Synthesis and properties of β-d-glucosidasephenolic copolymers as analogues of soil humic-enzyme complexes

Several β-d-glucosidase-phenolic copolymers were synthesized and three examined in detail: those containing l-tyrosine, pyrogallol and resorcinol. These copolymers were similar to naturallyoccurring soil humic-enzyme complexes in many ways: E₄/E₆ ratios, C, H, N and S content and IR spectra. The enzyme activity of the copolymers showed varying degrees of resistance to proteolysis, organic solvents, and storage at high temperatures. All immobilized enzymes had increased K_m values and decreased V_max values in comparison with soluble β-d-glucosidase (9.3 mM, 190μmol p-nitrophenol mg^?1h^?1); the β-d-glucosidase-resorcinol copolymer was the most active (10.5 mM, 104μmol p-nitrophenol mg^?1h^?1). β-d-Glucosidase activity was completely resistant to protease when the copolymer was fixed to bentonite clay but V_max values were reduced further (e.g. β-d-glucosidase-resorcinol-bentonite complex, 58.5μmol p-nitrophenol mg^?1 h^?1). On addition to soil, soluble β-d-glucosidase was rapidly inactivated (38% loss in 3 days, 80% loss in 21 days) whereas β-d-glucosidase-resorcinol/pyrogallol and β-d-glucosidase-L-tyrosine copolymers were comparatively stable (no loss in 9 days, 25% and 44% loss in 21 days). It is suggested that the copolymerization of enzyme during humic matter formation is a major factor leading to the stabilisation of soil enzymes and that adsorption and entrapment are comparatively insignificant.