title: Biochemical, kinetic, and in silico characterization of DING protein purified from probiotic lactic acid bacteria Pediococcus acidilactici NCDC 252.
abstract: DING proteins are intriguing proteins characterized by conserved N-terminal sequence. In spite of unusually high sequence conservation even between distantly related species, DING proteins exhibit outstanding functional diversity. An extracellular caseinolytic alkaline enzyme was purified to homogeneity from a probiotic lactic acid bacteria Pediococcus acidilactici NCDC 252 using a simple procedure involving ammonium sulphate precipitation and gel filtration chromatography. This was purified 45.72-fold with a yield and specific activity of 43.5 % and 250 U/mg, respectively. The calculated molecular weight was 38.7 and 38.9 kDa by MALDI and SDS-PAGE, respectively, and pI was 7.77. The enzyme exhibited optimal activity at pH 8.0 and 40 °C. It was considerably stable up to pH 12. For casein, the enzyme had K m of 20 μM with V max of 26 U/ml. The enzyme was resistant to organic solvents but sensitive to DTNB and EDTA that confirmed it as thiol protein with involvement of metal ions in catalysis. Its tryptic peptide fragments showed 95 % similarity with eukaryotic DING, i.e., human phosphate binding protein (HPBP). Homology-based structure evaluation using HBPB as template revealed both to be structurally conserved and also possessing conserved phosphate binding motifs.
authors.affiliation: Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Attri
authors.forename: Tejinder P
authors.initials: TP
authors.lastname: Khaket
authors.forename: Drukshakshi
authors.initials: D
authors.lastname: Jodha
authors.forename: Jasbir
authors.initials: J
authors.lastname: Singh
authors.forename: Suman
authors.initials: S
authors.lastname: Dhanda
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000096:biosynthesis*; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010376:Pediococcus / Q000201:enzymology / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010802:Phylogeny; D019936:Probiotics / Q000378:metabolism*; D011487:Protein Conformation; D012997:Solvents / Q000494:pharmacology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008670:Metals; D012997:Solvents; D019344:Lactic Acid; D010447:Peptide Hydrolases