval of 67.5–78 ◦ C to investigate the thermal stability of free Aureobasidium pullulans fructosyltransferase. Inactivation experiments were carried out also for a saccharide-free solution in the temperature interval of 53.1–71 ◦ C so that a stabilizing effect of saccharides contained in the reaction mixture could be assessed. A model of enzyme inactivation kinetics based on a two-step sequential mechanism was developed and verified for both media using the multitemperature evaluation of inactivation data. The model was used to predict the operational lifetime and optimal operational temperature of the biocatalyst. It was found that the use of the concentrated sucrose solution increases the operational temperature by about 15 ◦ C compared to a dilute aqueous solution. The value of 47.5 ◦ C was estimated as an optimal temperature at which the enzyme operational lifetime would be 1 year. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Fructosyltransferase (FTase, EC 2.4.1.9) from Aureobasidium pullulans [1,2] is one of the most common biocatalysts used for industrial production of fructooligosaccharides. Fructooligosaccharides are regarded as functional additives with acknowledged beneficial health effects that are produced via a fructosyl-transfer action of FTase on concentrated solutions of sucrose containing about 50–55% of saccharides and elevated temperatures of 50–55 ◦ C [3]. Immobilization of FTase is very important for the reduction of costs in a large-scale production owing to the process continualization [4] when some common immobilization methods do not have a strong stabilization effect on the enzyme [5]. A long-term operational stability of an enzyme preparation is a common prerequisite of successful large-scale operation of a biocatalytic process. This prerequisite implies a need of long-term laboratory experiments in the stage of biocatalyst development. It is possible to reduce the time needed for determination of operational stability by methods of accelerated testing either in continuous [6–10] or batch [11–14] systems when biocatalysts are exposed to more severe conditions than those on the process scale. This leads to an accelerated activity loss and the biocatalyst operational stability is predicted from the mathematical model derived from the accel-
∗ Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52493198. E-mail address: [email protected] (M. Polakoviˇc). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.06.009
erated activity loss experiments. The potential of this approach has unfortunately been underexploited probably due to non-trivial mathematical modelling required for its application. Key factors for the operational stability of FTase are temperature and the total concentration of saccharides in a reaction mixture [1]. Short-term experiments were carried out at 55 ◦ C and initial sucrose concentration ranging from 0 g dm−3 to 700 g dm−3 in order to investigate the effect of total saccharide concentration on the thermostability of whole-cell FTase of A. pullulans [1]. The rate of inactivation of the enzyme was slow at the overall saccharide concentration above 600 g dm−3 . During 1-h experiments, the total activity loss was less than 5%. On the other hand, the inactivation rate quickly grew with a decrease of saccharide concentration below this threshold. FTase lost 75% of initial activity during 1 h in a saccharide-free solution. Due to the limited time of inactivation, a simplified model of thermal inactivation was obtained in the form of a single-step reversible reaction kinetics and exponential relationship of rate constants on sucrose concentration. The objective of this study was to investigate the stabilizing effect of saccharides and to develop a kinetic model of thermal inactivation for the isolated enzyme that could be used for reactor design. In order to achieve these goals, inactivation batch experiments were performed at different temperatures in a reaction mixture and saccharide-free solution and the selected model was verified using a simultaneous fit of the inactivation data obtained.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139 2. Experimental 2.1. Preparation of enzyme A crude extract of the cells A. pullulans [15] was prepared by high-pressure homogenization [16]. The extract was then dialyzed through a membrane SpectraPor, MWCO 50,000 (Spectrum Medical Industries, USA) at 6 ◦ C against a sodium phosphate–citrate buffer with pH 5.5 (0.02 mol dm−3 sodium hydrogen phosphate and 0.01 mol dm−3 citric acid in the volumetric ratio 4.1:3). The retentate was further purified by ion-exchange chromatography using an anion-exchange resin Sepabeads FP-DA with the particle size of 150–300 m (Mitsubishi Chemical Co., Japan) [17]. 2.2. Determination of enzyme activity One unit of the FTase activity was defined as the number of micromoles of kestose (trisaccharide having two fructosyl units bound to a glucosyl moiety) produced from sucrose per 1 min. 0.5 cm3 of an enzyme sample was added into 5.5 cm3
135
of a sucrose solution in the sodium phosphate–citrate buffer (pH 5.5) at 55 ◦ C so that the final concentration of sucrose in the reaction mixture was 700 g dm−3 (2.045 M). Samples of the reaction mixture were withdrawn in predetermined time intervals. The reaction was stopped by boiling in a water bath for 2 min. The concentration of kestose was determined by HPLC (Knauer, Germany) using the column Watrex IEC Pb form as described previously [18].
2.3. Inactivation experiments The measurements of FTase inactivation were performed in thermostatted stirred glass reactors. One cubic centimeter of enzyme was added to 29 cm3 of a preheated sucrose solution in the phosphate–citrate buffer with pH 5.5 or to this buffer not containing sucrose. The influence of temperature was studied in the interval of 67.5–78 ◦ C for the reaction mixture with the initial sucrose concentration of 700 g dm−3 and of 53.1–71 ◦ C for the saccharide-free solution. In appropriate time intervals, 1 cm3 samples were withdrawn from the reactor and assayed for the residual activity as described above.
Fig. 1. Thermal inactivation of free Aureobasidium pullulans FTase in the (a, b) reaction mixture and (c–e) saccharide-free solution, respectively. The symbols represent the experimental activity data at different temperatures and the lines their fits with the model (Eqs. (2) and (3)).
