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| Cloning of Stenotrophomonas maltophilia Keratinase Gene (KerF) and Its Expression in Escherichia coli |
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Abstract To investigate the mechanisms of feather degradation by Stenotrophomonas maltophilia YHYJ-1, the keratinase gene (KerF) which included complete sequence of 1 981 bp and a 1 734 bp open reading frame encoded 580 amino acid was cloned from S. maltophilia (GenBank Accession No. HM590650) and ligated into pET28a(+) expression vector. A recombinant plasmid pET28-KerF was constructed and transformed into Escherichia coli BL21(DE3), and finally the recombinant strain was obtained. Recombinant KerF with a molecular mass of 50 kD was purified to electrophoretical homogeneity after nickel affinity chromatography and had an optimal pH and temperature of 9.0 and 50℃, respectively. It was strongly inhibited by Mn2+, Co2+, Mg2+, Ni+, Ba2+, Zn2+, Ca2+ , Fe3+ and phenylmethanesulfonyl fluoride (PMSF), but stimulated by ethylene diamine tetraacetic acid (EDTA). These results indicated that keratinase KerF gene from S. maltophilia is expressed successfully and the KerF is considered to be a alkaline serine protease.
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Received: 27 July 2010
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