Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

    Authors

    P. R. Salamone;R. J. Wodzinski

    Comments

    Authors: contact us about adding a copy of your work at STARS@ucf.edu

    Abbreviated Journal Title

    Appl. Microbiol. Biotechnol.

    Keywords

    SODIUM DODECYL-SULFATE; ERWINIA-CHRYSANTHEMI; ESCHERICHIA-COLI; GEL-ELECTROPHORESIS; CRYSTAL-STRUCTURE; SIGNAL PEPTIDE; PROTEASE; GENE; CLONING; SECRETION; Biotechnology & Applied Microbiology

    Abstract

    The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 mu g/ml) and not inhibited by antipain dihydrochloride (120 mu g/ml), aprotinin (4 mu g/ml), bestatin (80 mu g/ml), chymostatin (50 mu g/ml), E-64 (20 mu g/ml), leupeptin (4 mu g/ml), Pefabloc SC (2000 mu g/ml), pepstatin (4 mu g/ml), phosphoramidon (660 mu g/ml), or phenylmethylsulfonyl fluoride (400 mu g/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.

    Journal Title

    Applied Microbiology and Biotechnology

    Volume

    48

    Issue/Number

    3

    Publication Date

    1-1-1997

    Document Type

    Article

    Language

    English

    First Page

    317

    Last Page

    324

    WOS Identifier

    WOS:A1997YA95000006

    ISSN

    0175-7598

    Share

    COinS