| Pepsin,a typical aspartic protease,is widely used in food processing and agricultural by-product processing because of its excellent performance with respect to protein degradation.These applications include,but are not limited to,clarification of juice,improvement of the wine yield,and cheese making,feed additives and environmentally friendly high quality leather manufacturing.Owing to its extensive application and robust performance,the demand for pepsin is continually increasing.However,the production scale limited by raw materials and the lack of mutations with higher activity hinder its extensive application.Meanwhile,the poor thermostability of pepsin limits the development of its application.Therefore,it is invaluable for industrial application to promote the industrial production of pepsin and improve its activity and thermostability.In this work,the screening of heterologous expression strains with high yield,the design of improving pepsin's thermostability and activity were studied,so as to promote the its industrial application.In this study,a heterologous expression system of pepsin in Pichia pastoris was constructed and a screening protocol that involves a combination of the P.pastoris system and skim milk solid plates for recombinant strains with high activity was developed.Finally,the highest soluble yield of pepsin reported now was achieved.Subsequently,based on this efficient heterologous expression system,the rational designs of pepsin activity and thermostability were explored.In the thrmostability study,an in-depth analysis of the structural characteristics of pepsin was firstly performed,and possible thermodynamic weaknesses were predicted.By systematically testing a variety of software programs,it was found that mutations with good prediction scores were not associated with thermodynamic weakness and performed poorly during experimental verification.The major structural area of weakness in pepsin was determined to be the flexible regions on its surface.Several mutations were identified based on this structural weakness.Among them,two excellent mutants,D52N and S129A,were obtained,and their half-lives at 70°C were found to increase by 200.0%and 66.3%,respectively.This study showed that the guide of thermodynamic weakness promotes fast and accurate thermostability design using prediction software programs.Based on the structural characteristics and catalytic mechanism of aspartic proteases,the structural similarities between aspartic proteases were identified,and a novel scissor-like model was established.According to the model,the conserved domain—the antiparallel interdomain?-sheet connecting the two lobes to form a catalytic dyad—is important.Rational design was implemented based on the assumption that pepsin activity is increased by enhancing the stability of the conserved domain.The potential mutants were screened by measuring the binding free energy between the two lobes,and the catalytic properties of the mutants were determined using bovine hemoglobin.Finally,two mutants,D3R and S163R—with significantly increased activity—were identified,and their activity was increased by921 and 961 U·mg-1,respectively.Structural analysis confirmed the stability of the conserved domain of the two mutants was enhanced.According to the scissor-like model and catalytic mechanism,the nucleophilic attack of the water at the active site to initiate the catalysis is critical.Based on the hypothesis that shortening the distance between O atom of polarized water and amide C atom of cleavage peptide bond to promote nucleophilic attack,the more stable hydrogen bonding environment of the system was applied by the polarized protein-specific charge(PPC)force field.Potential mutations were achieved by O-C diatance and preliminary screened by VTAFWGKV.The catalytic properties of the excellent mutant were determined by bovine hemoglobin and the mutant S172P with activity increased by 3635 U·mg-1 was obtained.Finally,the shortening of the O-C distance in S172P system and the enhancement of Coulomb force between O and C atoms were confirmed by dynamic simulation and charge analysis under PPC force field. |
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