Application of bacterial cellulose pellets in enzyme immobilization

Application of bacterial cellulose pellets in enzyme immobilization

(Parte 2 de 2)

EDC coupling method) 82.6

Table 2 Effects of particle size on the relative activity of immobilized glucoamylase

Particle size (m) Relative activity (%) Activity (U/g-carrier)

3.2. Effects of drying condition and particle size

The cultivation of A. xylinum produced different sizes of bacterial cellulose beads based on the cultivation time. Hence, the preparationofbacterialcellulosebeadswasdiscussedfirst.After a suitable cultivation time, particle size with different ranges 0.5–1.5, 2–3 and 4–5mm were selected and used to test the immobilization of the enzyme. The results are shown in Table 2. The relative activity was significantly higher when the smaller beadswereused.Theresultswerelogicalsincethesmallerbeads provided a greater surface area and larger functional groups to connect with the enzymes.

The bacterial cellulose beads with different water contents were also investigated. The three kinds of bacterial cellulose beads, namely, wet, simply dried at room temperature for 24h, and oven dried at 50 ◦C for 24 h, were tested for their activities after immobilization of the enzyme. From the results shown in Table 3, the wet particles showed a significantly higher relative activity than the others. The immobilized efficiency calculated as the ratio of bound protein (measured activity) and amount of protein (activity) used for immobilization is about 38.3% which is common value in chemical adsorption.

Thus, the wet bacterial cellulose beads with the smallest size were used in the following experiments to test the characteristics of the immobilized enzyme.

3.3. The time of immobilization

Experiments were performed using different immobilization times from 15min to 120min with intervals of 15min and the results are shown in Fig. 1. The maximum relative activity was

Table 3 Effects of drying condition on the relative activity of immobilized glucoamylase

Drying condition Dry weight (g) Syneresis (%) Relative activity (%) Activity (U/g-carrier)

Fig. 1. Effect of the immobilization time on the relative activity of immobilized glucoamylase.

achieved in 60min of immobilization time. Above that time, the relative activity would slightly decrease. Hence, 1h was selected as the best length of time for enzyme immobilization.

3.4. Effect of pH on enzyme activity

The effect of pH on the relative activity of glucoamylase immobilized on the bacterial cellulose beads was studied by varyingthepHofthereactionmediumfrom2to6.5ataninterval of 0.5 and the pH profile is shown in Fig. 2. After the immobilization, the optimum pH was slightly shifted from 5 to 4.5 when compared to the free enzyme. The similar tendency for pH shift to acid was found in other research [14,15]. The better operating range, i.e. when the relative activities of the enzyme were above 90%, was also slightly increased from 3.5–5.5 to 3.0–5.5. In other words, the immobilized enzyme activity was better in acid when compared to the free enzyme. Moreover, the relative activities of the immobilized glucoamylase were still above 7% at pH 2.0. When compared with the highest value 83% at pH 2.5 in the recent research by Chang and Juang [15],i t is also the same value, i.e. 83%. Therefore, the immobilization enzymes in bacterial cellulose have a broader pH range of high activity than free enzymes.

Fig. 2. Effect of substrate pH on the relative activity of free and immobilized glucoamylase.

Fig. 3. Effect of reaction temperature on the relative activity of free and immobilized glucoamylase.

3.5. Effect of temperature on enzyme activity

The temperature dependence of the hydrolytic activity of free and immobilized glucoamylase is shown in Fig. 3. The optimum reaction temperature of the glucoamylase was at 60◦C for both cases. The better operating range, i.e. when the relative activities oftheenzymewereabove90%,wasat50–70◦Cbyimmobilization which was wider than that of the free enzyme (60–70◦C). The 10◦C decrease in the optimum activity temperature combined with thermal stability exhibited by the bacterial cellulose beads was an interesting finding of the present work. A similar behavior was also obtained recently [15–17]. The industrial applications of glucoamylases no longer required temperatures around 60–70◦C to reach the optimum catalytic activity temperature. Moreover, the relative activities of the immobilized glucoamylase were still above 68% at 20◦C in the present work. That means that the relative activities were more than 68% in the temperature range of 20–70◦C. This result is closed to the highest value 74% in the recent research by Chang and Juang [15]. This ability takes the advantage of the energy consumption in industry process since the substrate will not be needed to preheat.

The fast decrease in the relative activities at the high temperature range for both cases was due to the thermal denaturation. Thus, the immobilized glucoamylase on the bacterial cellulose beads could increase the enzyme activation but could not protect the enzyme for the thermal denature effect.

