Improving the catalytic activity of a detergent‐compatible serine protease by rational design

Abstract

Serine proteases are among the most important biological additives in various industries such as detergents, leather, animal feed and food. A serine protease gene, Fgapt4, from Fusarium graminearum 2697 was identified, cloned and expressed in Pichia pastoris. The optimal pH and temperature of FgAPT4 were 8.5 and 40°C, respectively. The relative activity was >30% even at 10°C. It had a wide range of pH stability (4.0-12.0) and detergent compatibility. To improve the catalytic activity, a strategy combining molecular docking and evolutionary analysis was adopted. Twelve amino acid residue sites and three loops (A, B and C) were selected as potential hot spots that might play critical roles in the enzyme's functional properties. Twentyeight mutants targeting changes in individual sites or loops were designed, and mutations with good performance were combined. The best mutant was FgAPT4-M3 (Q70N/D142S/A143S/loop C). The specific activity and catalytic efficiency of FgAPT4-M3 increased by 1.6 (1008.5 vs. 385.9 U/mg) and 2.2fold (3565.1 vs. 1106.3/s/mM), respectively. Computational analyses showed that the greater flexibility of the substrate pocket may be responsible for the increased catalytic activity. In addition, its application in detergents indicated that FgAPT4-M3 has great potential in washing.

CRISPR/Cas9 System-Mediated Multi-copy Expression of an Alkaline Serine Protease in Aspergillus niger for the Production of XOD-Inhibitory Peptides

“…4 AoproS8 displayed an optimal pH at pH 9.0 (Figure 4A), which is the same as that expressed in P. pastoris. 12 The optimal pH is little higher than that in many reported alkaline serine proteases from fungi, such as Thermoascus aurantiacus (pH 8.0) and Fusarium graminearum (pH 8.5) 26,27 but lower than that of A. niger (pH 10.0) and Penicillium chrysogenium (pH 10.0). 28,29 Moreover, AoproS8 showed stability over the pH range of 5.0−8.5 (Figure 4B), which is wider than that expressed in P. pastoris (pH 7.0− 9.0).…”

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides

“…Ta proA1 was purified to homogeneity by QSSF with a recovery yield of 52.8% (Table S2 ). Compared with other proteases, the purification efficiency of Ta proA1 was higher than that of the aspartic proteases RmproA (16.8%) (Sun et al 2018 ) and RmproB (18.8%) (Wang et al 2021 ) but lower than that of the serine protease FgAPT4 (59.6%) (Wang et al 2023 ) and the aspartic protease Apa1 (72%) (Wei et al 2023 ). Generally, aspartic proteases have optimal pH and pH stability under acidic conditions (Table S4 ).…”

Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

“…The proteolytic activity of the protease Th APT3 and its mutants was determined based on the report by Wang et al, without modification . Casein sodium salt was used as a substrate.…”

“…The proteolytic activity of the protease ThAPT3 and its mutants was determined based on the report by Wang et al, without modification. 15 Casein sodium salt was used as a substrate. One unit (U) of serine protease activity was defined as the amount of enzyme that released 1 μg/min of tyrosine under standard conditions (pH 9.5, 60 or 65 °C, and 20 min).…”

“…MD simulations were performed using the AMBER 14 software package for 30 ns at 333 K under an ff99SB force field. These parameters were set according to Wang et al 15 DSSP 3.0 was used to identify and define the secondary structures.…”