136
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
2.4. Mathematical modelling
The inactivation process was described by a set of the following differential equations:
A kinetic model [19] was examined in this study for the description of the courses of the relative enzyme activity a, defined as the ratio of cN , the concentration of native enzyme form N in time t, and cN0 , its initial concentration. This model was based on a mechanism,
k+1
k2
N R−→I
dcN = −k+1 cN + k−1 cR dt
(2a)
dcR = k+1 cN − k−1 cR − k2 cR dt
(2b)
t = 0, (1)
k−1
where N undergoes a reversible transformation into an intermediate inactive form R, which then reacts irreversibly to an inactive form I. The symbols k+1 , k−1 , and k2 are the rate constants of individual reactions. The mechanism has the same form as the well-known mechanism of Lumry–Eyring [20] but the interpretation is different. In the Lumry–Eyring mechanism, the intermediate is considered to be a fully unfolded enzyme and rapid equilibrium is typically assumed for the first reaction.
cN = cN0 ,
cR = 0
(2c)
The temperature dependence of the rate constants was expressed in the form of a rearranged Arrhenius equation, ki = eln ki0 e[(Ei /RT0 )(1−T0 /T )] ,
i = +1, −1, 2
(3)
where Ei is the activation energy of the ith reaction, R is the universal gas constant, and ki0 the value of the rate constant ki at the reference temperature T0 . The so-called multitemperature evaluation [11], where inactivation data for all temperatures are fitted simultaneously using non-linear regression, was applied. Mathematical modelling software Athena Visual Workbench (Stewart & Associates, Madison, WI, USA)
Fig. 2. Thermal inactivation experiments for the saccharide-free solution from Fig. 1 plotted in semi-logarithmic scale: (a) 53.1 ◦ C, (b) 57 ◦ C, (c) 61 ◦ C, (d) 64 ◦ C, (e) 70 ◦ C, and (f) 71 ◦ C. The straight-lines delimit the interval of first-order kinetics rate constants corresponding to individual experimental activity vs. time values.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139 was used for the parameter estimation. It used a gradient non-linear least square method and parameter uncertainties were calculated from the variance–covariance matrix.
3. Results and discussion Fig. 1 presents the results of FTase inactivation experiments carried out in the reaction mixture and saccharide-free solution, respectively. The experiments were designed to span very different intervals of the rates of activity loss, from relatively slow ones with enzyme half-lives of about 1 h (in both media) up to fast inactivation with an almost complete loss of activity in less than 5 min. In order to achieve the same window of observable inactivation rates in both environments, different temperature ranges had to be chosen. The width of this range was 18 ◦ C for the saccharide-
137
free solution (53–71 ◦ C) but only 10.5 ◦ C for the reaction mixture (67.5–78 ◦ C). Some inactivation experiments presented in Fig. 1 clearly demonstrate a biphasic character of enzyme inactivation when the first, faster phase is followed by the second, slower phase [21]. The best example is the experiment carried out in the reaction mixture at 67.5 ◦ C (Fig. 1a) where the activity loss was much slower after 1 h when the residual activity reached the value of about 50%. The biphasic inactivation is however not so apparent for most experiments. For that reason, Figs. 2 and 3 present individual experiments in a semi-logarithmic form of activity–time relationships where a deviation from a straight line trend defines a degree of departure from first-order kinetics. The deviations from first-order kinetics demonstrate an interesting trend with temperature which is completely opposite as
Fig. 3. Thermal inactivation experiments for the reaction mixture from Fig. 1 plotted in semi-logarithmic scale: (a) 67.5 ◦ C, (b) 69 ◦ C, (c) 70 ◦ C, (d) 75 ◦ C, and (e) 78 ◦ C. The straight-lines delimit the interval of first-order kinetics rate constants corresponding to individual experimental activity vs. time values.
138
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
Table 1 Model parameters of the inactivation kinetics for free Aureobasidium pullulans FTase in the reaction mixture with the initial sucrose concentration of 700 g dm−3 and in a saccharide-free solution. Parameter
Reaction mixture
Saccharide-free solution
k+10 (10−4 s−1 ) k−10 (10−4 s−1 ) k20 (10−4 s−1 ) E+1 (105 J mol−1 ) E−1 (105 J mol−1 ) E2 (105 J mol−1 ) H (105 J mol−1 ) S (J mol−1 K−1 )
7.72 ± 0.77 7.87 ± 4.88 11.5 ± 6.8 3.74 ± 0.15 6.04 ± 2.14 16.5 ± 6.1 −2.30 −670
1160 ± 180 999 ± 310 146 ± 47 3.53 ± 0.11 7.04 ± 0.61 14.9 ± 2.6 −3.51 −1020
Reference temperature T0 = 343.15 K. The values after the plus/minus sign represent the half-widths of 95% confidence intervals.
