SciELO - Scientific Electronic Library Online

vol.17 issue4-7A simplified kinetic model for the side reactions occurring during the enzymatic synthesis of ampicillinEvaluation of inorganic matrixes as supports for immobilization of microbial lipase author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




Related links


Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol.17 n.4-7 São Paulo Dec. 2000 



L.P.V.Calsavara, F.F.Moraes and G.M.Zanin
Chemical Engineering Department, State University of Maringá
Av. Colombo, 5790, Bloco D90, Maringá, CEP 87020-900
Phone: +(55) (44) 263-2652, Fax: +(55) (44) 263-3440, PR - Brazil


(Received: October 10, 1999 ; Accepted: April 18, 2000)



Abstract - Commercial cellobiase has been immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67% and 13.7%, respectively. Thermal stability of the free and immobilized enzyme (IE) was determined with 0.2% w/v cellobiose solution, pH 4.8, temperatures from 40 to 70°C for free enzyme and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability retaining its activity in the same test up to 60°C. Half-lives for free enzyme were 14.1, 2.1 and 0.17 h at 60, 65 and 70°C, respectively, whereas the IE at the same temperatures had half-lives of 245, 21.3 and 2.9 h. The energy of thermal deactivation was 80.6 kcal/mol for the free enzyme and 85.2 kcal/mol for the IE, confirming stabilization by immobilization.
Keywords: Thermal stability, immobilized enzyme, cellobiase, cellobiose.




The proposal of biomass as a renewable source of energy suffers from the difficulty that cellulose in biomass is difficult to hydrolyse because lignocellulose contains cellulose closely associated with hemicellulose and lignin, as well as with other biomass constituents (Busto et al., 1995).

The cellulase enzyme is formed by a set of different enzymes that hydrolyses cellulose, which is a linear polymer of D-glucose units linked by 1,4-b-D-glucosidic bonds, initially to glucose oligomers and finally to glucose. The cellulase enzyme system contains: endo-1,4-b-glucanase that attacks the cellulose chain randomly, cellobiohydrolase that cut the cellulose chain at the end liberating the dimmer cellobiose, and b-glucosidase (cellobiase) that hydrolyses cellobiose to glucose. Cellulolytic enzymes in conjunction with b-glucosidase act sequentially and cooperatively to degrade crystalline cellulose to glucose. The cellobiase is generally responsible for the regulation of the entire cellulolytic process and is a rate-limiting factor during enzymatic hydrolysis of cellulose, since both endoglucanase and cellobiohydrolase activities are often inhibited by cellobiose. Thus, the cellobiase not only produces glucose from cellobiose but also reduces cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently (Saha et al., 1994).

The idea to supplement cellulases with cellobiase aiming at increasing the rate of reaction and achieving greater final conversion of cellulose to glucose was implemented with success by various authors (Sundstrom, et al, 1981; Klei, et al, 1981; Woodward and Wiseman, 1982). Since cellobiose is water soluble, whereas cellulose is not, it occurred that to a greater advantage cellobiase could be supplemented in an immobilized form, being added as a fixed-bed side reactor, to the main batch hydrolysis reactor, and the liquid produced by hydrolysis would continually circulate from the main reactor to the fixed-bed one, up to the end of the reaction (Venardos, et al, 1980, Zanin and Moraes, 1985; Woodward et al., 1993). The advantage would be to use less supplemented cellobiase by ton of cellulose converted, since the half-life of immobilized enzymes are usually longer than for the free enzymes, that is, the immobilization of an enzyme into an insoluble support will result in an enhanced stability against the denaturing effect of heat. However, the quantification of this effect and its relation to the method of immobilization still needs further studies (Lenders et al., 1985). Cellobiose hydrolysis rates depend on both reaction conditions and enzyme activity; therefore, the knowledge of the cellobiase thermal deactivation through time is a prerequisite to obtain useful design equations (Aguado et al., 1995).

The present article reports experimental data concerning the thermal stability and energy of deactivation of a commercial cellobiase enzyme in the form of either being free in solution or immobilized.




Cellobiose from Sigma (St. Louis, USA) was used as substrate. This material had a very small quantity of glucose as impurity (1.33 mg of glucose/g).


The enzyme used is a b-glucosidase, Novozym 188, kindly supplied by Novo Nordisk (Denmark), and produced by the microorganism Aspergillus niger. It contained 170 mg/mL of protein and a specific activity of 9.5 mmol glucose/ protein at 50°C, pH 4.8.


Controlled pore silica (CPS) having mean pore size of 37.5 nm and average particle diameter of 0.351 mm, was used as the support for immobilization of cellobiase. It was kindly donated by Corning Glass Works (USA).


