Expression and Characterization of Endochitinase C from<i>Serratia marcescens</i>BJL200 and Its Purification by a One-Step General Chitinase Purification Method

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  • SYNSTAD Bjørnar
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • VAAJE-KOLSTAD Gustav
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • CEDERKVIST F. Henning
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • SAUA Silje F.
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • HORN Svein J.
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • EIJSINK Vincent G. H.
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
  • SØRLIE Morten
    Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences

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  • Expression and Characterization of Endochitinase C from Serratia marcescens BJL200 and Its Purification by a One-Step General Chitinase Purification Method

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In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher kcat and 15 fold-lower Km than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-β-D-N-N′-N″-triacetylchitotrioside, while BJL200-ChiC1 was 10–15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of β-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)2 (main product), GlcNAc, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.

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