Open AccessPurification and Characterization of Tilapia ( Oreochromis mossambicus ) Deoxyribonuclease I
Author(s) -
Hsiao YiMin,
Ho HengChien,
Wang WenYi,
Tam Ming F.,
Liao TaHsiu
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.t01-2-00786.x
Subject(s) - oreochromis mossambicus , tilapia , deoxyribonuclease , fishery , characterization (materials science) , fish <actinopterygii> , biology , chemistry , zoology , biochemistry , dna , materials science , nanotechnology
DNase I of tilapia ( Oreochromis mossambicus ) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg 2+ as activator. The Ca 2+ /Mg 2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55°C, but is not inactivated by trypsin or 2‐mercapto‐ethanol under alkaline conditions, with or without CaCl 2 . Its isoelectric point is 6.0. The 258‐amino‐acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys→Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar‐chain length. The major form has a molecular mass of 30914 Da. A 1061‐bp nucleotide sequence for the cDNA of tilapia DNase I, obtained by gene cloning and DNA sequencing, contains an ORF coding for a putative 26‐residue transmembrane peptide and the mature DNase I polypeptide.