Cellobiase was immobilized in CPS by the covalent method of Weetall (1993) with the following steps: (a) silanization of the carrier, with a 0.5% v/v solution of g-aminopropyltrietoxisilane, for 3h at 75°C; (b) washing with distilled water and drying for 15h at 105°C; (c) activation with a 2.5% v/v solution of glutaraldehyde, pH 7.0 for 45 min at 20°C; (d) wash with distilled water; (e) contact the activated carrier with a solution of the enzyme for 15h at 20°C; and, (f) wash the immobilized enzyme with distilled water, and stock it under sodium acetate buffer (0.2M) pH 4.5 at 4°C. The quantity of enzyme offered for immobilization in step (e) was 111.6 mg of protein/g of support.

Thermal Deactivation Test

Free cellobiase and the immobilized enzyme (IE) were incubated in a 0.2% w/v solution of cellobiose at pH 4.8 and temperatures from 40 to 70ºC for free enzyme, and 40 to 75ºC for the IE. Samples of the enzyme were collected each 40 min to measure the residual enzymatic activity. For free cellobiase, at 70ºC, sampling time interval was shortened to 5 min because of the rapid thermal deactivation observed at this temperature.

Enzymatic Activity

A jacketed glass batch thermo-controlled microreactor, equipped with magnetic stirring was used for the enzyme activity test. Free cellobiase, at the final concentration of 95 mL of enzyme/L of solution was incubated at 50ºC, pH 4.8 (acetate buffer at 50 mM), in a volume of 20 mL of 0.2% w/v cellobiose solution containing 1 mg/mL of sodium benzoate. In the case of the immobilized cellobiase, the same microreactor was used with 0.06g dry weight immobilized enzyme inside a stainless steel screen basket. Half-milliliter samples were collected each 3 min, for a total time of 18 min, boiled and then stocked at 4ºC for later glucose assay. The method of initial rate of Dixon and Webb (1979), was used with these data for the determination of enzyme activity.

Glucose and Protein Assays

Glucose was assayed with the enzymatic method GOD-PAP (Trinder, 1969), and protein was measured according to Lowry et al. (1951) using BSA as standard.

Energy of Thermal Deactivation

The Energy of Thermal Deactivation of free and immobilized cellobiase was calculated from the data collected with the thermal deactivation tests. It was assumed that enzyme thermal denaturation is a reaction, in which, the rate of enzyme deactivation (rd) is first order in relation to the concentration of the active enzyme (E):

rd = - Kd E


and the deactivation constant (Kd ) is a function of temperature as given by Arrhenius equation (Chaplin and Bucke, 1992):

Kd = Kd0 exp (-Ed / RT)


where Ed is the energy of thermal deactivation, R the universal gas constant (1.987 cal/mol K) and T the absolute reaction temperature.

For a batch reactor of constant liquid density the rate of reaction equals the time derivative of the concentration, and therefore it follows from Eq. (1) that:


which integrated with the initial condition E = E0 at t = 0, gives:

E = E0 exp (-Kd t)


where E0 is the initial active enzyme concentration, and t is the time elapsed during reaction.

When the enzyme is present in catalytic quantities, that is, in low concentrations, the residual enzyme activity (Ar) is directly proportional to the concentration of the active enzyme (E):


where A0 is the initial enzyme activity observed with the initial enzyme concentration (E0).

Combining Eqs. (4) and (5), the residual enzyme activity results as:

Ar = A0 exp (-Kd t)


This result is the exponential decay model. Therefore, by plotting residual activity data in the form of log of Ar /A0 against time, the deactivation constant (Kd) is obtained as the angular coefficient of the adjusted straight line.

From Eq. (2) it can be seen that the deactivation constant increases with temperature. Values obtained for Kd for various test temperatures are plotted in the form of Arrhenius plot, that is ln of Kd as a function of the inverse of absolute temperature, yielding the energy of deactivation (Ed), as the angular coefficient of the adjusted straight line, times R, the universal gas constant.

It is also of interest to calculate the enzyme half-life (t1/2), i.e., the time period necessary for the residual enzymatic activity to decrease to half of its initial value. If the enzyme thermal denaturation follows Eq. (6), then there is an inverse relation between the half-life of the enzyme and the deactivation constant (Zanin and De Moraes, 1998):

t1/2= ln 0.5 / (-Kd) = 0.693 / Kd




Immobilized Enzyme (IE)

The immobilized cellobiase particles contained 74.9 mg protein/g dry support; therefore the protein fixed by immobilization, i.e. the protein yield, corresponds to 67.1% of the protein offered in the immobilization protocol. The IE activity at 50ºC, pH 4.8 was 1.9 mmol glucose/ protein; hence the activity recovered by immobilization, i.e. the activity yield, corresponds to 20.0% of the activity offered in the immobilization procedure.

