Amorphous enzyme aggregates: stability toward heat and aqueous-organic cosolvent mixtures

Abstract

Chemical aggregates of four enzymes, i.e., polyphenol oxidase, acid phosphatase, β-glucosidase, and trypsin have been prepared by chemical cross-linking with glutaraldehyde. These aggregates were characterized for their stability under stress conditions such as high temperature and exposure to organic cosolvents. All the enzyme aggregates exhibit enhanced thermal stability. The concentration of cosolvents required to inactivate the enzymes by 50% was determined for these aggregates in acetonitrile, dimethylformamide, tetrahydrofuran, and dioxane. In most of the cases, the aggregates showed greater stability in the presence of organic cosolvents. Both in the case of free enzymes as well as the aggregates, the irreversible inactivation is less in organic cosolvents when acetonitrile and dimethylformamide are used (i.e., the solvents with polarity index greater than 5.8); however, all aggregates do not show better stability as compared to free soluble enzyme in organic cosolvent mixtures. Scanning electron micrographs (SEM) of polyphenol oxidase aggregates after exposure to 50% (v/v) dimethylformamide or tetrahydrofuran and after incubation at 60°C for 3 h were recorded. The extent of irreversible inactivation correlated well with the morphological changes observed in the case of these aggregates. The correlation of the nature of the cosolvent, loss of biological activity, and different appearance as seen by SEM may be useful in understanding the effect of nonaqueous microenvironment on enzyme structure.