the one observed in previous works [11,19,21]. The rate of FTase inactivation in saccharide-free solution at 53.1 ◦ C was governed by first-order kinetics (Figs. 1c and 2a). At higher temperatures, a departure from first-order kinetics is evident (Fig. 2b–f) when the deviation increases with temperature. The deviation is demonstrated by a phase with a significant deceleration of inactivation rate, which start is best visible at the residual activity of 40–50% for the experiments carried out at 70 ◦ C and 71 ◦ C (Figs. 1e, 2e and f). Deviations from first-order kinetics occur also for the inactivation experiments carried out in the reaction mixture at the temperatures from 67 ◦ C to 70 ◦ C (Figs. 1a and 3a–c). The deviation however disappears at higher temperatures of 75 ◦ C and 78 ◦ C (Figs. 1b, 3d and e). The trend was exactly opposite as for inactivation in the saccharide-free solution. Based on these observations, it was concluded that the simplest mechanism, which could describe, the inactivation behavior of FTase in both media is a two-step sequential mechanism (Eq. (1)) [19]. Each of the two sets of inactivation data presented in Fig. 1 was fitted simultaneously with the model (Eqs. (2) and (3)). Fig. 1 shows that a good match was achieved between the measured and fitted activity data both in the reaction mixture and saccharide-free solution when the values of the mean square error are equal to 3.2% and 4.3%, respectively. Table 1 presents the values of the estimated model parameters and their uncertainties represented by the half-widths of 95% confidence intervals. Since all uncertainties are lower than the corresponding mean values, all parameters are statistically significant and the model is adequate. The estimated parameters were used to calculate the dependence of the rate constants k+1 , k−1 , and k2 and the equilibrium constant, K1 = k+1 /k−1 , on temperature (Fig. 4a and b).
The results presented in Table 1 and Fig. 4 nicely illustrate the similarities and differences between the inactivation processes occurring in the reaction mixture and saccharide-free solution. The differences between the estimated activation energy values in the reaction mixture and saccharide solution are not statistically significant because their confidence intervals overlap. This compatibility gives a further credibility to the validated model. The differences by one or two orders of magnitude in the rate constants at the reference temperature of 70 ◦ C reflect the stabilization effect of saccharides on the inactivation process. The stabilization effect of saccharides on the FTase inactivation is thus of entropic character [22] which means that saccharide molecules probably interact primarily with water molecules surrounding the enzyme not with the enzyme molecule itself. Fig. 4a shows that the second reaction is so slow below 65 ◦ C that the inactivation process is completely controlled by the first, equilibrium reaction, which was also concluded previously for short-term inactivation experiments in the reaction mixture at 55 ◦ C [1]. This inactivation behavior is well demonstrated in Fig. 1d. The equilibrium constant strongly decreases with temperature but it is so high below 60 ◦ C that the first reaction becomes essentially irreversible (Fig. 4b). Therefore, the kinetics of inactivation at 53.1 ◦ C appears to be of first-order (Figs. 1c and 2a). Fig. 4a further shows that all three rate constants are of the same order of magnitude in the temperature interval of approximately 65–70 ◦ C for both the reaction mixture and saccharide-free solution. This means that the sequential character of the inactivation reactions is the most pronounced in this interval which is demonstrated by the biphasic character of inactivation curves (Figs. 1a, e, 2e–2f, and 3a–c). The most significant feature of the inactivation process at the temperatures above 70 ◦ C is that the values of the rate constant k2 are much higher than those of k+1 and k−1 (Fig. 4a). Consequently, the concentration of the intermediate form R is negligible in spite of that the equilibrium of the first reaction is shifted towards the native form N (Fig. 4b). The inactivation process is again governed by the forward reaction of the first reversible step, its kinetics is of first-order and the inactivation curves are monophasic (Figs. 1b, 4d and e). A noticeable fact is the decrease of the equilibrium constant K with temperature. Applying the van’t Hoff theory, the values of the standard reaction enthalpy H and standard reaction entropy S (Table 1) were calculated from the kinetic parame ters: H = E+1 − E−1 ; S = R ln k+10 /k−10 + ((E+1 − E−1 )/T0 ). In compliance with the theory, H is negative in both media. The exothermic character of the first reaction means that this step cannot be interpreted as a denaturation one. It implies that associa-
Fig. 4. Temperature dependence of (a) rate and (b) equilibrium constants of FTase inactivation plotted with reciprocal horizontal and logarithmic vertical axes. The lines are calculated from the model (Eqs. (2) and (3)) using the estimated parameters from Table 1. The points correspond to the temperatures of inactivation experiments. Solid symbols represent the saccharide-free solution and open symbols the reaction mixture.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
139
of rate constants. The model gave a plausible explanation for the observations that the inactivation process was controlled by apparent first-order kinetics at low and high inactivation temperatures whereas it had a biphasic character at medium temperatures. At the low inactivation temperatures, the key rate-controlling reaction had about 150 times lower rate in the reaction mixture than that in the saccharide-free solution whereas the activation energy values differed only by 5%. The stabilization effect of saccharides was thus of entropic character. Acknowledgements This study was supported by the Science and Technology Assistance Agency APVV (LPP-0234-06) and Slovak Grant Agency for Science VEGA (Grant No. 1/0655/09). References Fig. 5. Lifetime of free Aureobasidium pullulans FTase vs. temperature in the reaction mixture (solid line) and saccharide-free solution (dashed line) calculated using the parameters presented in Table 1.