Cellobiase Thermal Deactivation Data

The experimental results obtained with the thermal deactivation test for free enzyme are shown in Fig. 1, and for IE in Fig. 2.




It can be observed in Fig. 1 that up to 55°C, and within a period of 240 min of thermal denaturation, free cellobiase maintained its activity practically constant, whereas at 70°C the enzyme was almost totally deactivated within 40 min. The IE has shown higher stability (Fig. 2), maintaining its activity practically constant within a period of 240 min, for temperatures up to 60°C. At 70°C, 40% of the IE activity was still observed after 240 min.

Modeling Cellobiase Thermal Deactivation

Table 1 shows the experimental results for the deactivation constant (Kd) determined with Eq. (6) applied to the residual activity data, which was obtained with the thermal deactivation test, applied to the free and IE. The column labeled as "Fitting equation" contains the linearized form of Eq. (6) adjusted to thermal deactivation data for each test temperature. The range of temperatures selected in this Table corresponds to the temperatures in which the deactivation observed was sufficient to permit a good fit of the exponential decay model (Eq. (6)). These temperatures were for free cellobiase, from 55 to 70ºC, and for the IE, from 60 to 75ºC. In the adjusted equations -Kd is the slope of the straight line. This value of Kd was used with Eq. (7) to calculate the enzyme half-life. The deactivation constant (Kd) and the enzyme half-life (t1/2) calculated by these procedures are shown in the next column, labeled as "Adjusted for a single temperature".

Figure 3 is a plot of the deactivation constant for free and immobilized cellobiase, in the form of the Arrhenius plot, i.e. ln(Kd) as a function of the inverse of the absolute temperature. The straight lines shown in this figure correspond to Eq. (2) adjusted to these data:



Free cellobiase:

Kd = 5.7280 . 1051 exp [ - 80,573 / RT]

r = 0.9789


Immobilized cellobiase:

Kd = 3.1749 . 1053 exp [ - 85,238 / RT]

r = 0.9885


Thus, the experimentally observed energy of deactivation (Ed) is approximately 80.6 kcal/mol for free cellobiase and 85.2 kcal/mol for the IE. There is a 5.7% increase in the energy of deactivation for the IE.

From Eqs. (8) and (9), new values for the deactivation constant can be calculated, and these are shown in the last column of Table 1, which is labeled as "Adjusted for all temperatures", because Eqs. (8) and (9) were adjusted for all values of Kd obtained in the above mentioned temperature ranges, respectively used for the free and IE. The new values of Kd were used with Eq.(7) to calculate new values for the enzyme half-life, now adjusted for all temperatures. These results are shown in the last column of Table 1. It can be observed that owing to the scatter found in Fig. 3, for each test temperature, the parameter values for Kd and t1/2 of the last two columns of Table 1 agree mostly only in order of magnitude.

Now, comparing the experimental values of t1/2 , for free and IE, of the column "Adjusted for a single temperature" in Table 1, the immobilized cellobiase presented half-lives about 19 times greater, on average, than half-lives shown by free cellobiase, confirming that immobilization confers more stability to the enzyme. The ratio of the half-lives decreased from 20.8 to 16.9 as the temperature was reduced from 60 to 70ºC. This showed that stabilization by immobilization was more effective at the lower temperatures.

Bisset and Sternberg (1978) obtained for the cellobiase derived from Aspergillus phoenicis QM 329, half-lives: 216 h (55ºC), 8.6 h (60ºC), 0.5 h (65ºC) and 0.04 h (70ºC), using cellobiose 7.5 mM as substrate at pH 4.8. Comparing these results with the experimental half-lives for free enzyme presented in Table 1, it can be observed that the commercial cellobiase is more stable than the enzyme derived from Aspergillus phoenicis QM 329, at temperatures between 60 and 70ºC.



Cellobiase enzyme free in solution when thermally denaturated in 0.2% w/v cellobiose, pH 4.8, is stable up to 55°C for a period of 4 h.

When immobilized in controlled pore silica by the silane-glutaraldehyde covalent method, the same enzyme is stable up to 60°C, in the same conditions.

For higher temperatures, the enzyme thermal deactivation can be described by the exponential decay model giving: Kd = 5.7280 . 1051 exp [ - 80,573 / RT] for free cellobiase, and Kd = 3.1749 . 1053 exp [ - 85,238 / RT] for the immobilized enzyme.

