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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 421–429, 2003.