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

The hexagonally ordered cell surface layer (S-layer) from Thermoanaero bacter thermohydrosulfuricus L111-69 was used as a matrix for the immo bilization of naringinase (EC 3.2.1.40, alpha-L-rhamnosidase) (M(r) 11 0 000), beta-glucosidase (M(r) 66 000) and peroxidase (M(r) 44 000). N aringinase 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. T he enzyme was immobilized as a monolayer at the outermost surface of t he S-layer lattice and retained 60-80% of its original activity. Becau se of its smaller molecular size, beta-glucosidase was capable of pene trating the central funnel-shaped depression and, therefore, could fol low the topography of the S-layer lattice to a greater extent. A tenfo ld increase in activity from 16 to 160% was observed when beta-glucosi dase 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-la yer protein was also available in monomeric form, soluble S-layer prot ein-peroxidase conjugates with a molar ratio of 3:1 could be prepared in which 40% of activity from the native enzyme was preserved. These c omparable studies with soluble and crystalline S-layer protein confirm ed that rather than the physicochemical properties of the immobilizati on matrix, the entrapping of the peroxidase molecules inside the pores is responsible for the high activity loss. In summary, the results ob tained with a crystalline immobilization matrix show that the activity loss of immobilized enzymes correlates with the extent of interaction s with the matrix.