tion reactions can play a major role. Large activation energy values however indicate that the identified model may disguise a more complex inactivation mechanism and kinetics. A typical operational lifetime of industrial biocatalysts is determined by the period during which the biocatalyst activity drops to about 10% of the initial value. It determines a cycle needed for replacement of spent biocatalyst and therewith the biocatalyst cost per unit amount of product. In order to avoid an exorbitant biocatalyst cost, an efficient operational lifetime should be about 1 year for intracellular fungal glycoenzymes such as FTase [4]. The biocatalyst operational lifetime effectively determines the operational temperature because thermal inactivation is the main cause of biocatalyst activity loss. The operational temperature subsequently determines the reactor size and total load of biocatalyst in the reactor. Fig. 5 shows the lifetime of FTase in both media as a function of temperature calculated using the parameters presented in Table 1. Both dependences have the same trend with a temperature shift of about 12–15 ◦ C that expresses the stabilizing effect of saccharides in a different way. The predicted FTase lifetime in the reaction medium increases rapidly below the temperature of 55 ◦ C and the recommended value of 1 year is found at 47.5 ◦ C. This temperature is however by 20 ◦ C lower than the lowest temperature used in the experiments. The estimated lifetime is thus an extrapolation which is however a good starting point for long-term experiments. It is evident from Fig. 5 that the operational lifetime sharply changes around the optimal temperature when 1 ◦ C difference may result in a change of lifetime of about 100 days. For that reason the operational temperature should be determined with the accuracy of at least 0.5 ◦ C. 4. Conclusions It was found that the thermal inactivation of free fructosyltransferase from A. pullulans had a biphasic character in both a saccharide-free solution and reaction mixture containing a high concentration of saccharides. The kinetics of these processes in the investigated temperature intervals could be reliably described by a model derived from a two-step sequential mechanism, which employed the Arrhenius equation for the temperature dependence
[1] Madlová A, Antoˇsová M, Polakoviˇc M, Báleˇs V. Thermal stability of fructosyltransferase from Aureobasidium pullulans. Chem Pap 2000;54:339–44. [2] Hayashi S, Nonoguchi M, Shimokawa Y, Tubouchi M, Takasaki Y. Purification and properties of extracellular -fructofuranosidases from Aureobasidium sp. ATCC 20524. J Ind Microbiol 1992;9:145–7. [3] Yun JW. Fructooligosaccharides—occurrence, preparation, and application. Enzyme Microb Technol 1996;19:107–17. ˇ [4] Vanková K, Onderková Z, Antoˇsová M, Polakoviˇc M. Design and economics of industrial production of fructooligosaccharides. Chem Pap 2008;62:375–81. [5] Onderková Z, Bryjak J, Polakoviˇc M. Properties of fructosyltransferase from Aureobasidium pullulans immobilized on an acrylic carrier. Chem Pap 2007;61:359–63. [6] Roels JA, Van Tilberg R. Temperature dependence of the stability and the activity of immobilized glucose isomerase. ACS Symp Ser 1979;106:147–72. [7] Boy M, Dominik A, Voss H. Fast determination of biocatalyst process stability. Process Biochem 1999;34:535–47. [8] Bommarius AS, Broering JM. Established and novel tools to investigate biocatalyst stability. Biocatal Biotransform 2005;23:125–39. [9] Gibbs PR, Uehara CS, Neunert U, Bommarius AS. Accelerated biocatalyst stability testing for process optimization. Biotechnol Progr 2005;21:762–74. [10] Rogers TA, Bommarius AS. Utilizing simple biochemical measurements to predict lifetime output of biocatalysts in continuous isothermal processes. Chem Eng Sci 2010;65:2118–24. ˇ [11] Vrábel P, Polakoviˇc M, Stefuca V, Báleˇs V. Analysis of mechanism and kinetics of thermal inactivation of enzymes: evaluation of multitemperature data applied to inactivation of yeast invertase. Enzyme Microb Technol 1997;20:348–54. [12] De Cordt S, Avila I, Hendrickx M, Tobback P. DSC and protein-based timetemperature integrators: case study of ␣-amylase stabilized by polyols and/or sugar. Biotechnol Bioeng 1994;44:859–65. [13] Peterson ME, Daniel Roy M, Danson MJ, Eisenthal R. The dependence of enzyme activity on temperature: determination and validation of parameters. Biochem J 2007;402:331–7. [14] Rogers TA, Daniel RM, Bommarius AS. Deactivation of TEM-1 -lactamase investigated by isothermal batch and non-isothermal continuous enzyme membrane reactor methods. ChemCatChem 2009;1:131–7. [15] Vandáková M, Platková Z, Antoˇsová M, Báleˇs V, Polakoviˇc M. Optimisation of cultivation conditions for production of fructosyltransferase by Aureobasidium pullulans. Chem Pap 2004;58:15–22. ˇ [16] Platková Z, Polakoviˇc M, Stefuca V, Vandákova M, Antoˇsová M. Selection of carrier for immobilization of fructosyltransferase from Aureobasidium pullulans. Chem Pap 2006;60:469–72. [17] Antoˇsová M, Illeová V, Vandáková M, Druˇzkovská A, Polakoviˇc M. Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans. J Biotechnol 2008;135:58–63. [18] Antoˇsová M, Polakoviˇc M, Slovinská M, Madlová A, Illeová V, Báleˇs V. Effect of sucrose concentration and cultivation time on batch production of fructosyltransferase by Aureobasidium pullulans CCY 27-1-1194. Chem Pap 2002;56:394–9. [19] Polakoviˇc M, Bryjak J. Modelling of the kinetics of thermal inactivation of glucoamylase from Aspergillus niger. J Mol Catal B: Enzym 2002;19–20:443–50. [20] Lumry R, Eyring H. Conformation changes of proteins. J Phys Chem 1954;58:110–20. [21] Polakoviˇc M, Vrábel P. Analysis of the mechanism and kinetics of thermal inactivation of enzymes: critical assessment of isothermal inactivation experiments. Process Biochem 1996;31:787–800. [22] Xie G, Timasheff SN. The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem 1997;64:25–43.
