A Metalloaminopeptidase from Flavobacterium breve: Characterization and Molecular Cloning | Zendy

Sreekumar Othumpangat | Zendy; Kiyoshi Hayashi | Zendy

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A Metalloaminopeptidase from Flavobacterium breve: Characterization and Molecular Cloning

Author(s) -

Sreekumar Othumpangat,

Kiyoshi Hayashi

Publication year - 2019

Publication title -

science documents

Language(s) - English

Resource type - Journals

eISSN - 2574-1721

pISSN - 2573-1882

DOI - 10.32954/synsdocs.2019.001.04

Subject(s) - aminopeptidase , flavobacterium , fast protein liquid chromatography , amino acid , leucyl aminopeptidase , enzyme , enzyme kinetics , biochemistry , microbiology and biotechnology , biology , molecular mass , chemistry , chromatography , leucine , bacteria , genetics , active site , pseudomonas

Aminopeptidase from Flavobacterium breve, was purified by a three step FPLC column chromatography to homogeneity from theculture filtrate. The aminopeptidase gene was cloned by using TAIL-PCR technique. The gene encodes for a polypeptide composed of497 amino acids with a theoretical molecular weight of 58 kDa. SDS-PAGE detection revealed that the protein is of 52 kDa. Thenative enzyme showed high affinity to Leu-pNA (km 0.0515 mM), and kcat /km of 88.8 s-1mM-1. The enzyme had an optimum pH 7.5and was stable from pH 6 to 9. The purified aminopeptidase was stable up to 60 oC and the optimum temperature for the maximumactivity was at 70 oC. The amino acid sequence showed 47% identity to aminopeptidase of Aeromonas caviae (family M14), a Zn2+dependent metallozyme.

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