Modification of β‐lactoglobulin by microbial transglutaminase under high hydrostatic pressure: Localization of reactive glutamine residues

Abstract

Microbial transglutaminase (mTG) mediated modification of bovine beta-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 degrees 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 degrees 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.

Cross-linking of Hen Egg White Lysozyme by Microbial Transglutaminase under High Hydrostatic Pressure: Localization of Reactive Amino Acid Side Chains

“…HEWL was incubated at a concentration of 20 mg/mL in 50 mM Tris-HCl buffer (pH 7.5) in the absence or presence of mTG (40 U/g HEWL) for 30 min at 40 °C at atmospheric or high hydrostatic pressure (600 MPa) as described in ref . Pressure treatment was performed with a high-pressure plant (Bernd Dieckers, Willich, Germany) using a mixture of water/ethylene glycol (50:50 v/v) for pressure transduction.…”

“…Lyophilized HEWL samples (0.1% w/v) were digested with TPCK-treated trypsin in 50 mM Tris-HCl, pH 7.5, at an enzyme/substrate ratio of 1:100 at 37 °C for 6 h. Afterward, reduction was performed with DTT (final concentration of 1%, w/v) incubated overnight at 6 °C. Tryptic peptides were analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization−time-of-flight mass spectrometry (ESI-TOF-MS) using equipment and general conditions according to ref . A Zorbax 300 Extend-C18 column (Agilent, Waldbronn, Germany) was used.…”

“…This observation was traced back to the fact that the active site of the enzyme, which is surrounded by β strands, is nearly incompressible . The whey protein β-lactoglobulin can be converted to oligomers by mTG under high hydrostatic pressure in the range from 300 to 500 MPa, indicating that the simultaneous pressure/enzyme treatment may open new fields for the functionalization of food proteins , .…”

Effect of polymerization with transglutaminase on in vitro digestion and antigenicity of β-lactoglobulin

“…In its native state, b-Lg has no TGreactive glutamine residues (Partschefeld, Richter, Schwarzenbolz, & Henle, 2007), and therefore needs to be denatured either by high hydrostatic pressure (Lauber, Krause, Klostermeyer, & Henle, 2003), heat, or by chemical denaturation (i.e., cleavage of disulphide bonds) to be susceptible to crosslinking by TG (Eissa et al, 2006).…”

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety

“…However, after incubation of the protein with mTG for 1 hr at 40 • C at 400 MPa, four out of nine glutamine residues were identified as accessible for the mTG-catalyzed reaction. This indicated partial unfolding of bLGL under pressure and exposure of previously inaccessible glutamine residues (Partschefeld et al, 2007). Similarly, hen egg white lysozyme (HEWL) does not represent a substrate for mTG at atmospheric pressure.…”