A NEW APPLICATION OF HUMIC SUBSTANCES: ACTIVATION OF SUPPORTS FOR IMMOBILIZATION OF INVERTASE ENZYME

André Henrique Rosa¹, Alexandre Aparecido Vicente², Julio Cesar Rocha¹ and Henrique Celso Trevisan²

¹Dept.^o of Analytical Chemistry –

²Dept.^o of Biochemistry and Technological Chemistry

Institute of Chemistry, Araraquara (UNESP)

CP. 355, CEP 14800 - 900, Araraquara-SP, Brazil

e-mail: andrrosa@iq.unesp.br

“Keywords”: humic substances, invertase, immobilized enzyme

The most important class of naturally occurring complex agents is that of humic substances (HS). They comprise a complex mixture of physically heterogeneous substances which show a relatively high molecular weight with a large number of oxygen-containing functional groups. Alternative methodology for their extraction have been published recently (ROCHA et al., 1999). The HS are formed by secondary reactions during the decay process and by transformation of biomolecules originating from dead organisms and microbial activity. These compounds are important in soil conservation, water holding capacity and complexation of metals species in aquatic systems (ROCHA et al., 2000). Due the presence in their structure of great amount of carboxylic, hydroxylic and amide groups, they are becoming a very important candidates for the activation of enzyme immobilization supports.

Immobilized enzymes have been extensively used in analytical chemistry and industry. The success of the biocatalysis is mainly related to the support used and for the activating reagent, that allow the generation of suitable groups for the linking enzyme.

In this work the enzyme invertase was immobilized on aminopropyl silica (APTS-SiO₂) activated with humic substances (APTS-SiO₂-HS) as well as on silica activated with glutaraldehyde (APTS-SiO₂-GA), using a traditional method (KENNEDY & WHITE, 1985). The activity of the invertase in both systems has been compared.

HS used in the activation of the silica was extracted from soil of Cananéia, São Paulo State, Brazil, using the procedure recommended by IHSS (WERSHAW et al., 1992). It was carried out as follows: 1.0 g of silica with particle size in the range 150-300 mm and pore diameter 450Å, was activated with 50 mg of HS dissolved in 0.1 mol/l NaOH, with the final pH adjusted at 7.0 by adding 0.1 mol/l HCl solution. Slurry was stirred for 24 hs and centrifuged at 3000 g during 15 min. Then the supernatant was carefully removed and the support (APTS-SiO₂-SH) washed exhaustively with acetate buffer at pH 4.5. In order to "stabilize" the adsorbed and covalently bonded APTS-SiO₂-SH, the above dry product was cured at 120 °C for 5 hs according to KOOPAL, et al., 1998. After curing, it was washed with the same acetate buffer. For the immobilization (EC 3.2.1.26) 20 ml (5 mg/ml concentration) were added and the material left 48 hs under stirring at room temperature.

Invertase activity was determined by measuring the rate of formation of reduced sugars during hydrolysis of 0.146 mol/l sucrose in 0.05 mol/l acetate buffer at pH 4.5 and temperature 45°C. Total reduced sugar was measured colorimetrically, using the reaction with dinitrosalicylic acid DNS (SUMNER & HOWELT, 1935).

Table 1 shows main characteristics of humic substances extracted from Cananéia soil.

Table 1 – Elemental and functional characteristics of humic substances extracted from Cananéia soil, Brazil.

The amount of humic substances bound on the APTS-SiO₂ was equal to 50 mg. The infrared spectrum of the APTS-SiO₂-HS and of the enzyme linked to this support, showed prevalence of covalent linking of NH₂ groups with carboxylic groups of the support activated. The maximum amount of invertase immobilized in APTS-SiO₂-HS was 15232 U/g while in the system APTS-SiO₂-GA, it was equal to 13357 U/g. The experimental enzymatic activity was 3706 and 3274 U/g, for the systems APTS-SiO₂-HS and APTS-SiO₂-GA, respectively. These results are mainly due to the high percentage of carboxylic groups (55.0%) present in the sample of humic substances that favor reactions with the enzyme amino groups.

Considering the increased amount and activity of immobilized enzyme compared with the glutaraldehyde method, we may conclude that this work opens new perspective in the preparation of supports for enzymes immobilization employing humic substances.

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ROCHA, J.C. et al.; Aquatic humus from an unpolluted Brazilian dark-brown stream: general characterization and size fractionation of bound heavy metals. J. Environ. Monit., v. 2, p. 39, 2000.

KENNEDY, J.F. & WHITE, C.A. Principles of immobilization of enzymes. In: WISEMAN, A. (Ed.), “Handbook of Enzyme Biotechnology”, 2^nd ed., Chichestester, Wiley & Sons (1985).

WERSHAW, R.L. et al.; NMR characterization of humic acid fractions from different Philipine soils and sediments. Anal. Chim. Acta., 232, 31, 1992.

KOOPAL, L.K. et al.; Chemical immobilization of humic acid on silica, J. Coll. Surf., 141, 385, 1998.

SUMMER, J.B. & HOWELT, S.F. A method for determination of invertase activity. J. Biol. Chem., 108, 51, 1935.

Acknowledgments: FAPESP/ CNPq