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1 Department of Chemical and Biochemical Engineering, Technical University of Denmark 2 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark 3 University of Southern Denmark 4 University of Copenhagen
<i>N</i>-terminal processing and substrate cleavage pattern of <i>Pectobacterium carotovorum</i> Prt1
Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, KM was 1.81 mg/mL and kcat was 1.82 × 107 U M-1. The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn2+ addition caused regain of the activity, but Zn2+addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1′ at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing. © 2014 Springer-Verlag Berlin Heidelberg.
Applied Microbiology and Biotechnology, 2014, Vol 98, Issue 24, p. 10077-10089
Potato lectin; Plant extension; Glycan microarray; Proline preference; Thermal stability; Autolytic processing; Applied Microbiology and Biotechnology; Biotechnology
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