Proline iminopeptidase from Bacillus megaterium: purification and characterization

Journal of Biochemistry
Tadashi YoshimotoD Tsuru

Abstract

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.

Citations

Sep 16, 2016·World Journal of Microbiology & Biotechnology·Hongyu YangYaping Tian
Jan 7, 2003·Applied and Environmental Microbiology·Tomás BolumarFidel Toldrá
Nov 19, 2019·Journal of Experimental Botany·Abi S GhifariMonika W Murcha
Jul 1, 1996·FEMS Microbiology Reviews·T Gonzales, J Robert-Baudouy
Sep 14, 2020·The Plant Journal : for Cell and Molecular Biology·Abi S GhifariMonika W Murcha
May 23, 1998·The International Journal of Biochemistry & Cell Biology·D F Cunningham, B O'Connor
May 1, 2010·Biochimie·Neil D Rawlings

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