Beany flavor affects sensory experience of soy protein-based food. Deamidation is an effective approach to improve the flavor of soy protein, but excessive hydrolysis has a negative effect on its molecular structure and processing property. In this study, protein-glutaminase (PG; EC 3.5.1.44) was heterologous expressed in E. coli Nissle 1917 (EcN) and used to specifically modify soy protein isolate (SPI) for improved flavor properties of textured protein. A fluorescence-based enzyme activity screening method was developed and optimized for fermentation conditions. After screening for optimal fermentation conditions based on cell fluorescence intensity, 3.01 U/mL of PG activity was achieved. SPI was treated with PG (0–24 h), and the degree of deamidation (DD) rapidly increased to 47.4% within 0–2 h. There was no significant decrease in the molecular weight of deamidated SPI (DSPI) after specific deamidation of PG within 0–10 h. The contents of main beany flavor substances (e.g., hexanal, benzaldehyde, and 2-pentylfuran) in DSPI decreased significantly with increasing DD. Compared with soy protein, moderate deamidation did not affect the processing properties and fibrous structure of DSPI in preparing high-moisture extrudates, while the structure of the extrudate decreased after a deamidation time of more than 6 h. This finding developed a safe expression system for PG and provided a promising method to address beany flavor in soy-based meat analogues produced by high moisture extrusion.