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Biotechnol. Prog. 1995, 11, 104-106
Thermal Deactivation of Free and Immobilized fl-Gluooddaseh m Penicillium ficlaicuhsum Jose Aguado,' M.Dolores Romero, Lourdes M g u e z , and Jose A. Calles Department of Chemical Engineering, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid, Spain
Thermal deactivation of free and immobilized /3-glucosidase from Penicillium funiculosum has been studied in the temperature range 30-70 "C.Immobilization results in a remarkable increase in /%glucosidase thermal stability. A two-step series deactivation mechanism has been used to describe the experimental results for both free and immobilized B-glucosidases. The kinetic equations resulting from this model fit our experimental results with a mean deviation of less than 10%.
Introduction The enzymatic hydrolysis of cellulose and cellulosic substrates to glucose is a multistep process catalyzed by the three major components of the cellulase complex, endogluwrases, exoglucanases, and @-glucosidases,working in a synergistic mechanism (Ryu et al., 1984; Klyosov et al., 1986). /?-Glucosidaseplays an important role: it is essential for the conversion of the intermediate cellobiose, which is a strong inhibitor of the other cellulase components (Gong et al., 1977). Cellobiose hydrolysis rates depend on both reaction conditions and catalyst activity; therefore, the knowledge of the /?-glucosidase thermal deactivation through time is a prerequisite to obtain useful design equations. Despite this interest, relatively few studies have been published on the thermal deactivation of @-glucosidase (Gianfreda et al., 1985; Yazdi et al., 1989; Woodward et al., 1992) and, in particular, on the cellobiose hydrolysis reaction kinetics considering thermoinactivation of the catalyst (Bravo et al., 1990; Caminal et al., 1985). In this paper, we report studies of the thennoinactivation of the /?-glucosidase activity in a commercial cellulase from Penicillium funiculosum. A thermoinactivation model was developed, and its parameters were estimated for both free and immobilized /?-glucosidases. Materials and Methods /I-GlucosidaseImmobilization. Commercial cellulase from Penicillium funiculosum in powder form (4.3 unitdmg of solid) from Sigma was used as the source of /?-glucosidase. The procedure for /?-glucosidaseimmobilization on nylon powder is based on that previously described by Morris et al. (1975) and adequately modified for B-glucosidase immobilization (Aguado et al., 1993). The yield of the immobilization procedure was 67%. Thermoinactivation Assays. Free or immobilized cellulases from P. funiculosum were placed in citrate buffer (pH 4.8)at a fixed temperature. Periodically, samples were removed, cooled, and assayed for @-glucosidase activity as previously described (Aguado et al., 1993). Results and Discussion Free /I-Glucosidase Deactivation. Experiments were carried out in the temperature range 30-70 "C. For
* Author to whom all correspondence should be addressed.
h
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2
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h
c, .r(
P
.d
0.8
m
u
.I
!z0 Q,
g0.4 m
bl
0
0
500 1000 Time (minutes)
I
1500
b
DO
Time (minutes) Figure 1. Thermal deactivation of free &glucosidase from Penicillium funiculosum. (a) 0, T = 30 "C;0,T = 40 "C;0,T = 45 "C; A, T = 50 "C. (b) T = 55 "C;A, T = 60 "C;m, T = 65 "C;0, T = 70 "C.
