Immobilization of soybean peroxidase on its natural support

MAGRI, MARÍA L; V. MIRANDA, MARIA; OSVALDO CASCONE

Santiago de Chile

Congreso; 12th International Biotechnology Symposium; 2004

  SOYBEAN SEED COAT PEROXIDASE IMMOBILIZATION ON ITS NATURAL SUPPORT   María L. Magri, María V. Miranda and Osvaldo Cascone Facultad de Farmacia y Bioquímica (UBA) Buenos Aires, Argentina     INTRODUCTION   Enzymes have a great appeal in chemical processes as “green chemistry” reagents that will allow future sustainable developments[1]. In this sense, enzyme technology has become a multidisciplinary field with applications in diverse processes. Peroxidases in particular have been studied extensively and show many attractive properties for biocatalysis, such as wide specificity, high stability in solution and easy accessibility from plant materials. Soybean seed coat peroxidase (SBP) has arisen great interest due to its different activity and stability profile as compared with its widespread counterpart, horseradish peroxidase isoenzyme C (HRP-C)[2]. SBP conformational stability is 43.3 kJ/mol under physiological conditions, which is significantly higher than that reported for HRP-C (17 kJ/mol). Thermal stability is also higher (Tm = 86°C vs. 74°C, at pH 7.0) and SBP has a 20-fold higher catalytic efficiency than HRP-C (with ATBS as the substrate and at its optimum pH). Phenolic compounds are discharged in the wastewater streams of various industries and most of them are toxic and have been classified as hazardous pollutants. Physicochemical treatment processes are not very selective in terms of the number of pollutants removed, so that in highly concentrated wastewaters they become very expensive, even if the target pollutant is present at a low concentration. Besides, selectivity for the removal of certain compounds may be important in order to meet strict regulatory criteria or to facilitate further treatments[3]. Enzymatic peroxidase treatment of phenol involves the catalytic oxidation of the aromatic ring by hydrogen peroxide to form aromatic free radicals that combine to create higher molecular weight products, which precipitate due to their low solubility. SBP is obtained from soybean seed coats, which is an agricultural by-product, turning it into a very profitable option for biotechnological applications. Since recent research showed that immobilised SBP can be efficiently utilised for phenol removal from wastewater[4], immobilisation of SBP directly on its natural support is a very attractive option for a cost-effective wastewater treatment. Purification processes involve several steps that increase the cost of the product to be immobilised, so if the enzyme can be used as it is in nature, the problem of the treatment costs can be solved from an integrated point of view. In this work we study the immobilisation of SBP on its natural support, that is, soybean seed coats, as a way to extend the half-life of the enzyme thus making cost-effective the wastewater treatment.     MATERIALS AND METHODS Materials: Soybean seed coats were a courtesy of local agricultors. Sodium periodate, glutaraldehyde, guaiacol, potassium ferricyanide, 4-aminoantipyrine, hydrogen peroxide and buffer salts were all of analytical grade. Methods: Immobilisation chemistry was performed in glass containers. Soaked seed coats were weighed and submerged in a periodate or glutaraldehyde solution of different concentrations, at various pH. After 2 h, seed coats were thoroughly washed to eliminate soluble SBP. Treated seed coats were assayed for peroxidase activity. Peroxidase activity assay: a fast-screening method for check peroxidase immobilisation was performed by using guaiacol as the substrate and hydrogen peroxide as the electron donor. A 1-g aliquot of treated seed coats was added to 5 ml of the reaction mixture, which included 8 mM guaiacol, 0.5 mM hydrogen peroxide and 50 mM sodium acetate buffer, pH 5.5. After 1-min agitation, the supernatant absorbance at 470 nm was measured. Phenol removal assay: a 1-g aliquot of treated seed coats was added to a reaction mixture which typically contained 2.7 mM phenol, 2.7 mM hydrogen peroxide and 50 mM sodium phosphate