Preparation of silica with controlled pore sizes for enzyme immobilization

Preparation of silica with controlled pore sizes for enzyme immobilization

H.C. Trevisan¹, L.H.I. Mei² and G.M. Zanin³

¹ Depto. de Bioquímica e Tecnologia Química, IQ-UNESP, R. Prof. Francisco Degni, s/n, Araraquara - SP,

Brazil, CEP 14800-900; E- mail: [e-mail]

² Depto. de Tecnologia de Polímeros, Faculdade de Engenharia Química, UNICAMP,

Campinas - SP, Brazil, CEP 13083-970

³ Depto. de Engenharia Química, FUEM, Av. Colombo, 5790, Bloco D-90,

CEP 87020-900, Maringá - PR, Brazil,

(Received: September 17, 1998; Accepted: September 3, 1999)

Abstract - A simple method for the preparation of silica with controlled pore size, for use as a support for the immobilization of enzymes, is described in this article. Using sodium silicate and hydrochloric acid, a microporous silica was obtained that was then submitted to a hydrothermal treatment, resulting in macroporous silica suitable for enzyme immobilization. Suitability of the macroporous silica as a support depends on the method chosen for its preparation, which will determine pore volume and the effect of hydrothermal treatment on pore size. The pore volume of the support was 0.8-0.9 cc/g and the average pore size, controlled by the hydrothermal treatment, was in the range of 16 to 75 nm. The enzyme amyloglucosidase was used for the immobilization studies.

Keywords: silica, preparation, enzyme immobilization, amyloglucosidase.

INTRODUCTION

Enzyme immobilization is now a well-established alternative in the development of processes. The variety of methods and supports allows a great number of combinations, such that practically every enzyme can be successfully immobilized.

Although a large number of supports have been used for enzyme immobilization, only those with good mechanical properties and chemical stability will be favored in process applications, especially inorganic ones. For this reason, the use of siliceous materials such as glass with controlled porosity and silica has been widespread.

It is already known that the support should have the largest possible pore volume, which does not compromise its mechanical strength and simultaneously a pore size suitable for fitting the enzyme with its optimal configuration to catalyze the substrate transformation. With the aim to avoid the high cost of CPG (controlled pore glass, Sigma 1997) and to obtain supports with suitable characteristics, silicas of different pore sizes were prepared and tested for each enzyme-substrate pair of interest, for the purpose of finding the best pore size for application in processes.

As hydrothermal treatment has little effect on the pore volume of the silica (Leboda et al., 1995a), the method developed in this article was conducted in such a way that the pore volume of the microporous silica was controlled by time and gelling temperature and aging (Leboda et al., 1995; Leboda et al., 1995a). Pore size was adjusted by the temperature of the hydrothermal treatment.

MATERIALS AND METHODS

Enzyme and Chemicals.

Amyloglucosidase (AMG 200L) was kindly supplied by NOVO; sodium silicate was a gift from ICI; 3-aminopropyltriethoxy silane was obtained from Aldrich; glutaraldehyde, Coomassie Brilliant Blue G and bovine serum albumin were from Sigma; and the HCl used was of a technical grade. All the other reagents were commercially available products with a high degree of purity.

Support preparation.

A sol was obtained by mixing diluted sodium silicate (13.3% SiO₂, 4.4% Na₂O) with a 5% excess of HCl (21.4%). The sol was gelled in a plastic tray at a constant temperature, cut into pieces of about 2 cm and washed until salt free. The gel was immersed in distilled water, aged overnight at a constant temperature and dried at 150 ° C. The dried microporous silica obtained was ground in a ball mill and sieved; only the fraction in the 0.5 to 0.7 mm range was used in this work. Portions of about 5g were poured into water and the air removed from the pores by vacuum. Those silica suspensions were treated in the autoclave at a constant temperature of up to 270° C during 5 hrs. After that the samples were cooled and the gel was filtered and dried at 120° C; then they were assayed for suitability as a support for AMG immobilization.

Assays and Support Characterization

As a preliminary assay, pore volume was determined by weighing the water mass necessary to fill the pores. Excess water outside the pores of the samples was removed with filter paper. The immobilization capacity of the support was evaluated by immobilizing the amyloglucosidase enzyme (AMG). For this purpose, aminopropyl silica was prepared (Trevisan and Mei, 1992) and the enzyme was linked by the glutaraldehyde method (Weetall, 1976). The protein loaded was calculated based on the Coomassie assay (Sedmak and Grossberg, 1977). The activity of the immobilized enzyme was measured with 5% soluble starch as the substrate, in a citrate buffer of 0.1 M, pH 4.5 at 45° C, and it was expressed as µ mol of glucose liberated per minute, based on the o-toluidine assay (Cooper and McDaniel, 1970). Further characterization of the supports was performed by the mercury porosimetry method.

RESULTS AND DISCUSSION

After considering the influence of gelling and aging times on pore volume, a series of samples were prepared and submitted to hydrothermal treatment. For practical reasons and taking in account the results, the gelling and aging times were standardized at 16 hrs and washing time at 7 hrs. Conditions for preparing these samples as well as the pore volume analysis are summarized in Table 1.

