Engineering of protein glutaminase for highly efficient modification of fish myofibrillar protein through...

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp³¹, Ser⁷², Asn¹²¹, Asp⁴⁷¹, and Glu⁴⁸⁵ were crucial for maintaining PG5-myosin interaction, with Ser⁷² and Asn¹²¹ making significant energy contributions.