buffer, pH 6.0. The reaction was carried out in glass vessels aerated by constant agitation at room temperature. After 24 h phenol concentration was measured in the supernatant. Phenol quantitation assay: the sample was properly diluted to 800 μl and treated with 100 μl 84 mM potassium ferricyanide in 250 mM sodium carbonate and 100 μl 21 mM 4-aminoantipyrine in 250 mM sodium carbonate. The absorbance at 510 nm was determined. Statistical data analysis: All phenol assays were performed in duplicate and the relative percent error was less than 5%. Each experiment included a control (untreated seed coats) and different blanks (Bi, blank without seed coats; Bh, blank with heat-inactivated seed coats; Ba, blank with azide-treated seed coats; and Bw, blank with thoroughly-washed seed coats) to ensure the validity of data. Bi´s phenol concentration was taken as the reference value to calculate phenol removal due to the presence of seed coats. In all experiments the confidence interval for blank values agreed with the confidence interval for Bi values.   RESULTS   Two immobilisation methods were assayed: the periodate and the glutaraldehyde chemistry. A screening experiment was performed where different periodate concentrations (0.1, 1, 5, 10 or 20 mM) were used at different pH values (5, 7 or 9). Different glutaraldehyde concentrations (0.05, 0.1, 0.2 or 0.5 mM) were also assayed at different pH values (7 or 9). Conditions were selected from bibliography to cover the range of higher reactivity for each immobilisation reagent. Periodate did not lead to a significant immobilisation extent in any condition, as determined by the screening peroxidase assay and the phenol removal assay. In contrast, glutaraldehyde was effective in enzyme immobilisation at pH 7 at a concentration of 1 % or higher. The optimum concentrations of glutaraldehyde were 0.1 mM and 0.2 mM for the phenol removal assay, and in order to minimise reagent costs the lower concentration was chosen for further experiments. For seed coats treated with 0.1 mM glutaraldehyde, the molar relationship of phenol to hydrogen peroxide was assayed in the range 0.5:1, 1:1 and 1.5:1 and was optimal at 1:1, thus indicating that an enzymatic reaction was taking place. The positive control showed the same pattern, thus indicating that it was working as a control of the enzymatic activity. Immobilisation did not change the free enzyme optimum activity pH, which was 6.0. Treated seed coat stability (stored soaked in water at 4-8 °C) was assessed over a four-week period and the immobilised enzyme maintained its activity, whereas the free-enzyme control stored under the same conditions became inactive after 24 h. To determine the maximum phenol removal capacity of the treated seed coats, phenol and hydrogen peroxide concentrations in the reaction mixture were concurrently varied and made to react with a 1-g aliquot of treated seed coats: around 50 % of the total phenol was removed overnight at a phenol concentration of 0.08 mM or lower, and the percentage decreased with the increase in phenol concentration. A final experiment was conducted to determine the reusability of treated seed coats. After a 24-h standard phenol removal assay, seed coats were washed three times and the excess of water was removed. A fresh reaction mixture was added to the already used seed coats and left overnight to react. It was shown that used seed coats could be reused just once, since in the second time of reuse the seed coats lost all their activity, probably due to the precipitation of phenol polymers on the surface of the seed coat that hinder the access of substrate to the active site. [1] "Enzyme technology: an overview" Beilen, J. and Li, Z. Curr. Opin. in Biotech. 13 (2002), 338-344. [2] "Activity, stability and conformational flexibility of soybean seed hull peroxidase" Kamal, J. and Behere, D. J Inorg Biochem. 94 (2003), 236-242. [3] “Removal of phenolic compounds from synthetic wastewater using soybean peroxidase” Caza, N. et al. Wat Res. 33 (1999), 3012-3018. [4] "Characterization of soybean peroxidase for the treatment of aqueous phenols" Wright, H. and Nicell, J. Bioresource Technology. 70 (1999), 69-79.