| Keratinase capable of hydrolyzing chicken feather keratin is a serine protease produced from Bacillus licheniformis PWD-1.;A biotechnology using streptavidin-biotin binding for the immobilization of proteins of interest has been developed. Fusion genes of streptavidin and a protein of interest can be genetically constructed and expressed to produce a bifunctional fusion protein. Isolation and immobilization, therefore, can be achieved in a single step from cell lysates by mixing with a biotinylated solid matrix. However, in this organism they form insoluble inclusion bodies requiring complicated renaturation procedures.;Keratinase gene (kerA) from Bacillus licheniformis PWD-1 genomic DNA and streptavidin gene (stp) from pETSA7 in E. coli was amplified by PCR. Both full ( stp) and core (stpc) strepavidin genes were fused with kerA and introduced into the pUB18-P43 vector creating pJB and pJC plasmids. Two plasmids (pJBD and pJCD) carrying the kerA-stp and kerA-stpc with modification of linker sequence were constructed and expressed. The kerA containing pro- and prepro-sequences was conjugated with stp and stpc creating plasmids pPROKSTP, pPREPROKSTP, pPROKSTPC and pPREPROKSTPC. Only kerA carrying the pro-region in the fusion protein was overexpressed intracellularly and accumulated as insoluble inclusion bodies in E. coli BL21(DE3) pLysS. Renaturation efficiency was approximately 50% for fusion proteins using optimal refolding conditions.;Immobilization of keratinase was accomplished by mixing biotinylated solid matrix in the growth medium. Compared with the E. coli system, the B. subtilis system is much simpler and gives a higher yield.;Heat stability, durability, and pH tolerance of the immobilized enzyme was improved as was observed for the chemically immobilized keratinase. Kinetic parameters of immobilized fusion protein, including Vmax, Km, and kcat , were also determined. (Abstract shortened by UMI.). |
