Soil Bid. Biochem. Vol. 25, No. 6, pp. 671-677, 1993 Printed in Great Britain. All rights reserved 0038-07 I7/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd INTERACTIONS OF INVERTASE WITH TANNIC ACID, HYDROXY-ALUMINIUM (OH-Al) SPECIES OR MONTMORILLONITE L. GIANFREDA, M. A. RAO and A. VIOLANTE Dipartimento di Scienze Chimico-Agrarie, Universitl di Napoli “Federico II”, Via Universitl 100, 80055 Portici, Napoli, Italy (Accepted I5 January 1993) Summary-Interactions between invertase, tannic acid and hydroxy-aluminium (OH-Al) polymers were investigated with regard to their influence on enzyme activity and the formation of active enzymatic complexes. OH-Al species slightly inhibited invertase activity (inhibition constant, K, = 133 mM) whereas a marked inhibition (190%) was detected at a tannic acid concentration greater than 1.25 mM. The V,, and K,,, values indicated a pure non-competitive and a mixed-type inhibition mechanism of OH-Al species and tannic acid, respectively. Depending on the incubation time and the tannic acid-to-enzyme ratio, the interaction between invertase and tannic acid yielded both soluble and insoluble complexes, which displayed reduced activity levels. The decrease of invertase activity was a function of both the tannic acid-to-invertase ratios and the contact time. Bonding between invertase and tannic acid as well as the residual activity of the immobilized enzyme were greatly increased by the presence of OH-Al polymers during the complexation process. Many more active invertase molecules were removed from solution and an enhanced precipitation of active invertase-tannic acid complexes was observed. Clearly not only OH-Al ions facilitated flocculation of tannateinvertase complexes with different structural characteristics but their interaction with tannic acid molecules gave rise to Al precipitation products having a different charge and sorption sites. Furthermore, an invertas&H-Al-tannic acid complex was anchored on montmoril- lonite surfaces and the complex formed showed a relatively high enzymatic activity. OH-Al species acting as bridges between tannate molecules and montmorillonite surfaces also facilitated the immobilization of soluble invertase_OH-Al-tannate complexes. INTRODUCTION In soil, all of the extracellular enzymes are more or less permanently immobilized on organic, inorganic or organomineral soil components (Burns, 1982; Boyd and Mortland, 1990; Burns, 1986). The exact nature of the association of enzymes with soil con- stituents still remains to be clarified, since several mechanisms and numerous starting materials can be involved in this very complicated process. Several studies have been carried out, mainly, on the interactions of enzymes with humic acids or pure crystalline aluminosilicates (Ladd and Butler, 1975; Theng, 1979; Burns, 1986). However, in soils various hydrolytic products of Al and Fe coat the clay minerals, modifying the physicochemical properties of soil minerals (Bamhisel and Bertsch, 1989). The influence of OH-Al species, held by the surfaces of phyllosilicates, on the activity of enzymes has received only little attention. Gianfreda et al. (1991, 1992) found that the partial coating of montmoril- lonite with Al(OH), species, to simulate the “dirty clays” usually present in soil environments, markedly affected the amount of adsorbed invertase or urease enzymes as well as their residual activities. In soils, Al species undergo numerous reactions which play an important role in pedogenesis, formation, stabilization and turnover of humic substances as well as on aggregation and related soil physical properties. Furthermore, soluble monomers and polymers of Al as well as short-range ordered or crystalline Al hydroxides and oxyhydroxides may interact with humic and fulvic acids, low-molecular weight organic acids and phenolic compounds and form OH-Al-organic species. In turn, these species may be held on clay surfaces forming much more complex organo-mineral associations (Huang and Violante, 1986). Phenolic acids and polyphenols, which are ubiqui- tous in soils, seem to play a very important role in immobilizing extracellular enzymes (Ladd and Butler, 1975; Bums, 1986). In fact, it has been recognized that a significant proportion of immobilized soil enzymes is associated with humic fraction by the for- mation of enzyme-polyphenolic copolymers during the genesis of humic substances (Bums, 1986). The abiotic polymerization of phenolic compounds and the formation of humic substances are catalysed by clay minerals, Fe, Mn and Al oxides and hydroxides, primary minerals and even OH-Al polymers (Kumada and Kato, 1970; Shindo and Huang, 1985; McBride ef al., 1988; Huang, 1990). Many studies have been carried out both on the interactions between hydrolytic products of Al and phenolic compounds (mainly tannic acid) in the absence or presence of clay minerals (Violante and 671