*,
temperatures up to 50 "C, an initial increase in @-glucosidase activity was observed (Figure la). These initial maxima, as well as the final activities ( A r 1500 mid, decrease as the temperature increases. For deactivation temperatures higher than 50 "C (Figure lb), the maxi-
@ 1995 American Chemical Society and American Institute of Chemical Engineers 8756-7938/95/3011-0104$09.00/0
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Table 1. Values of ai,kl,and k2 Obtained by Fitting the Experimental Data of Free Enzyme Deactivation to Equation 3
temperature ("C) 30 40 45 50 55 60 65 70
ai (IU/mg of protein)
k1 (min-l) 2.10 x 10-2 f 1.5 10-3 1.3 10-3 2.00 x 10-1 5.07x lo-' f 0.5 x 1.39 f 0.106 3.60f 0.36 9.34f 0.615 23.3f 2.3 56.5ic 4.1
1.31f 0.039 1.24f 0.022 1.20f 0.189 1.18f 0.027 1.15f 0.051 1.12f 0.075 1.08f 0.073 1.07f 0.068
1.49 x 3.04 4.07x 6.68 x 9.88 2.01 x 6.00 1.23
*
kz (min-1) 10-4 f 4.25 10-5 10-4 f 4.5 10-5 10-4 6.71x 10-5 f 8.22x 10-4 f 1.2 10-4 10-3 6.9 x 10-4 10-3 9.09 10-4 10-2 f 1.01 10-3
* **
Table 2. Values of Ai, kl,and k2 Obtained by Fitting the Experimental Data of Immobilized Enzyme to Equation 7 temperature ("C) Ai (IU/g of support) k l (min-') k2 (min-') 30 17.79f 0.145 3.41 10-3 f 2.3 10-4 3.59 x 10-8 17.71 ic 0.38 5.70 10-3 ic 5.3 10-4 3.51 x f 3.2 x 40 17.15 f 0.32 9.65 10-3 f 1.1 10-3 2.35 x f 2.6 x 50 1.40 x io-'2 f 2.2 x 10-3 3.81 10-4 3.01 10-5 60 13.33f 0.375 2.42 x f 6.1 x 1.17 10-3 f 9.9 x 10-5 70 10.25f 0.876
mum was not observed. For temperatures above 60 "C, the /?-glucosidase activity falls to zero after a short incubation time. The experimental results presented in the preceding paragraph lead us to consider a two-step deactivation mechanism (Gianfreda et al., 1984; Henley and Sadana, 1984, 1985, 1986; Greco and Gianfreda, 1984):
20
I
I
/\,\I
kl
kz
E -.E* -.E,
(1)
where E represents the native /?-glucosidase structure, with specific activity a, (IU/mg of protein), Ed represents the final deactivated structure without specific activity, and E* represents a distribution of intermediate active structures whose average specific activity, ai, may be either higher or lower than the native structure specific activity, a,, depending on the temperature level. Then, the overall specific activity, varying with time, can be expressed as a = a,-
[El [E"] + aiEo Eo
where Eo is the initial /?-glucosidase concentration. By assuming first-order kinetics for both deactivation steps, eq 2 can be expressed as follows:
The experimental results were fit to eq 3 with a, = 1.17 IU/mg, previously determined. Table 1shows the values of the kinetic parameters obtained at different temperatures. For temperatures up to 50 "C,the intermediate structure (ai) has a higher activity than the native structure (a, = 1.17 IU/mg). This is in agreement with the maximum observed for the activity in Figure l a . However, for temperatures higher than 50 "C, ai values are lower than a,, and therefore, the maximum in the overall specific activity was not observed (Figure lb). Moreover, Fzl values are significantly higher than Fzz values over the full temperature range. Similar behavior has been previously described in the literature for other free enzymes (Hennecke and Boecke, 1974; Sasaki et al., 1975). The kinetic parameters in Table 1were correlated with temperature as follows ai = 0.2186 exp(543.92lT)
K , = 7.59
x
lo2' exp(-41001.75/RT)
312
(4)
(5)
gz 0 0
500
1000
1500
Time (minutes) Figure 2. Thermal deactivation of P-glucosidase from Penicillium funiculosum immobilized on nylon powder: 0, T = 30 "C; 0,T = 40 "C; A, T = 50 "C; A, T = 60 "C; 0 , T = 70 "C.
k2 = 4.69 x
1 O I 2 exp(-23200.87/RT)
(6)
The combination of eqs 3-6 fit our experimental results with a deviation of less than 10%. ImmobilizedB-Glucosidase Deactivation. Figure 2 shows that immobilization increases the stability of the /?-glucosidase. So for temperatures up to 50 "C, there is not an appreciable loss of activity after 1500 min, and the maximum observed when free enzyme was used does not appear. It is noteworthy to remark the high remaining activities after 1500 min at 60 and 70 "C,in contrast to the free /?-glucosidase fully deactivated at the same temperatures. Equation 3 was also applied to the experimental data from immobilized /?-glucosidase by replacing the free overall specific activity, a (IU/mg of protein), with the overall immobilized activity A (IU/g of support). Thus, the change in the immobilized activity with time could be expressed as follows:
The fit of our experimental results to eq 7, with the previously determined value A, = 17.51 IU/g of support, leads to the values ofAi, k l , and k2 summarized in Table 2. In contrast with the results obtained using free /?-glucosidase, Ai values for temperatures up to 50 "C are
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practically coincident with the immobilized activity of the native form (A, = 17.51 IU/g of support). Thus, the covalent bond of the enzyme to the support prevents the formation of the more active intermediate structures responsible for the initial maximum observed in the activity values occurring when free /?-glucosidase was used. For temperatures higher than 50 "C, the activities of the intermediate species decrease as the temperature increases. This fact can explain the decay in /?-glucosidase activity with time observed at 60 and 70 "C. The kinetic parameters Ai, KI, and K Z were correlated with temperature using the following expressions:
exp(2850.33/T) T L 323 K (8)
Ai = 2.53 x
Ai = 17.51 IU/g of support T 5 323 K
k, = 5 x lo4 exp(-9939.27/RT) k, = 4.73 x
exp(-43143.73/RT) (10) The combination of eqs 8-10 reproduces our experimental results with a deviation lower than 10%. It is interesting to note that the immobilization process decreases the activation energy of the first deactivation step (41 kcdmol for free /?-glucosidaseversus 10 k c d mol for immobilized /3-glucosidase). This fact provides evidence that the immobilization process promotes the formation of the partially deactivated intermediate structures (E*), which are less active than the native enzyme due to the linkage with the support. On the other hand, the immobilization increases the activation energy of the second step (23 versus 43 kcdmol, respectively); in other words, the formation of the final deactivated structure (E*)is strongly hindered. Literature Cited Aguado, J.; Romero, M. D.; Rodriguez, L. Immobilization of B-glucosidasefrom Penicillium funiculosum on Nylon Powder. Biotechnol. Appl. Biochem. 1993, 17, 49-55. Bravo, V.; Camacho, F.; Paez, M. P. Loss of ~-1,4-Glucosidase Activity in the Cellulases from Trichoderma reesei. An. Quim. 1990,86 (2), 208-212. Caminal, G.; Lopez Santin, L.; Sol& C. Kinetic Modelling of the Enzymatic Hydrolysis of Pretreated Cellulose. Biotechnol. Bioeng. 1985,27 (91, 1282-1290.
Gianfreda, L.; Marruci, G.; Grizzuti, N.; Gseco, G. Acid Phosphatase Deactivation by a Series Mechanism. Biotechnol. Bweng. 1984,26, 518-522. Gianfreda, L.; Modderi, M.; Greco, G. Enzyme Stabilization towards Thermal, Chemical and Proteolitic Deactivation. Enzyme Microb. Technol. 1985, 7, 78-82. Gong, C. S.; Ladisch, M. R.; Tsao, T. Cellobiase from T. viride: Purification, Properties and Mechanism. Biotechnol. Bioeng. 1977,19, 959-964. Greco, G.; Gianfreda, L. An Experimental Technique for the Discrimination between Series and Parallel Mechanism of Enzyme Deactivation. Biotechnol. Lett. 1984, 6, 693-698. Henley, J. P.; Sadana, A. Mathematical Analysis of Enzyme Stabilization by a Series-Type Mechanism: Influence of Chemical Modifiers. Biotechnol. Bweng. 1984,26,959-969. Henley, J. P.; Sadana, A. Categorization of Enzyme Deactivation Using a Series-Type Mechanism. Enzyme Microb. Technol. 1985, 7,50-60. Henley, J. P.; Sadana, A. Deactivation Theory. Biotechnol. Bioeng. 1986,28 (8),1277-1285. Hennecke, H.; Boecke, A. Modification of Phenylalanyl-tRNA synthetase from Escherichia coli by Histidine-specific Reagents. Effects on Structure and Function. Eur. J.Biochem. 1974,50,151-166. Klyosov, A. A.; Kude, J.; Goldskin, G. C.; Meyer, D. Role of the Components of the Cellulase Complex in Hydrolysis of Insoluble Cellulose. &tu Bwtechnol. 1986,6,369-375. Morris, D. L.; Campbell, J.; Hornby, W. E. A Chemistry for the Immobilization of Enzymes on Nylon. Biochem. J. 1975,147, 593-603. Ryu, D. D.; Kim, C.; Mandels, M. Competitive Adsorption of Cellulase Components and ita Significance in a Synergistic Mechanism. Biotechnol. Bioeng. lBEM, 26, 488-495. Sasaki, R.; Ikura, K.; Sugimoto, E.; Chiba, H. Purification of Biophosphoglyceromutase-2,3-bisph~hoglyceratephoaphataee and Phosphoglyceromutase from Human Erythrocyte. Eur. J. Biochem. 1975,50,681-693. Woodward,J. R.; Radford, A.; Rodriguez, L.; Aguado, J.; Romero, M. D. Immobilization of &glucosidase from Penicillium funiculosum from different Sources on Nylon Tube. Acta Biotechnol. 1992, 5, 329-336. Yazdi, M. T.; Woodward,J. R.; Radford, A.; Keen, J. N. Cellulase Production by Neurospora crassa. Purification and Characterization of cellulolyticenzymes. Enzyme Microbwl. Technol. 1989,12,116-119. Accepted September 9, 1994." Abstract published in Advance ACS Abstracts, October 1, 1994. @
