Rational Design of Pepsin for Enhanced Thermostability via Exploiting the Guide of Structural Weakness on Stability

Abstract

Enzyme thermostability is an important parameter for estimating its industrial value. However, most naturally produced enzymes are incapable of meeting the industrial thermostability requirements. Software programs can be utilized to predict protein thermostability. Despite the fast-growing number of programs designed for this purpose; few provide reliable applicability because they do not account for thermodynamic weaknesses. Aspartic proteases are widely used in industrial processing; however, their thermostability is not able to meet the large-scale production requirements. In this study, through analyzing structural characteristics and modifying thermostability using prediction software programs, we improved the thermostability of pepsin, a representative aspartic protease. Based on the structural characteristics of pepsin and the experimental results of mutations predicted by several energy-based prediction software programs, it was found that the majority of pepsin’s thermodynamic weaknesses lie on its flexible regions on the surface. Using computational design, mutations were made based on the predicted sites of thermodynamic weakness. As a result, the half-lives of mutants D52N and S129A at 70°C were increased by 200.0 and 66.3%, respectively. Our work demonstrated that in the effort of improving protein thermostability, identification of structural weaknesses with the help of computational design, could efficiently improve the accuracy of protein rational design.

Methods for improving meat protein digestibility in older adults

“…Pepsin requires an optimum temperature of 37 °C, similar to body temperature, and an optimum pH of 1.8 (Fig. 1) [34]. The enzyme exhibits strong proteolytic activity, preferentially hydrolyzing peptide bonds involving the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) at pH > 2.0 [35].…”

Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

“…For instance, an engineered version of xylanase from Gloeophyllum trabeum was demonstrated to have an improved temperature optimum up to 80 o C and enhanced stability at low pH (2 -7.5) compared to the wild-type enzyme enabling applications in feed and brewing industries 20 . In another study, mutations D52N and S129A were introduced into pepsin based on G calculations and the half-life of these mutants was increased significantly 21 . However, so far there have been no reports on improving the thermostability of Cas effectors for diagnostic purposes.…”