title: Origin of D-amino acids detected in the acid hydrolysates of purified Escherichia coli β-galactosidase.
abstract: In previous report, we detected D-amino acids in the acid hydrolysates of purified recombinant β-galactosidase. Here, we employed a deuterium-hydrogen exchange method to discriminate innate D-amino acids from those generated during hydrolytic incubation. After hydrolysis of β-galactosidase in DCl/D2O, amino acids were derivatized with NBD-F and separated on a reverse-phase column, followed by liquid chromatography-tandem mass spectrometry equipped with a chiral column. Our results show an absence of innate D-amino acid residues in the protein and suggest that the protein undergoes isomerization during a very early stage of hydrolytic incubation.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Masae
authors.initials: M
authors.lastname: Sekine
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hidaka
authors.affiliation: Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address: hommah@pharm.kitasato-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Homma
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: amasaki@mail.ecc.u-tokyo.ac.jp.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Masaki
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D011492:Protein Hydrolysates / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D011492:Protein Hydrolysates; D001616:beta-Galactosidase
keywords: Acidic hydrolysis; Escherichia coli; Isomerization; d-Amino acid; β-Galactosidase