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Kinetics of thermal inactivation of free Aureobasidium pullulans fructosyltransferase a ˇ Zdenka Onderková a , Jolanta Bryjak b , Katarína Vanková , Milan Polakoviˇc a,∗ a Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic b Department of Bioorganic Chemistry Faculty of Chemistry Wroclaw University of Technology, Norwida 4/6, 50-373 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 15 February 2010 Received in revised form 21 June 2010 Accepted 23 June 2010 Keywords: Fructosyltransferase Thermal stability Biocatalyst operational lifetime Inactivation Kinetics Modelling
a b s t r a c t A batch catalytic process of fructooligosaccharide production with the initial sucrose concentration of 700 g dm−3 was carried out in the temperature interval of 67.5–78 ◦ C to investigate the thermal stability of free Aureobasidium pullulans fructosyltransferase. Inactivation experiments were carried out also for a saccharide-free solution in the temperature interval of 53.1–71 ◦ C so that a stabilizing effect of saccharides contained in the reaction mixture could be assessed. A model of enzyme inactivation kinetics based on a two-step sequential mechanism was developed and verified for both media using the multitemperature evaluation of inactivation data. The model was used to predict the operational lifetime and optimal operational temperature of the biocatalyst. It was found that the use of the concentrated sucrose solution increases the operational temperature by about 15 ◦ C compared to a dilute aqueous solution. The value of 47.5 ◦ C was estimated as an optimal temperature at which the enzyme operational lifetime would be 1 year. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Fructosyltransferase (FTase, EC 2.4.1.9) from Aureobasidium pullulans [1,2] is one of the most common biocatalysts used for industrial production of fructooligosaccharides. Fructooligosaccharides are regarded as functional additives with acknowledged beneficial health effects that are produced via a fructosyl-transfer action of FTase on concentrated solutions of sucrose containing about 50–55% of saccharides and elevated temperatures of 50–55 ◦ C [3]. Immobilization of FTase is very important for the reduction of costs in a large-scale production owing to the process continualization [4] when some common immobilization methods do not have a strong stabilization effect on the enzyme [5]. A long-term operational stability of an enzyme preparation is a common prerequisite of successful large-scale operation of a biocatalytic process. This prerequisite implies a need of long-term laboratory experiments in the stage of biocatalyst development. It is possible to reduce the time needed for determination of operational stability by methods of accelerated testing either in continuous [6–10] or batch [11–14] systems when biocatalysts are exposed to more severe conditions than those on the process scale. This leads to an accelerated activity loss and the biocatalyst operational stability is predicted from the mathematical model derived from the accel-
∗ Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52493198. E-mail address: [email protected] (M. Polakoviˇc). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.06.009
erated activity loss experiments. The potential of this approach has unfortunately been underexploited probably due to non-trivial mathematical modelling required for its application. Key factors for the operational stability of FTase are temperature and the total concentration of saccharides in a reaction mixture [1]. Short-term experiments were carried out at 55 ◦ C and initial sucrose concentration ranging from 0 g dm−3 to 700 g dm−3 in order to investigate the effect of total saccharide concentration on the thermostability of whole-cell FTase of A. pullulans [1]. The rate of inactivation of the enzyme was slow at the overall saccharide concentration above 600 g dm−3 . During 1-h experiments, the total activity loss was less than 5%. On the other hand, the inactivation rate quickly grew with a decrease of saccharide concentration below this threshold. FTase lost 75% of initial activity during 1 h in a saccharide-free solution. Due to the limited time of inactivation, a simplified model of thermal inactivation was obtained in the form of a single-step reversible reaction kinetics and exponential relationship of rate constants on sucrose concentration. The objective of this study was to investigate the stabilizing effect of saccharides and to develop a kinetic model of thermal inactivation for the isolated enzyme that could be used for reactor design. In order to achieve these goals, inactivation batch experiments were performed at different temperatures in a reaction mixture and saccharide-free solution and the selected model was verified using a simultaneous fit of the inactivation data obtained.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139 2. Experimental 2.1. Preparation of enzyme A crude extract of the cells A. pullulans [15] was prepared by high-pressure homogenization [16]. The extract was then dialyzed through a membrane SpectraPor, MWCO 50,000 (Spectrum Medical Industries, USA) at 6 ◦ C against a sodium phosphate–citrate buffer with pH 5.5 (0.02 mol dm−3 sodium hydrogen phosphate and 0.01 mol dm−3 citric acid in the volumetric ratio 4.1:3). The retentate was further purified by ion-exchange chromatography using an anion-exchange resin Sepabeads FP-DA with the particle size of 150–300 m (Mitsubishi Chemical Co., Japan) [17]. 2.2. Determination of enzyme activity One unit of the FTase activity was defined as the number of micromoles of kestose (trisaccharide having two fructosyl units bound to a glucosyl moiety) produced from sucrose per 1 min. 0.5 cm3 of an enzyme sample was added into 5.5 cm3
135
of a sucrose solution in the sodium phosphate–citrate buffer (pH 5.5) at 55 ◦ C so that the final concentration of sucrose in the reaction mixture was 700 g dm−3 (2.045 M). Samples of the reaction mixture were withdrawn in predetermined time intervals. The reaction was stopped by boiling in a water bath for 2 min. The concentration of kestose was determined by HPLC (Knauer, Germany) using the column Watrex IEC Pb form as described previously [18].
2.3. Inactivation experiments The measurements of FTase inactivation were performed in thermostatted stirred glass reactors. One cubic centimeter of enzyme was added to 29 cm3 of a preheated sucrose solution in the phosphate–citrate buffer with pH 5.5 or to this buffer not containing sucrose. The influence of temperature was studied in the interval of 67.5–78 ◦ C for the reaction mixture with the initial sucrose concentration of 700 g dm−3 and of 53.1–71 ◦ C for the saccharide-free solution. In appropriate time intervals, 1 cm3 samples were withdrawn from the reactor and assayed for the residual activity as described above.