3.6. Stability of the immobilized enzyme

The stability of the relative activity of the free and the immobilized glucoamylase with pH is shown in Fig. 4. Free enzyme was stable at pH 4 and the relative activities were maintained at 90% in the pH range of 3–6. The immobilized glucoamylase was stable at pH 7 and the relative activity was maintained above 80%inthepHrangeof5–7.Thisresultindicatedthattheenzyme activity had more influence on the pH after immobilization.

The thermal stabilities of the free and the immobilized glucoamylase in terms of the relative activities are compared in

Fig. 4. pH stability of free and immobilized glucoamylase.

Fig. 5. The enzyme was found to be stable up to a temperature of50◦Cforthefreeenzyme,andupto60◦Cfortheimmobilized enzyme which was slightly better than the free enzyme. Hence, the effect of thermal deactivation for the immobilized enzyme was not significant as compared to the free enzyme state.

3.7. Reuse stability of immobilized enzymes

Glucoamylase immobilized in the bacterial cellulose beads was used repeatedly to hydrolyze starch since reusability was important for repeated applications in a batch or a continuous reactor. The relative activity of the immobilized glucoamylase when repeatedly used is shown in Fig. 6. After 14 repeated usages,theimmobilizedglucoamylaseretained60%oftheresidual activity. The relative activity that significantly decreased by at least 20% after the first reaction could be due to the imperfect washing step. The activity of the repeated usage of immobilized glucoamylase was almost constant from cycle 4 to 14. Thus, the immobilized enzyme has a potential for industrial applications.

3.8. Kinetics of hydrolysis

The Michaelis–Menten kinetics of the hydrolytic activity of the free and the immobilized glucoamylase was investigated

Fig. 5. Thermal stability of free and immobilized glucoamylase.

Fig. 6. Effect of repeated use on the relative activity of immobilized glucoamylase.

by initial rate method. After measuring the changes of product concentrations against time at different initial substrate (soluble starch) concentrations, the initial production rate can be calcu- lated. The Michaelis constant, Km, and the maximum reaction velocity, Vmax, were evaluated from the double reciprocal plot of initial production rate and substrate (Lineweaver–Burk plots).

TheVmax valueof1.75g/L/minexhibitedbytheimmobilized glucoamylasetothebacterialcellulosebeadswaslowerthanthat ofthefreeenzyme(2.62mg/mL/min).TheKm valuedetermined for the immobilized glucoamylase (3.27mg/mL) was slightly increased than that of the free glucoamylase (3.03mg/mL), and indicated a similar affinity behavior toward the substrate. The phenomena in the Km value shows that the structure of enzyme does not change in this immobilized support. This result was similar as the immobilization of glucoamylase in the other natural polymers such as chitosan-clay composite [15], activated charcoal [17] and montmorillonite [18].

4. Conclusions

Glucoamylase was immobilized to bacterial cellulose beads with the use of some activated methods and the epoxy method withglutaraldehydecouplingwasthebestone.Thewetbacterial cellulosebeadswiththesmallestsizewerethebestchoiceforthe different drying conditions and particle sizes. The immobilized glucoamylase enhanced the enzyme abilities against changes in thepHvalueandtemperatureespeciallyinthelowertemperature region. The relative activity of the immobilized glucoamylase was still above 7% at pH 2.0 and it was the highest value in the literature. The relative activities were more than 68% in the lower temperature region even at 20◦C. Therefore, the bacterial cellulose beads are a promising support for the preparation of immobilized glucoamylase for industrial applications.

References

[3] D. Norouzian, A. Akbarzadeh, J.M. Scharer, M.M. Young, Biotechnol.

[5] M. Iguchi, S. Yamanaka, A. Budhiono, J. Mater. Sci. 35 (2000) 261–270.

[6] R. Jonas, L.F. Farah, Polym. Degrad. Stab. 59 (1998) 101–106.

[8] F. Yoshinaga, N. Tobouchi, K. Watanabe, Biosci. Biotechnol. Biochem. 61 (1997) 219–224. [9] S.K. Cousins, R.M. Brown Jr., Polymer 38 (1997) 903–912.

[10] M. Shoda, Y. Sugano, Biotech. Bioproc. Eng. 10 (2005) 1–8.

[14] J. Bryjak, Biochem. Eng. J. 16 (2003) 347–355.

[15] M.Y. Chang, R.S. Juang, Enzym. Microb. Technol. 36 (2005) 75– 82. [16] R.N. Silva, E.R. Asquier, K.F. Fernandes, Proc. Biochem. 40 (2005) 15–1159. [17] A.S. Rani, M.L.M. Das, S. Satyanarayana, J. Mol. Catal. B: Enzym. 10 (2000) 471–476. [18] G. Sanjay, S. Sugunan, Catal. Commum. 6 (2005) 525–530.

(Parte 2 de 2)

Comentários