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Optimization of the immobilization parameters on chitosan microparticles

= ∑ d obs d pred d pred

3.3. Covalent immobilization of β- D -galactosidase on chitosan supports

3.3.2. Optimization of the immobilization parameters on chitosan microparticles

Chitosan microparticles were obtained by the emulsion cross-linking method and the parameters influencing the preparation process of microspheres were already optimized by experimental design, as well as by sequential optimization (see Sections 3.1 and 3.2). In this study, the optimization process was continued with the parameters influencing the immobilization: enzyme loading, amount of glutaraldehyde and immobilization time. In this respect, two aspects must be considered. Firstly, the glutaraldehyde concentration was included in the experimental design for optimizing the particle size of chitosan microspheres. As it was previously stated, glutaraldehyde has a double role in this process, cross-linker and activator, and the number of free aldehyde functions available for covalent binding is a key issue for efficient immobilization. In this respect, study of influence of

glutaraldehyde concentration was imperious, although for the experimental design the enzyme-linking ability was not a topic. Secondly, chitosan concentration used in the preliminary experiments for evaluation of different chitosan-based carriers (presented in Sub-section 3.3.1) was 2% (w/v, in acetic acid 2%, v/v solution). Since the experimental design results showed that concentration of chitosan is the most important parameter to obtain small-sized chitosan microspheres by the emulsion cross-linking method and the value of optimal concentration was stated as 1.5%, this concentration has been utilized in the immobilization experiments.

To characterize the overall efficiency of the immobilization process, total activity yield was calculated as percentage of the total enzymatic activity recovered following immobilization (activity of the immobilized enzyme multiplied by the amount of the immobilized enzyme), related to the total β-D-galactosidase activity introduced in the immobilization process (activity of the free enzyme multiplied by the amount of free enzyme subjected to immobilization).

3.3.2.1. Influence of the loaded protein concentration

In order to maximize the amount of immobilized β-D-galactosidase onto chitosan beads, the protein amount subjected to immobilization was varied between 0.37 mg and 18.67 mg, on 0.2 g wet chitosan beads (chitosan beads were kept in buffer solution). These amounts led to a protein/carrier ratio between 1.85 and 93.35 mg/g wet carrier (Table 3.9). The enzyme loading capacity was evaluated in relation with the immobilization yield of the protein, calculated as the difference between the initial protein subjected to immobilization and the protein remained in the supernatant after immobilization. The binding capacity of microparticles did not change too much with increasing amounts of available enzyme. The loaded protein values were, excepting the last entrance in Table 3.9, in a very narrow interval, between 3.6-4.0 mg/g wet chitosan. As the enzyme has been immobilized on chitosan gel beads that contained a large amount of water, the enzyme/carrier ratio on these hydrated gel beads was obviously much lower than related to the dry carrier. 1 g chitosan microspheres obtained by emulsion cross-linking lost 86.4% of their weight by drying at room temperature until constant weight. Consequently, the enzyme loading values, related to dry carrier, are much higher, between 27-29 mg/g. The results presented in Table 3.9 show that increasing the amount of enzyme subjected to immobilization resulted in

enhancement of loaded protein only at low protein/carrier ratios. The explanation is that the number of active groups of support has a certain value, and if the bound positions are saturated, increasing the amount of enzyme in the immobilization medium cannot raise the amount of loaded enzyme.

Table 3.9. Influence of loaded protein on activity of immobilized β-D-galactosidase Protein/carrier

The loaded protein data were satisfactorily correlated with the activities of immobilized

β-D-galactosidase, which also exhibited close values. However, the activity differences were higher than observed for the loaded protein, probably because the activity of an immobilized enzyme depends not only on the attached protein but also on other factors, like steric hindrances and diffusional effects.

The efficiency of the immobilization process can be better evaluated using the total activity yield, which allows a global assessment of the obtained results. As it can be seen in Table 3.9, covalent immobilization on chitosan microspheres was accomplished with moderate efficiency, the total activity yield remaining below 15%. It must be pointed out that covalent binding often results in decrease of enzyme activity, but this drawback is generally compensated by increased stability of the immobilized enzyme.

As it was mentioned in section 1.4.1. β-D-galactosidase has tetrameric structure composed of 4 identical subunits which form two identical dimers with two active sites, one in each dimer (Pereira-Rodriguez et al., 2012). The reasons for decline of enzyme activity during or following the immobilization process could be numerous, and some of them were mentioned previously. Another explanation could be that native enzyme loses its activity when it is diluted or dialyzed, because its tetrameric structure is disturbed in aqueous medium. During a control experiment, the enzyme diluted 4 fold in 0.02 mol/L pH 7.5 potassium phosphate buffer was shacked during 16 h. After that interval, the diluted enzyme retained only 47.6% of the initial activity.

The commercially available β-D-galactosidase (MAXILACT LX 5000) is stabilized with 47% glycerol because addition of polyols to aqueous solutions of enzyme strengthens the hydrophobic interactions among non-polar amino acid residues leading to protein rigidification (Iyer and Ananthanarayan, 2008). To check the behaviour of unstabilized enzyme, it was dialyzed for 24 h in 0.02 mol/L pH 7.5 potassium phosphate buffer. The dialyzed enzyme retained only 36.6% of its activity. Taking out the enzyme from the stabilized environment leads to decrease of its activity, probably due to dissociation of the tetrameric structure in aqueous medium in absence of the glycerol polyolic stabilizer (Becerra et al., 1998; Pilipenko et al., 2007)

One of the possibilities of multimeric enzymes stabilization is immobilization by multisubunit covalent attachment (Fernandez-Lafuente, 2009). As glutaraldehyde is also participating in the cross-linking of chitosan microspheres, probably only a few -CHO functional groups are remaining for covalent binding of the enzyme, not enough to implement multipoint attachment of the enzyme. Another explanation could be the partial dissociation of the native enzyme in aqueous medium prior to fulfil the covalent binding, leading to activity decrease, since monomer and trimer forms are inactive (Becerra et al., 1998; Pilipenko et al., 2007).

