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. Twenty-eight 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.

FIGURE 1

Schematic diagram of amino acid sequence of protease FgAPT4 in different processing stages.

FIGURE 2

Characterization of purified recombinant FgAPT4‐WT and mutant FgAPT4‐M3. (A) pH‐activity profile. (B) pH‐stability profile after incubation at 25°C for 1 h. (C) temperature‐activity profile. (D) temperature‐stability profile after incubation at different temperatures for various durations.

FIGURE 3

The design strategy of protease FgAPT4 mutants. (A) Evolutionary analysis. Column labels indicate the 40 sites that make up the substrate pocket, and the row labels show the 20 possible amino acids, in addition to a gap at that site. The colour of and number in each cell in the chart indicates the frequency of the occurrence at that amino acid residue (or a gap) at the indicated position. Cells without any annotation indicate that these amino acids had a minimal probability (<1%) of occurring at the site. The FgAPT4 amino acid sequence was shown to the left. Small grey squares indicate residues that were consistent with evolutionary trends; small red squares marked with “M” represent potential mutational hot spots in FgAPT4. (B) Details of the substrate pocket conformation of protease FgAPT4. The boundary between the outer and inner surfaces was shown as dotted line. The substrate was shown in yellow. (C) Detailed compositional information, evolutionary analysis and mutation targets for the three loops. WT, wild type.

FIGURE 4

Mutants screening and superposition effect of protease FgAPT4. (A) preliminary screening of mutants and superimposition schemes. (B) catalytic activity of key mutants in the superposition process. (C) the Michaelis–Menten plots of FgAPT4‐WT and the final generation mutant FgAPT4‐M3.

FIGURE 5

Analysis of MD simulation for FgAPT4‐WT and FgAPT4‐M3. (A) the RMSF value of MD simulations over the last 10 ns. (B) Hydrogen bond interaction and occupancy rate analysis. (C) the conformation analysis of substrate pocket for FgAPT4‐WT. (D) the conformation analysis of substrate pocket for FgAPT4‐M3.

FIGURE 6

Stability of protease FgAPT4‐WT and FgAPT4‐M3 in washing environment. (A) 10% organic solvents (B) 0.5% surfactant (C) commercial liquid and solid detergents.

FIGURE 7

The removal ability of protease FgAPT4‐M3 to various stains. CK, treatment with water; Liquid, treatment with 1% liquid detergent (heat deactivation); Solid, treatment with 7 mg/ml solid detergent (heat deactivation); Enzyme, treatment with FgAPT‐M3 (200 U/ml); L + E, treatment with 1% liquid detergent (heat deactivation) and FgAPT‐M3 (200 U/ml); S + E, treatment with 7 mg/ml solid detergent (heat deactivation) and FgAPT‐M3 (200 U/ml).