Fig. 1. Thermal inactivation of free Aureobasidium pullulans FTase in the (a, b) reaction mixture and (c–e) saccharide-free solution, respectively. The symbols represent the experimental activity data at different temperatures and the lines their fits with the model (Eqs. (2) and (3)).
136
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
2.4. Mathematical modelling
The inactivation process was described by a set of the following differential equations:
A kinetic model [19] was examined in this study for the description of the courses of the relative enzyme activity a, defined as the ratio of cN , the concentration of native enzyme form N in time t, and cN0 , its initial concentration. This model was based on a mechanism,
k+1
k2
N R−→I
dcN = −k+1 cN + k−1 cR dt
(2a)
dcR = k+1 cN − k−1 cR − k2 cR dt
(2b)
t = 0, (1)
k−1
where N undergoes a reversible transformation into an intermediate inactive form R, which then reacts irreversibly to an inactive form I. The symbols k+1 , k−1 , and k2 are the rate constants of individual reactions. The mechanism has the same form as the well-known mechanism of Lumry–Eyring [20] but the interpretation is different. In the Lumry–Eyring mechanism, the intermediate is considered to be a fully unfolded enzyme and rapid equilibrium is typically assumed for the first reaction.
cN = cN0 ,
cR = 0
(2c)
The temperature dependence of the rate constants was expressed in the form of a rearranged Arrhenius equation, ki = eln ki0 e[(Ei /RT0 )(1−T0 /T )] ,
i = +1, −1, 2
(3)
where Ei is the activation energy of the ith reaction, R is the universal gas constant, and ki0 the value of the rate constant ki at the reference temperature T0 . The so-called multitemperature evaluation [11], where inactivation data for all temperatures are fitted simultaneously using non-linear regression, was applied. Mathematical modelling software Athena Visual Workbench (Stewart & Associates, Madison, WI, USA)
Fig. 2. Thermal inactivation experiments for the saccharide-free solution from Fig. 1 plotted in semi-logarithmic scale: (a) 53.1 ◦ C, (b) 57 ◦ C, (c) 61 ◦ C, (d) 64 ◦ C, (e) 70 ◦ C, and (f) 71 ◦ C. The straight-lines delimit the interval of first-order kinetics rate constants corresponding to individual experimental activity vs. time values.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139 was used for the parameter estimation. It used a gradient non-linear least square method and parameter uncertainties were calculated from the variance–covariance matrix.
3. Results and discussion Fig. 1 presents the results of FTase inactivation experiments carried out in the reaction mixture and saccharide-free solution, respectively. The experiments were designed to span very different intervals of the rates of activity loss, from relatively slow ones with enzyme half-lives of about 1 h (in both media) up to fast inactivation with an almost complete loss of activity in less than 5 min. In order to achieve the same window of observable inactivation rates in both environments, different temperature ranges had to be chosen. The width of this range was 18 ◦ C for the saccharide-
137
free solution (53–71 ◦ C) but only 10.5 ◦ C for the reaction mixture (67.5–78 ◦ C). Some inactivation experiments presented in Fig. 1 clearly demonstrate a biphasic character of enzyme inactivation when the first, faster phase is followed by the second, slower phase [21]. The best example is the experiment carried out in the reaction mixture at 67.5 ◦ C (Fig. 1a) where the activity loss was much slower after 1 h when the residual activity reached the value of about 50%. The biphasic inactivation is however not so apparent for most experiments. For that reason, Figs. 2 and 3 present individual experiments in a semi-logarithmic form of activity–time relationships where a deviation from a straight line trend defines a degree of departure from first-order kinetics. The deviations from first-order kinetics demonstrate an interesting trend with temperature which is completely opposite as
Fig. 3. Thermal inactivation experiments for the reaction mixture from Fig. 1 plotted in semi-logarithmic scale: (a) 67.5 ◦ C, (b) 69 ◦ C, (c) 70 ◦ C, (d) 75 ◦ C, and (e) 78 ◦ C. The straight-lines delimit the interval of first-order kinetics rate constants corresponding to individual experimental activity vs. time values.
138
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
Table 1 Model parameters of the inactivation kinetics for free Aureobasidium pullulans FTase in the reaction mixture with the initial sucrose concentration of 700 g dm−3 and in a saccharide-free solution. Parameter
Reaction mixture
Saccharide-free solution
k+10 (10−4 s−1 ) k−10 (10−4 s−1 ) k20 (10−4 s−1 ) E+1 (105 J mol−1 ) E−1 (105 J mol−1 ) E2 (105 J mol−1 ) H (105 J mol−1 ) S (J mol−1 K−1 )
7.72 ± 0.77 7.87 ± 4.88 11.5 ± 6.8 3.74 ± 0.15 6.04 ± 2.14 16.5 ± 6.1 −2.30 −670
1160 ± 180 999 ± 310 146 ± 47 3.53 ± 0.11 7.04 ± 0.61 14.9 ± 2.6 −3.51 −1020
Reference temperature T0 = 343.15 K. The values after the plus/minus sign represent the half-widths of 95% confidence intervals.