Some samples were submitted to hydrothermal treatment in order to study the range of temperatures and their suitabilities as supports for AMG immobilization. As mentioned previously (Eaton, 1974), the optimum pore size for AMG is around 35 nm. Assays done in this work also provided an evaluation of average pore size in relation to temperature, [e.g., in Table 2, the best conditions for immobilization were obtained at 200° C, so this temperature should result in a 35 nm silica].

Tables 3 and 4 show the amount of immobilized protein and the improvement in immobilized activity, which is the most important parameter, including a comparative sample of CPS 50 nm (controlled pore silica, Corning Glass Works). From Tables 2, 3 and 4, it can be seen that the best silica for AMG immobilization was obtained by treatment at 200-210°C. As pore volume increases, there is an increase in the amount of immobilized enzyme due to the larger surface available and a decrease in the number of micropores, inaccessible to the enzyme by steric hindrance. The results obtained for enzyme immobilization on silica V (Table 4) were good enough to consider it a suitable support.

The silica V was analyzed by mercury porosimetry in order to relate pore size with the amount of AMG immobilized and with the temperature of the hydrothermal treatment. In Table 4 and Figure 1 it can be seen that optimum pore size for AMG immobilization was around 35 nm, as already reported (Eaton, 1974). For smaller average pore diameters, there was a sharp decrease in immobilized activity due to size exclusion of the enzyme molecule. For bigger pore sizes, the decrease corresponded with the reduction in the surface area of the support (Figure 1).

The pore size distribution of four samples of silica V is shown in Figure 2 together with the distribution for CPS 50 nm (Corning). Silica V has a relatively narrow pore size distribution and its microstructure also seems to be suitable for chromatography, in addition to its use in enzyme immobilization.

In our attempt to correlate the parameters of hydrothermal treatment with average pore diameter, it was found that pore diameter versus treatment pressure data falls on a straight line. The pressure considered here is that of water vapor saturation at equilibrium, obtained from thermodynamic tables, at the same temperature as that of treatment (Figure 3). This relationship suggest that pore size may be controlled by selecting the pressure (and temperature) of hydrothermal treatment. It is worth pointing out that the effect of the treatment on microporous silica will depend on the method of its preparation, and the line shown in Figure 3 will be affected by it. Thus, to control pore size conditions for preparing the microporous silica and the effect of hydrothermal treatment must be considered together.

CONCLUSION

In this work it was shown that selecting the preparation conditions for the silica can result in a suitable product for immobilization of enzymes. Optimization of the method led to a support of a quality comparable to that of the imported commercial ones, which was easy to prepare and cheap. An advantage of this method is that the average size of the pores can be controlled, depending on the enzyme to be immobilized. Several enzymes, such as b-galactosidase, invertase and lipases, are being immobilized with success in the silica obtained by this process.

Commercial silicas are also being tested to verify the response to hydrothermal treatment. Continuing this research, we are now working on the preparation of spherical particles with the same structure for enzyme immobilization and chromatography, in addition to the immobilization of other enzymes on this support.

ACKNOWLEDGEMENTS

We thanks NOVO Industri for the AMG, ICI for the sodium silicate, Prof. Dr. José Arana Varela for the porosimetry analyses and FAPESP, CNPq and PADC/FINEP for their financial support.

REFERENCES

Cooper, G.R., McDaniel, V., The Determination of Glucose by the o-Toluidine Method. Clinical Chemistry 6: 159-170 (1970)

Eaton, D.L., Immobilized Biochemicals and Affinity Chromatography. 42 (Advances in Experimental Medicine and Biology): 241-258 (1974)

Leboda, R., Mendyk, E., Gierak, A., Tertykh, V.A., Hydrothermal Modification of Silica Gels (Xerogels) 1. Effect of Treatment Temperature on their Pore Structure. Colloids Surfaces A: Physicochem. Eng. Aspects. 105: 181-189 (1995)

Leboda, R., Mendyk, E., Gierak, A., Tertykh, V.A., Hydrothermal Modification of Silica Gels (Xerogels) 2. Effect of Duration of Treatment on Their Pore Structure. Colloids Surfaces A: Physicochem. Eng. Aspects. 105: 191-197 (1995a)

Sedmak, J.J., Grossberg, S.E., A Rapid, Sensitive and Versatile Assay for Protein Using Coomassie Brilliant Blue G250. Anal. Biochem. 79: 544-552 (1977)

Sigma Chemical Co. Catalog, p.1879 (1997)

Trevisan, H.C., Mei, L.H.I., Supports for Enzyme Immobilization. An. Acad. Bras. Ci. 64(2): 111-116 (1992)

Weetall, H.H., Covalent Coupling Methods for Inorganic Support Materials. Methods in Enzymology. 44 (Immobilized Enzymes): 134-148 (1976)

Publication Dates

• Publication in this collection
  24 Apr 2000
• Date of issue
  Mar 2000

History

• Accepted
  03 Sept 1999
• Received
  17 Sept 1998