The immobilized cellobiase has shown half-lives nearly 20 times greater on average, than the half-lives observed with the free enzyme, confirming that immobilization confers more thermal stability to this enzyme, and showing that this stabilization is more pronounced at the lower temperatures in the range of 60 to 70ºC.



We would like to thank Novo Nordisk for kindly providing the enzyme samples. We are also thankful to the financial support given by CAPES, CNPq, PADCT and the University of Maringá.



A0 initial enzymatic activity (mmol glucose/ protein)
Ar residual enzymatic activity (mmol glucose/ protein)
C1 constant (Table 1)
E active enzyme concentration (g/L)
E0 initial active enzyme concentration (g/L)
Ed energy of thermal deactivation (cal/mol)
Kd deactivation constant (h-1)
R universal gas constant (1.987 cal/mol K)
T absolute temperature (K)
t time (h)
t1/2 enzyme half-life (h)



Aguado, J., Romero, M. D., Rodríguez, L. and Calles J. A., Thermal Deactivation of Free and Immobilized b-Glucosidase from Penicillinum funiculosum. Biotechnology Progress, 11, 4-106 (1995).        [ Links ]

Bisset, F. and Sternberg, D., ., Immobilization of Aspergillus b-Glucosidase on Chitosan. Applied and Environmental Microbiology, 35, 750-755 (1978).        [ Links ]

Busto, M. D., Ortega, N. and Perez-Mateos, M., Studies on Microbial b-D-Glucosidase Immobilized in Alginate Gel Beads. Process Biochemistry, 30, No. 5, 421-426 (1995).        [ Links ]

Chaplin, M. F. and Bucke, C., Enzyme Technology. Cambridge University Press, Cambridge, 18-23 (1992).        [ Links ]

Dixon, M. and Webb, E. C., Enzyme. 3rd Ed., Longman Group Limited, London, 138-168 (1979).        [ Links ]

Klei, H. E., Sundstrom, D. W., Coughlin, R. W. and Ziolkowski, K., Hollow-fiber Enzyme Reactor in Cellulose Hydrolysis. Biotechnology and Bioengineering Symp., No. 11, 593-601 (1981).        [ Links ]

Lenders, J.-P., Germain, P. and Crichton, R. R., Immobilization of a Soluble Chemically Thermostabilized Enzyme. Biotechnology and Bioengineering, 27, 572-578 (1985).        [ Links ]

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein Measurement with the Folin Phenol Reagent. Journal Biological Chemistry, 193, 265-275 (1951).        [ Links ]

Saha, B. C., Freer, S. N. and Bothast, R. J., Production, Purification, and Properties of a Thermostable b-Glucosidase from a Color Variant Strain of Aureobasidium pullulans. Applied and Environmental Microbiology, 60, No.10, 3774-3780 (1994).        [ Links ]

Sundstrom, D. W., Klei, H. E., Coughlin, R. W., Biederman, G. J. and Brower, C. A., Enzymatic Hydrolysis of Cellulose to Glucose using Immobilized b-Glucosidase. Biotechnology and Bioengineering, 23, 473-485 (1981).        [ Links ]

Trinder, P., Determination of Glucose in Blood using Glucose Oxidase with an Alternative Oxygen Acceptor. Annals of Clinical Biochemistry, 6, 24-27 (1969).        [ Links ]

Venardos, D., Klei, H. E. and Sundstrom, D. W., Conversion of Cellobiose to Glucose using Immobilized b-Glucosidase Reactors. Enzyme Microbiology Technology, 2, 112-116 (1980).        [ Links ]

Weetall, H. H., Preparation of Immobilized Proteins Covalently Coupled through Silane Coupling Agents to Inorganic Supports. Applied Biochemistry and Biotechnology, 41, 157-188 (1993).        [ Links ]

Woodward, J. and Wiseman, A., Fungal and other b-D-Glucosidase – their Properties and Applications. Enzyme and Microbial Technology, 4, 73-79 (1982).        [ Links ]

Woodward, J., Koran Jr., L. J., Hernandez, L. J. and Stephan, L. M., Use of Immobilized b-Glucosidase in the Hydrolysis of Cellulose. American Chemical Society, 533, 240-250 (1993).        [ Links ]

Zanin, G. M. and de Moraes, F. F., Hidrólise do Bagaço de Cana em Microrreator de Leito Fluidizado. Revista Brasileira de Engenharia, 3, 45-56 (1985).        [ Links ]

Zanin, G. M. and de Moraes, F. F., Thermal Stability and Energy of Deactivation of Free and Immobilized Amyloglucosidase in the Saccharification of Liquefied Cassava Starch. Applied Biochemistry and Biotechnology, 70-72, 383-394 (1998).        [ Links ]

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License