the one observed in previous works [11,19,21]. The rate of FTase inactivation in saccharide-free solution at 53.1 ◦ C was governed by first-order kinetics (Figs. 1c and 2a). At higher temperatures, a departure from first-order kinetics is evident (Fig. 2b–f) when the deviation increases with temperature. The deviation is demonstrated by a phase with a significant deceleration of inactivation rate, which start is best visible at the residual activity of 40–50% for the experiments carried out at 70 ◦ C and 71 ◦ C (Figs. 1e, 2e and f). Deviations from first-order kinetics occur also for the inactivation experiments carried out in the reaction mixture at the temperatures from 67 ◦ C to 70 ◦ C (Figs. 1a and 3a–c). The deviation however disappears at higher temperatures of 75 ◦ C and 78 ◦ C (Figs. 1b, 3d and e). The trend was exactly opposite as for inactivation in the saccharide-free solution. Based on these observations, it was concluded that the simplest mechanism, which could describe, the inactivation behavior of FTase in both media is a two-step sequential mechanism (Eq. (1)) [19]. Each of the two sets of inactivation data presented in Fig. 1 was fitted simultaneously with the model (Eqs. (2) and (3)). Fig. 1 shows that a good match was achieved between the measured and fitted activity data both in the reaction mixture and saccharide-free solution when the values of the mean square error are equal to 3.2% and 4.3%, respectively. Table 1 presents the values of the estimated model parameters and their uncertainties represented by the half-widths of 95% confidence intervals. Since all uncertainties are lower than the corresponding mean values, all parameters are statistically significant and the model is adequate. The estimated parameters were used to calculate the dependence of the rate constants k+1 , k−1 , and k2 and the equilibrium constant, K1 = k+1 /k−1 , on temperature (Fig. 4a and b).
The results presented in Table 1 and Fig. 4 nicely illustrate the similarities and differences between the inactivation processes occurring in the reaction mixture and saccharide-free solution. The differences between the estimated activation energy values in the reaction mixture and saccharide solution are not statistically significant because their confidence intervals overlap. This compatibility gives a further credibility to the validated model. The differences by one or two orders of magnitude in the rate constants at the reference temperature of 70 ◦ C reflect the stabilization effect of saccharides on the inactivation process. The stabilization effect of saccharides on the FTase inactivation is thus of entropic character [22] which means that saccharide molecules probably interact primarily with water molecules surrounding the enzyme not with the enzyme molecule itself. Fig. 4a shows that the second reaction is so slow below 65 ◦ C that the inactivation process is completely controlled by the first, equilibrium reaction, which was also concluded previously for short-term inactivation experiments in the reaction mixture at 55 ◦ C [1]. This inactivation behavior is well demonstrated in Fig. 1d. The equilibrium constant strongly decreases with temperature but it is so high below 60 ◦ C that the first reaction becomes essentially irreversible (Fig. 4b). Therefore, the kinetics of inactivation at 53.1 ◦ C appears to be of first-order (Figs. 1c and 2a). Fig. 4a further shows that all three rate constants are of the same order of magnitude in the temperature interval of approximately 65–70 ◦ C for both the reaction mixture and saccharide-free solution. This means that the sequential character of the inactivation reactions is the most pronounced in this interval which is demonstrated by the biphasic character of inactivation curves (Figs. 1a, e, 2e–2f, and 3a–c). The most significant feature of the inactivation process at the temperatures above 70 ◦ C is that the values of the rate constant k2 are much higher than those of k+1 and k−1 (Fig. 4a). Consequently, the concentration of the intermediate form R is negligible in spite of that the equilibrium of the first reaction is shifted towards the native form N (Fig. 4b). The inactivation process is again governed by the forward reaction of the first reversible step, its kinetics is of first-order and the inactivation curves are monophasic (Figs. 1b, 4d and e). A noticeable fact is the decrease of the equilibrium constant K with temperature. Applying the van’t Hoff theory, the values of the standard reaction enthalpy H and standard reaction entropy S (Table 1) were calculated from the kinetic parame ters: H = E+1 − E−1 ; S = R ln k+10 /k−10 + ((E+1 − E−1 )/T0 ). In compliance with the theory, H is negative in both media. The exothermic character of the first reaction means that this step cannot be interpreted as a denaturation one. It implies that associa-
Fig. 4. Temperature dependence of (a) rate and (b) equilibrium constants of FTase inactivation plotted with reciprocal horizontal and logarithmic vertical axes. The lines are calculated from the model (Eqs. (2) and (3)) using the estimated parameters from Table 1. The points correspond to the temperatures of inactivation experiments. Solid symbols represent the saccharide-free solution and open symbols the reaction mixture.
Z. Onderková et al. / Enzyme and Microbial Technology 47 (2010) 134–139
139
of rate constants. The model gave a plausible explanation for the observations that the inactivation process was controlled by apparent first-order kinetics at low and high inactivation temperatures whereas it had a biphasic character at medium temperatures. At the low inactivation temperatures, the key rate-controlling reaction had about 150 times lower rate in the reaction mixture than that in the saccharide-free solution whereas the activation energy values differed only by 5%. The stabilization effect of saccharides was thus of entropic character. Acknowledgements This study was supported by the Science and Technology Assistance Agency APVV (LPP-0234-06) and Slovak Grant Agency for Science VEGA (Grant No. 1/0655/09). References Fig. 5. Lifetime of free Aureobasidium pullulans FTase vs. temperature in the reaction mixture (solid line) and saccharide-free solution (dashed line) calculated using the parameters presented in Table 1.