The results of protein loading study showed that highest binding capacity for chitosan microparticles cross-linked with 3% glutaraldehyde was 27 mg protein/g dry support.

3.3.2.2. Influence of glutaraldehyde concentration

For optimization of the covalent binding process the influence of glutaraldehyde concentration was studied, keeping constant the other parameters which influence the process. Microparticles were synthesized by emulsion cross-linking method in an oil phase composed of 40% sunflower oil and 60% n-hexadecane, with a stirring rate of 1500 rpm, 2.5% Tween 80 and 1.5% chitosan solution concentrations (optimal values given by the experimental design).

Different glutaraldehyde concentrations were applied for testing the binding capacity of microparticles. The glutaraldehyde amount was varied between 1% and 5%, related to the chitosan solution volume. The immobilization process was carried out at 10ºC, for 16 hours. The amount of native enzyme amount subjected to immobilization was 27 mg protein/g dry chitosan. Samples have been taken at 2, 4, and 16 hours and were analyzed.

The immobilization efficiency, expressed as total activity yield, was calculated for all samples and the obtained values are displayed in Fig. 3.26. The highest efficiency was reached with 2% and 3% glutaraldehyde, after 4 hours coupling reaction time. The activities were lower in case of low and high glutaraldehyde concentrations.

0 2 4 6 8 10 12 14

Total activity yield [%]

1 2 3 4 5

Glutaraldehyde concentration [%]

2 h 4 h 16 h

Fig. 3.26. Effect of glutaraldehyde concentration on the catalytic efficiency of immobilized β-D-galactosidase

The glutaraldehyde being involved in the cross-linking of chitosan as well as in the enzyme binding process it is very difficult to evaluate how many free aldehyde groups are remaining for covalent binding of NH2 groups of the enzyme. In case of 1% glutaraldehyde concentration, the cross-linking degree was lower, resulting in softer particles and only the remaining glutaraldehyde amount was implied in the binding of enzyme. Applying higher concentrations of glutaraldehyde resulted in harder microparticles with high better cross-linking degree; outside of glutaraldehyde being involved in formation of particles, several CHO groups remained for enzyme binding. It can be also seen that activities after 16 h reaction time decreased, regardless to the glutaraldehyde concentration.

Glutaraldehyde was introduced solely during the particle preparation stage, as experiments to add part of glutaraldehyde simultaneously with the enzyme solution resulted in very low activities. Glutaraldehyde is not only cross-linking reagent, but also could be a denaturing reagent. Therefore, higher glutaraldehyde concentrations can directly influence the activity of immobilized enzyme. As shown in Fig. 3.26, when the concentration of glutaraldehyde was lower than 3%, the activity of immobilized enzyme reached the maximum value.

Increasing the concentration of glutaraldehyde at more than 3% resulted in decrease of

activity. The reason could be that at high concentrations glutaraldehyde can undergo aldol condensation, which affects the construction of holes on the surface of beads, as it was demonstrated by other authors (Zhang et al., 2010). This effect not only makes the immobilization more difficult, but will also likely change the conformation of enzyme, leading to decline of enzyme activity. As a result, the optimal concentration of glutaraldehyde was set as 3%.

3.3.2.3. Influence of immobilization time

To study the influence of immobilization time, the process has been carried out up to 10 h.

Samples were taken at every 2 h and the total activity yield was calculated as described above. The experiments were carried out with 3% glutaraldehyde concentration and the same enzyme/carrier ratio as in previous experiments.

From Fig. 3.27 results that at up to 6 h immobilization time the activity of immobilized enzyme increased in time, but prolonged time led to decrease of activity. These results are in accordance with the previous experiments, displayed in Fig. 3.26, where the same effect was noticed. Consequently, the optimal immobilizing time should be no more than 6 h.

8 9 10 11 12 13 14

0 200 400 600 800

Immobilization time [min]

Total activity yield [%]

Fig. 3.27. Influence of immobilization time the catalytic efficiency of immobilized β-D-galactosidase

3.3.2.4. Improvement of the catalytic efficiency by reduction with sodium borohydride

In order to increase the efficiency of the immobilization process, the Schiff bases obtained after covalent attachment of the enzyme were reduced by sodium borohydride. Compared to the process without reduction, an almost twofold increase of the activity has been noticed. The values of activity and total activity yield reached 3.39 U/g dry support and 23.5%, respectively, while in the same immobilization conditions, but without reduction, the values were 1.89 U/g (dry support) and 13.06%, respectively (data taken from Table 3.9). The explanation is that reduction with sodium borohydride allows transformation of the weak Schiff bases to stable secondary amino bonds, in the same time as the remaining free aldehyde groups on the solid support are converted into inert hydroxyl groups, inducing a favourable conformational change of the immobilized enzyme.

3.4. Sol-gel entrapment of β-D-galactosidase in organic-inorganic composite matrices