The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for alpha-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K(M) and E(a) values in silica-bound alpha-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.

The capability of binding enzymes adsorptively to unmodified and silanized silica and glass as well as modified polystyrene carriers was studied for alpha-amylase, beta-amylase, and alpha-chymotrypsin. In most cases a high percentage of protein was bound very firmly under considerable loss of activity. The leakage of protein from the carriers was studied by measuring the intrinsic protein fluorescence on beta-amylase adsorptively bound to aminopropyl silica, aminomethyl, and hexadecylaminomethyl polystyrene. It was compared with the leakage of beta-amylase covalently bound to the same carriers via glutaraldehyde, trichloro-triazine, or benzoquinone. In the absence and in the presence of substrate, at 25 and at 60 degrees C, the leakage rates of the adsorptively bound enzymes were not higher than in the covalently bound systems. The poorest binding stability was found in benzoquinone-coupled beta-amylase derivatives. It is even reduced at higher temperatures, whereas the temperature did not show any remarkable influence on the leakage of the other derivatives. In adsorptively as well as in all the covalently bound systems, the presence of substrate did not promote the protein leakage.

The formation of alkyl beta-D-fructofuranosides by invertase from sucrose in aqueous solutions of methanol, ethanol, or n-propanol is studied for the dependence on alcohol and invertase concentrations as well as on reaction time. The yield of alkyl beta-D-fructosides is shown to be controlled by three competitive reactions: the alcoholysis of sucrose, the hydrolysis of sucrose, and the hydrolysis of alkyl beta-D-fructosides. Both the conversion rate of sucrose and the fraction of alkyl beta-D-fructosides in the product mixture are dependent on the chain length of the alcohols. They decrease in the sequence methanol > ethanol > n-propanol. Alkyl beta-D-fructosides are also formed by invertase starting from alcoholic solutions of fructose.

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