Enhancing The Stability Of Thermophilic Protease Pyrolysin Through Rational Design

Pyrolysin was a cell envelope-associated protease originating from the hyperthermophilic archaeon Pyrococcus furiosus.As a typical hyperthermophilic protease,pyrolysin had perfect stability at high temperature,and its optimum reaction temperature was 115?.The half-life of the recombinant enzyme obtained from heterologous expression in E.coli was about 12 h under the condition of 95 ?,and the half-life of pyrolysin isolated from the original strains P.furiosus was about 4 h at the temperature of 100?.Pyrolysin has such excellent high temperature stability that it not only plays important physiological functions in P.furiosus,but also has broad application prospects in industrial production.In terms of proteases,stability is crucial for both evolutionary and industrial applications of them.Researchers often need to find a better stability of the proteases or modification of existing proteases to meet the needs of industrial production.There are usually two ways to improve the stability of proteins:directed evolution and rational design,and rational design include empirical rational design and system rational design.Empirical rational design refers to mutants construction of residues near the active sites and imitation the experience of transform of other thermophilic proteases;systematic rational design is to use some softwares to predict the stability of proteins,which can be used to predict the effect of point mutation on the stability of proteases.Finally,the prediction results are verified by experiments.In order to further improve the stability of pyrolysin,this study has used empirical rational design and systematic rational design.We have successfully constructed the mutants with improved stability such as mutants N311P,1338A,N907V and N907M,etc.Then we combined these mutants to obtain a further stable mutant N311P/N907M.Meanwhile,we combined the mutant N311P/N907M with mutant D818N/D820N and R249E/D818N/D820N to obtain 3 stable mutants,the latter two of which were previously constructed in our labotary,the 3 mutants includes N311P/N907M/R249E,N311P/N907M/D818N/D820N and N311P/N907M/R249E/D818N/D820N.Next,we systematically compared the stability of these mutants by comparing the residual activity after heat preservation at 95 ? and measuring the stability above 100?.Moreover,we also compared the stability of these mutants with chemical reagents urea,SDS and EDTA.The results showed that:the three mutants had good stability in the conditions mentioned above,and mutant N311P/N907M/R249E/D818N/D820N was the best,this mutant has improved the residual activity after heat preservation at 95 ? for 12 h from about 50%of the WT to 90%,and this result is also significantly higher than that of the mutant R249E/D818N/D820N,which was constructed in our laboratory before,the residual activity is 76%.This study has provided an experimental basis for the modification of other proteases,especially hyperthermophilic proteases,and laid the foundation for the industrial application of protease pyrolysin.