Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization

Abstract

Sucrose phosphorylase from Bifidobacterium adolescentis was recombinantly expressed in Escherichia coli and purified by use of a His-tag. Kinetic characterization of the enzyme revealed an optimal temperature for phosphorolytic activity of 58°C, which is surprisingly high for an enzyme from a mesophilic source. The temperature optimum could be further increased to 65°C by multipoint covalent immobilization on Sepabeads EC-HFA. The optimal immobilization conditions were determined by surface response design. The highest immobilization yield (72%) was achieved in a phosphate buffer of 0.04 mM at pH 7.2, irrespective of the temperature. The immobilized enzyme was able to retain 65% of its activity after 16 h incubation at 60°C. Furthermore, immobilization of the enzyme in the presence of its substrate sucrose, increased this value to 75%. The obtained biocatalyst should, therefore, be useful for application in carbohydrate conversions at high temperatures, as required by the industry.