Casein is a commonly used protein in the food industry, with its related products such as beverages and desserts. However, the further application of casein is limited by its solubility and stability. This study aimed to improve the functional of casein through protein-glutaminase (PG) deamination. The deamination of casein using PG was optimized through central composite design experiments, and its impact on the structure, solubility, and stability of casein was investigated. The results demonstrate that the optimal conditions for PG deamidation were determined at pH 6.0, E/S 15 U/g, and a temperature of 45 °C. The deamidation process alters the secondary structure of casein, resulting in a decrease in α-helix structure and an increase in β-sheet structure. The modification of casein improved emulsifying activity at pH 8.0 and 10.0, respectively, while significantly enhancing the solubility from 5.0 to 6.0. Furthermore, the deamidation of casein caused an increase in zeta potential and a decrease in particle size, resulting in improved stability of the protein solution due to reduced particle aggregation. The 3% deamidated casein-based beverage with carrageenan exhibited reduced precipitation rates compared to the control after sterilization at 121 °C for 15 min. In summary, PG deamidation offers a promising method for the modification and enhancement of the functional properties, including solubility, stability, and emulsifying activity of casein, thereby expanding its use of casein in the food industry.

The lack of fat in yogurt can lead to alterations in taste and whey separation, reducing consumer acceptance. In this study, the feasibility of enhancing the quality of skim milk yogurt through a combination of transglutaminase (TG) and protein-glutaminase (PG) was investigated. The combination of TG and PG resulted in simultaneous cross-linking and deamidated of casein micelles, with PG deamidation taking priority over TG cross-linking, leading to higher solubility and lower turbidity of milk proteins compared with TG alone. When 0.06 U/mL TG and 0.03 U/mL PG were added, firmness and viscosity indexes significantly increased by 38.26 and 78.59%, respectively as compared with the control. Microscopic images revealed increased cross-linking with casein and filling of cavities by smaller sub-micelles in the combination of TG and PG treatment. Furthermore, the combination of TG and PG resolved issues of rough taste and whey separation, leading to improved overall liking. This study highlights the benefits of using both enzymes in dairy production and has important implication for future research.

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670^T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu²⁺. The optimum PG substrate was Cbz-Gln-Gly, and the K_m and V_max values were 1.68 mM and 1.41 μM mg protein⁻¹ min⁻¹, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.