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Modification of β‐lactoglobulin by microbial transglutaminase under high hydrostatic pressure: Localization of reactive glutamine residues
Author(s) -
Partschefeld Claudia,
Richter Sandy,
Schwarzenbolz Uwe,
Henle Thomas
Publication year - 2007
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200600226
Subject(s) - glutamine , chemistry , tissue transglutaminase , hydrostatic pressure , substrate (aquarium) , enzyme , trypsin , biochemistry , chromatography , amino acid , biology , ecology , physics , thermodynamics
Microbial transglutaminase (mTG) mediated modification of bovine β‐lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP‐HPLC with ESI‐TOF‐MS. For bLG treated with mTG at ambient pressure for 1 h at 40°C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40°C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.