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

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 degrees C, respectively. The relative activity was > 30% even at 10 degrees 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 2.2 fold (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.