tion reactions can play a major role. Large activation energy values however indicate that the identified model may disguise a more complex inactivation mechanism and kinetics. A typical operational lifetime of industrial biocatalysts is determined by the period during which the biocatalyst activity drops to about 10% of the initial value. It determines a cycle needed for replacement of spent biocatalyst and therewith the biocatalyst cost per unit amount of product. In order to avoid an exorbitant biocatalyst cost, an efficient operational lifetime should be about 1 year for intracellular fungal glycoenzymes such as FTase [4]. The biocatalyst operational lifetime effectively determines the operational temperature because thermal inactivation is the main cause of biocatalyst activity loss. The operational temperature subsequently determines the reactor size and total load of biocatalyst in the reactor. Fig. 5 shows the lifetime of FTase in both media as a function of temperature calculated using the parameters presented in Table 1. Both dependences have the same trend with a temperature shift of about 12–15 ◦ C that expresses the stabilizing effect of saccharides in a different way. The predicted FTase lifetime in the reaction medium increases rapidly below the temperature of 55 ◦ C and the recommended value of 1 year is found at 47.5 ◦ C. This temperature is however by 20 ◦ C lower than the lowest temperature used in the experiments. The estimated lifetime is thus an extrapolation which is however a good starting point for long-term experiments. It is evident from Fig. 5 that the operational lifetime sharply changes around the optimal temperature when 1 ◦ C difference may result in a change of lifetime of about 100 days. For that reason the operational temperature should be determined with the accuracy of at least 0.5 ◦ C. 4. Conclusions It was found that the thermal inactivation of free fructosyltransferase from A. pullulans had a biphasic character in both a saccharide-free solution and reaction mixture containing a high concentration of saccharides. The kinetics of these processes in the investigated temperature intervals could be reliably described by a model derived from a two-step sequential mechanism, which employed the Arrhenius equation for the temperature dependence
[1] Madlová A, Antoˇsová M, Polakoviˇc M, Báleˇs V. Thermal stability of fructosyltransferase from Aureobasidium pullulans. Chem Pap 2000;54:339–44. [2] Hayashi S, Nonoguchi M, Shimokawa Y, Tubouchi M, Takasaki Y. Purification and properties of extracellular -fructofuranosidases from Aureobasidium sp. ATCC 20524. J Ind Microbiol 1992;9:145–7. [3] Yun JW. Fructooligosaccharides—occurrence, preparation, and application. Enzyme Microb Technol 1996;19:107–17. ˇ [4] Vanková K, Onderková Z, Antoˇsová M, Polakoviˇc M. Design and economics of industrial production of fructooligosaccharides. Chem Pap 2008;62:375–81. [5] Onderková Z, Bryjak J, Polakoviˇc M. Properties of fructosyltransferase from Aureobasidium pullulans immobilized on an acrylic carrier. Chem Pap 2007;61:359–63. [6] Roels JA, Van Tilberg R. Temperature dependence of the stability and the activity of immobilized glucose isomerase. ACS Symp Ser 1979;106:147–72. [7] Boy M, Dominik A, Voss H. Fast determination of biocatalyst process stability. Process Biochem 1999;34:535–47. [8] Bommarius AS, Broering JM. Established and novel tools to investigate biocatalyst stability. Biocatal Biotransform 2005;23:125–39. [9] Gibbs PR, Uehara CS, Neunert U, Bommarius AS. Accelerated biocatalyst stability testing for process optimization. Biotechnol Progr 2005;21:762–74. [10] Rogers TA, Bommarius AS. Utilizing simple biochemical measurements to predict lifetime output of biocatalysts in continuous isothermal processes. Chem Eng Sci 2010;65:2118–24. ˇ [11] Vrábel P, Polakoviˇc M, Stefuca V, Báleˇs V. Analysis of mechanism and kinetics of thermal inactivation of enzymes: evaluation of multitemperature data applied to inactivation of yeast invertase. Enzyme Microb Technol 1997;20:348–54. [12] De Cordt S, Avila I, Hendrickx M, Tobback P. DSC and protein-based timetemperature integrators: case study of ␣-amylase stabilized by polyols and/or sugar. Biotechnol Bioeng 1994;44:859–65. [13] Peterson ME, Daniel Roy M, Danson MJ, Eisenthal R. The dependence of enzyme activity on temperature: determination and validation of parameters. Biochem J 2007;402:331–7. [14] Rogers TA, Daniel RM, Bommarius AS. Deactivation of TEM-1 -lactamase investigated by isothermal batch and non-isothermal continuous enzyme membrane reactor methods. ChemCatChem 2009;1:131–7. [15] Vandáková M, Platková Z, Antoˇsová M, Báleˇs V, Polakoviˇc M. Optimisation of cultivation conditions for production of fructosyltransferase by Aureobasidium pullulans. Chem Pap 2004;58:15–22. ˇ [16] Platková Z, Polakoviˇc M, Stefuca V, Vandákova M, Antoˇsová M. Selection of carrier for immobilization of fructosyltransferase from Aureobasidium pullulans. Chem Pap 2006;60:469–72. [17] Antoˇsová M, Illeová V, Vandáková M, Druˇzkovská A, Polakoviˇc M. Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans. J Biotechnol 2008;135:58–63. [18] Antoˇsová M, Polakoviˇc M, Slovinská M, Madlová A, Illeová V, Báleˇs V. Effect of sucrose concentration and cultivation time on batch production of fructosyltransferase by Aureobasidium pullulans CCY 27-1-1194. Chem Pap 2002;56:394–9. [19] Polakoviˇc M, Bryjak J. Modelling of the kinetics of thermal inactivation of glucoamylase from Aspergillus niger. J Mol Catal B: Enzym 2002;19–20:443–50. [20] Lumry R, Eyring H. Conformation changes of proteins. J Phys Chem 1954;58:110–20. [21] Polakoviˇc M, Vrábel P. Analysis of the mechanism and kinetics of thermal inactivation of enzymes: critical assessment of isothermal inactivation experiments. Process Biochem 1996;31:787–800. [22] Xie G, Timasheff SN. The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem 1997;64:25–43.