A Rational Design For Improving The Pepsin Resistance Of Cellulase E4

When feeding enzymes are consumed by livestock and poultry,the stomach is the first organ to come in contact with those enzymes.As a result,the feeding enzymes will face two major hazards in the stomach:acidolysis and enzymatic hydrolysis.Therefore,it is very important to improve the tolerance of feeding enzymes to the gastric juice environment.We have been preliminarily established a rational design strategy of improving pepsin resistance for?-amylase isolated from Aspergillus based on the evaluation of the transition complex and molecular structure.Is this rational design strategy universal,and still effective for aglycosylated proteins?Due to the uncertainty of the degree of protein glycosylation.Therefore,a glycosylated cellulase E4 isolated from Thermonospora fusca,was selected as the study object.And we did a rational design to cellulase E4.Those mutants and wild type were expressed in Escherichia coli and Pichia pastoris,respectively,and analyzed the pepsin resistance of the aglycosylated cellulases and glycosylated cellulases.Results:?1?Designed candidate mutants with pepsin resistanceE4^L691C/L800R691C/L800R based on the evaluation of transition complex and molecular structure.?2?Expression and purification of aglycosylated wild-type and mutants proteins in E.coli.Those proteins were digested by simulated gastric juice for one hour.The result show that the half-life of E4^WTT is 1min.While the half-life of E4^L691C/L800R could last to 60min.?3?Expression and purification of glycosylated E4^WT and E4 ^L691C/L800R proteins in Pichia pastoris.After being digested by simulated gastric juice for one hour,the half-life of E4^WT is 1min,and the half-life of E4^L691C/L800R is more than 60min.?4?The enzymatic properties of E4^WT and E4^L691C/L800R revealed that the Km of aglycosylated E4^WT and aglycosylated E4^L691C/L800R691C/L800R are 0.01499 mg/mL,0.01488 mg/mL;the Kcat value were25.17s^-1,23.36 s^-1.The Km of glycosylated E4^WT and glycosylated E4^L691C/L800R691C/L800R are0.04099 mg/mL,0.04026 mg/mL;the Kcat value are 30.08 s^-1,26.13 s^-1.The optimum temperature of all of proteins are about 60°C,and the optimum pH are5.0-6.0.Those proteins can store in a buffer of pH 4.0-8.0 or at a temperature of30°C-40°C for a certain period of time,and maintain more than 80%of enzyme activity.Conclusions:?1?Obtained cellulase E4^L691C/L800R691C/L800R through rational design,its pepsin resistance has a significantly improved.?2?The rational design strategy based on the evaluation of transition complex and molecuar structure are suitable for the improvement of the pepsin resistance of cellulase E4.?3?Molecular design using aglycosylated celluase E4 protein three-dimensional structure still apply to improve pepsin resistance of glycosylated proteins?4?The cellulase E4^L691C/L800R has the similar enzymatic properties with E4^WT,such as the optimum temperature and pH,temperature stability,pH stability.?5?Glycosylation weakens the E4^WTT and E4^L691C/L800R affinity with the substrate,but doesn't reduce the ability to decompose substrates.