Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems.
Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.
Wang
Jeng-Jie
JJ
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695, USA.
Swaisgood
Harold E
HE
Shih
Jason C H
JC
D001412:Bacillus subtilis / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D004800:Enzymes, Immobilized / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013045:Species Specificity; D019809:Streptavidin / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D010447:Peptide Hydrolases; C035855:keratinase