IMMOBILIZATION-STABILIZATION OF KERASE, A SERINE-PROTEASE FROM STREPTOMYCES-FRADIAE, BY COVALENT ATTACHMENT TO POROUS-GLASS

Kerase, a serine protease from Streptomyces fradiae, was immobilized o n porous glass by covalent attachment of the enzyme through its amino groups. Chemical modification studies have shown that the amino groups can be used to establish covalent attachment of this protease to poro us glass, modification of four lysine residues (44.4% of the accessibl e amino groups) resulting in a 6.5% loss of enzymatic activity. After immobilization, the optimal reaction pH range changed from 7.5-8.5 to 9-10, the protease being stable over a broad pH range of 6-12, while t he soluble protease was irreversibly denatured in the alkaline pH rang e (pH > 8), Moreover, the optimal reaction temperature was changed fro m 55 to 65 degrees C, showing higher thermal stability, Kerase immobil ized onto porous glass was stable for at least 28 days when working in a repeated-batch process of three cycles per day, with an activity lo ss of 22.1 +/- 3.1%.