Binding Mechanisms And Properties Of Acid Phosphatase On Soil Active Particles

This paper studied the adsorption mechanisms of acid phosphatase, which is an important hydrolase for the transformation of phosphorus in soils, on two typical soil colloids and major soil clay minerals by means of equilibrium adsorption, fluorescence, circular dichroism and microcalorimetry. The catalytic kinetics, stability and thermodynamic characteristic of immobilized enzymes were also investigated. We intended to provide insight into the interactive mechanisms among soil components. The study would also be significant for the application of immobilized enzymes in regulating soil fertility and remediating polluted environments. The main results are outlined as follows:1. The adsorption of enzyme on examined soil colloids and minerals were spontaneous,△H_ads＞0,△S_ads＞0, indicating that adsorption was driven by an increase in entropy. The order for the absolute values of△G_ads was inorganic soil colloids＞organic soil colloids, red soil colloids＞brown soil colloids, goethite＞kaolinite＞montrnoriilonite. These implied that the tendency of enzyme adsorption was stronger on inorganic and red soil colloids than that on organic and brown soil colloids. Electrostatic repulsion and hydrophobic interaction were considered to be the main forces contributing to the higher adsorption heats of enzyme on organic colloids than on inorganic colloids.2. Fluorescence emission spectra of free enzyme and enzyme desorbed from organic brown soil colloids, red soil colloids, goethite and kaolinite (pH 7.0) showed the same maximum intrinsic fluorescence intensity at 336 nm, while enzyme desorbed from inorganic brown soil colloids, montrnorillonite and kaolinite (pH 5.5) produced a blue-shift by 3 nm for the maximum intrinsic fluorescence peak. Tryptophan (Trp) and tyrosine (Tyr) residues are naturally intrinsic fluorophores in protein molecule. The changes of fluorescence intensity and peak position indicated that the polarity of microenvironment surrounding the Trp and Tyr residues were altered.3. The adsorption/desorption process increased the content ofα-helix andβ-sheet, suggesting the changes of enzyme conformation from random to ordered structure. More ordered structure was remained on organic brown soil colloid than that on inorganic brown soil colloid, whereas inorganic red soil colloids favored the preservation of ordered structure. The random form was much more transformed intoα-helix for the enzyme immobilized by montmorillonite and goethite. The conformation of enzyme interacted with kaolinite transformed from random andβ-turn toβ-sheet at pH 5.5, but from random toβ-structures at pH 7.0.4. The affinities of immobilized acid phoshatase on soil colloids and clay minerals for substrate were different from that of free enzyme. The Michaelis-Menten constant K_m of kaolinite-enzyme complexes was smaller than that of free enzyme and other enzymatic complexes. Compared with free enzyme, the V_max values of immobilized enzyme decreased and the orders were: red soil＞brown soil, organic brown soil colloids＞inorganic brown soil colloids, inorganic red soil colloids＞organic red soil colloids. The heats released by free enzyme was the greatest (-13.8 Jg^-1), followed by enzyme on red soil colloids (-10.3～-11.0 Jg^-1) and brown soil colloids (-6.8～-9.9 Jg^-1). The heats produced from inorganic soil colloids-enzyme complexes (-9.9～-11.0 Jg^-1) were higher than that from organic soil colloids-enzyme complexes (-6.8～-10.3 Jg^-1). For goethite-enzyme complex, the V_max was 13.68μmol mg^-1 min^-1, which was higher than those of montrnorillonite- and kaolinite- enzyme complexes (0.96～9.52μmol mg^-1 min^-1). The heats released by goethite-enzyme complex was -13.6 Jg^-1, while only -4.9～- 12.7 Jg^-1 were detected for montmorillonite- and kaolinite-enzyme complexes.5. Acid phosphatase immobilized by organic brown soil colloids was more resistant to proteinase K than that by inorganic brown soil colloids. In contrast, the proteolytic stability was higher on inorganic red soil colloids than that on organic red soil colloids. Organic matter and iron oxides were considered to facilitate the activity of immobilized enzymes. Phosphatase adsorbed by goethite showed the highest proteolytic stability, followed by montmorillonite- and kaolinite- enzyme. More protection for phosphatase on kaolinite against hydrolysis by proteinase K was observed in acidic environments.6. To our knowledge, this is the first paper providing the proteolytic thermodynamic information on clay mineral-immobilized enzymes using microcalorimetry. The results were consistent with the proteolytic stability by the measurement of enzyme activity. The sequence of resistance to hydrolysis was goethite-enzyme (-267.5μJ)＞montrnorillonite-enzyme (-338.9μJ)＞kaolinite- enzyme(-430.1μJ).