A rational design for improving the pepsin resistance of cellulase E4 isolated from T. fusca based on the evaluation of the transition complex and molecular structure

Abstract

When feeding enzymes are consumed by livestock and poultry, the stomach is the first organ to come in contact with feeding enzymes. They will face major hazards: acidolysis and enzymatic hydrolysis. Therefore, it is very important to improve the tolerance of feed enzymes to the gastric juice such as pepsin. However, the hydrolysis of amino acids by pepsin is not specific, so improving the resistance of enzymes to pepsin is a great challenge. In this paper, we have selected cellulase E4 isolated from Thermonospora fusca as our study object, which exhibits unique properties because of its endo- and exocellulase characteristics, good synergistic property, and has great potential application value as feed addictive. So we propose a strategy to improve pepsin resistance of cellulose E4, including the following steps: 1) predicting amino acids that affect the formation of transitional complexes between pepsin and E4; 2) selection of candidate mutation sites; 3) prediction of optimal mutants. The mutant of E4L691H/L800H was predicted the best, and then we expressed and tested the characteristics of E4L691H/L800H, the results showed that the E4L691H/L800H had 25% more protein residual than the wild-type after 1-hour digestion, and the enzymatic properties were consistent with those of the wild-type. This study provides a useful molecular improvement strategy for protein pepsin resistance.