The crystalline cell surface layer from Thermoanaerobacter thermohydrosulfuricus L111-69 as an immobilization matrix: influence of the morphological properties and the pore size of the matrix on the loss of activity of covalently bound enzymes

摘要

The hexagonally ordered cell surface layer (S-layer) from Thermoanaerobacter thermohydrosulfuricus L111-69 was used as a matrix for the immobilization of naringinase (EC 3.2.1.40, α-L-rhamnosidase) (M_R 110000), β-glucosidase (M_r 66000) and peroxidase (M_r 44000). Naringinase is significantly larger than the 4-5 nm-sized pores passing through the S-layer lattice and could also span the 6 nm-wide central funnel-shaped depression in the centre of the hexameric unit cells. The enzyme was immobilized as a monolayer at the outermost surface of the S-layer lattice and retained 60-80% of its original activity. Because of its smaller molecular size, β-glucosidase was capable of penetrating the central funnel-shaped depression and, therefore, could follow the topography of the S-layer lattice to a greater extent. A tenfold increase in activity from 16 to 160% was observed when β-glucosidase was immobilized via spacers. Although the 4-5 nm-sized peroxidase could be immobilized via different functional groups in high density, the enzymic activity retained was always less than 3%. Since the S-layer protein was also available in monomeric form, soluble S-layer protein-peroxidase conjugates with a molar ratio of 3:1 could be prepared in which 40% of activity from the native enzyme was preserved. These comparable studies with soluble and crystalline S-layer protein confirmed that rather than the physicochemical properties of the immobilization matrix, the entrapping of the peroxidase molecules inside the pores is responsible for the high activity loss. In summary, the results obtained with a crystalline immobilization matrix show that the activity loss of immobilized enzymes correlates with the extent of interactions with the matrix.