Monday, May 4, 2009
5-33
Immobilization and Stabilization of Xylanase by multipoint covalent attachment on Glyoxyl Agarose Support
Anny Manrich, Paulo Waldir Tardioli, Wellington S. Adriano, and Raquel Lima Camargo Giordano. Chemical Engineering Department, Universidade Federal de São Carlos, Washington Luiz, Km 235, Monjolinho, São Carlos, SP, Brazil
Xylanases have important applications in industry, such as the bioconversion of lignocellulosic biomass to xylose and other fermentable sugars for the production of ethanol and xylitol. Immobilization and stabilization of enzymes may allow their reuse in many cycles of the reaction, decreasing the hydrolysis process costs. This work proposes the use of a rational approach to obtain immobilized commercial xylanase biocatalysts with optimized features. Xylanases NS50014 from Novozymes was characterized and immobilized on glyoxyl-agarose support. The activation of agarose was performed by etherification with glycidol, followed by oxidation with sodium periodate, to produce linear aldehyde groups in the support. When immobilizing xylanases (25oC, pH 10.05) on this activated support, only 5% of yield immobilization was reached after 24 hours and no significant stabilization of the immobilized enzyme was observed. The immobilization reaction occurs between aldehyde groups of the support and amine groups of the protein. Therefore, the low concentration of lysine groups in the enzyme molecule, measured by acid hydrolysis followed by amino acid analysis, could explain these poor results. In order to increase the concentration of amine groups on the enzyme surface, the protein was chemically modified with ethylenediamine (EDA). The modified enzyme was then immobilized on glyoxyl-agarose. The new enzyme derivatives were 20-fold more stable than the soluble, aminated and dialyzed enzyme (70°C, pH 7), with 100% of immobilization yield. Therefore, the increase of the number of amine groups in the enzyme surface was confirmed to be a good strategy to improve the properties of immobilized xylanases.