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Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems
Author(s) -
Wang JengJie,
Swaisgood Harold E.,
Shih Jason C. H.
Publication year - 2002
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10485
Subject(s) - bacillus subtilis , biotinylation , escherichia coli , chemistry , fusion protein , keratinase , chromatography , streptavidin , fusion , inclusion bodies , biochemistry , recombinant dna , biotin , biology , enzyme , bacteria , genetics , linguistics , philosophy , gene
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.