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Characterization of a bifunctional aminoacylase/carboxypeptidase from radioresistant bacterium Deinococcus radiodurans R1

Paper ID Volume ID Publish Year Pages File Format Full-Text
25385 43571 2007 13 PDF Available
Title
Characterization of a bifunctional aminoacylase/carboxypeptidase from radioresistant bacterium Deinococcus radiodurans R1
Abstract

The gene encoding a Deinococcus radiodurans R1 bifunctional aminoacylase/carboxypeptidase (DR_ACY/CP) was amplified by polymerase chain reaction and cloned into pQE-30 to generate pQE-DRAC. The cloned gene consists of an open reading frame of 1197 bp encoding a protein with a molecular mass of 42,729 Da. The predicted amino acid sequence shows high homology with those of Geobacillus kaustophilus aminoacylase, Geobacillus stearothermophilus aminoacylase, Pyrococcus horikoshii carboxypeptidase/aminoacylase and Thermoanaerobacter tengcongensis aminoacylase/carboxypeptidase. The expressed enzyme was purified from the crude extract of IPTG-induced Escherichia coli M15 (pQE-DRAC) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified enzyme was determined to be 43 kDa by SDS-PAGE. Maximal aminoacylase activity with N-acetyl-methionine as the substrate occurred at pH 8.0 and 40 °C in the sodium phosphate buffer. The aminoacylase activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations, such as Co2+, Mn2+ and Ni2+. The purified enzyme had broad specificity toward N-acetylated l-amino acids as well as N-CBZ-peptides. Carboxypeptidase activity of DR_ACY/CP to N-CBZ-Gly-Ala exhibited Km and kcat values of 4.3 mM and 28 s−1, respectively. The enzyme also had activity toward the cell wall-related substrates, d-Ala-Gly, d-Ala-Gly-Gly and l-Orn-l-Ala.

Keywords
Aminoacylase; Carboxypeptidase; Deinococcus radiodurans; Gene expression; Biochemical characterization
First Page Preview
Characterization of a bifunctional aminoacylase/carboxypeptidase from radioresistant bacterium Deinococcus radiodurans R1
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 2, 1 February 2007, Pages 322–334
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering