Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene 3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at . Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.
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