title: Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains.
abstract: After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.
authors.affiliation: Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schuh
authors.forename: Uwe
authors.initials: U
authors.lastname: Schwarzenbolz
authors.forename: Thomas
authors.initials: T
authors.lastname: Henle
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002645:Chickens; D004529:Egg White / Q000032:analysis*; D005260:Female; D006874:Hydrostatic Pressure; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011503:Transglutaminases / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D011503:Transglutaminases; C421864:hen egg lysozyme; D009113:Muramidase