Improving common vetch protein isolate flavor through glutaminase-mediated deamidation.

Common vetch protein, similar to pea protein, offers valuable qualities like being non-GMO, hypoallergenic, and nutritious. However, its strong beany flavor hinders consumer acceptance. This study explores enzymatic deamidation using glutaminase to address this issue. GC-MS analysis identified 54 volatile compounds in the raw material protein, with 2-pentylfuran, hexanal, and several nonenals contributing the most to the undesirable aroma. Principal component analysis (PCA) confirmed the effectiveness of glutaminase deamidation in removing these off-flavors. The study further reveals that deamidation alters the protein's secondary structure, with an increase in α - helix structure and a decrease in ß - sheet structure. The surface hydrophobicity increased from 587.33 ± 2.63 to 1855.63 ± 3.91 exposing hydrophobic clusters that bind flavor compounds. This disruption weakens the interactions that trap these undesirable flavors, ultimately leading to their release and a more pleasant aroma. These findings provide valuable insights for enzymatic deodorization of not only common vetch protein but also pea protein.

Purified protein glutaminase from Chryseobacterium proteolyticum enhances the properties of wheat gluten.

Protein glutaminase (PG), originating from Chryseobacterium proteolyticum, can catalyze the deamidation of glutamine residues in plant proteins into glutamic acid, thus enhancing its functional properties. However, the low yield of PG limits its industrial production. In this study, the yield of PG in C. proteolyticum TM1040 increased by 121 %, up to 7.30 U/mL in a 15 L fermenter after medium optimization. Subsequently, purified PG was obtained by cation exchange chromatography (CEX) coupled with hydrophobic interaction chromatography (HIC). The degree of deamidation (DD) of wheat gluten after purified PG deamidation was 87.11 %, which is superior to chemical deamidation in safety and DD. The emulsifying and foaming properties of deamidated wheat gluten were 2.67 and 18.86 times higher, and the water- and oil-holding properties were 4.23 and 18.77 times higher, respectively. The deamidated wheat gluten with enhanced functional properties was used to improve the flavor and texture in baking cakes.

Enhancing Solubility and Reducing Thermal Aggregation in Pea Proteins through Protein Glutaminase-Mediated Deamidation.

The limited solubility and stability of pea proteins hinder their utilization in liquid formulations. In this study, protein glutaminase (PG) was employed to modify pea protein isolates (PPIs) and obtain deamidated PPI with varying degrees of deamidation (DD, 10-25%). The solubility and thermal stability of these deamidated PPI samples were assessed, and a comprehensive analysis, including SDS-PAGE, zeta potential, FTIR, surface hydrophobicity, and intrinsic fluorescence, was conducted to elucidate the mechanism behind the improvement in their functional properties. The results reveal that PG modification greatly enhances the solubility and heat stability of PPI, with the most notable improvements observed at higher DD (>20%). PG modification increases the net charge of PPI, leading to the unfolding and extension of the protein structures, thus exposing more hydrophobic groups. These structural changes are particularly pronounced when DD exceeds 20%. This increased electrostatic repulsion between carboxyl groups would promote protein unfolding, enhancing interactions with water and hindering the aggregation of unfolded protein in the presence of salts at elevated temperatures (supported by high-performance size exclusion chromatography and transmission electron microscopy). Accordingly, PG-mediated deamidation shows promise in enhancing the functional properties of PPI.

L-Glutamine-, peptidyl- and protein-glutaminases: structural features and applications in the food industry.

L-Glutaminases are enzymes that catalyze the cleavage of the gamma-amido bond of L-glutamine residues, producing ammonia and L-glutamate. These enzymes have several applications in food and pharmaceutical industries. However, the L-glutaminases that hydrolyze free L-glutamine (L-glutamine glutaminases, EC 3.5.1.2) have different structures and properties with respect to the L-glutaminases that hydrolyze the same amino acid covalently bound in peptides (peptidyl glutaminases, EC 3.5.1.43) and proteins (protein-glutamine glutaminase, EC 3.5.1.44). In the food industry, L-glutamine glutaminases are applied to enhance the flavor of foods, whereas protein glutaminases are useful to improve the functional properties of proteins. This review will focus on structural backgrounds and differences between these enzymes, the methodology available to measure the activity as well as strengths and limitations. Production methods, applications, and challenges in the food industry will be also discussed. This review will provide useful information to search and identify the suitable L-glutaminase that best fits to the intended application.

Identification of Deamidated Peptides in Cytokine-Exposed MIN6 Cells through LC-MS/MS Using a Shortened Digestion Time and Inspection of MS2 Spectra.

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 ß-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed ß-cell proteins and can be involved in the autoimmune process in T1D.

Enhancing the Usability of Pea Protein Isolate in Food Applications through Modifying Its Structural and Sensory Properties via Deamidation by Glutaminase.

This study aimed to demonstrate the feasibility of improving the properties of pea protein isolate (PPI) related to food applications via deamidation with glutaminase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FT-IR) profiling revealed that the current glutaminase treatment did not change the basic protein subunit composition. However, it allowed a certain extent of protein unfolding and conformational reorganization to generate more flexible and extended proteins with reduced average particle size and more hydrophobic groups exposed. The underlying mechanisms might include the reduction of ß-sheets and antiparallel ß-sheets and the increase of the ß-turn structure. Moreover, the treatment time was of importance. A 12 h treatment was generally better than a 24 h treatment, and PPI treated with glutaminase at 50 °C for 12 h to a degree of deamidation of 56.1% exhibited significantly improved solubility, homogeneity, dispersibility, and suspendability with reduced beany flavor, grittiness, and lumpiness (compared to those of the untreated PPI). Thus, the glutaminase treatment offers a promising approach for enhancing the usability and applicability of pea proteins.