Structure-guided engineering of serratiopeptidase using constant-pH molecular dynamics and site-directed mutagenesis to improve pH-dependent functional resilience.

Serratiopeptidase is a metalloprotease enzyme mainly used for its anti-inflammatory and therapeutic applications. The functional performance of serratiopeptidase is compromised by the reduced enzymatic activity at extreme pH levels especially in oral administration. To address the problem, the present research employed the structure-based rational design strategy to assess the possibility of modifying the functional performance of serratiopeptidase by introducing a point mutation, N412D, which might improve the functional performance of the metalloprotease under extreme pH levels. Constant pH molecular dynamics simulations were used as an in-silico tool for the qualitative analysis of the wild type (WT) and the mutated serratiopeptidase under variable pH environments. To perform the functional evaluation, the mutated serratiopeptidase was expressed, refolded, and purified using the Escherichia coli expression host. Retained caseinolytic activity was analyzed at different pH levels ranging from 3.0 to 11.0 to evaluate the functional property of serratiopeptidase. It has been noted that the functional activity of mutated serratiopeptidase is slightly changed with respect to an optimal pH, coupled with high retained caseinolytic activity at variable pH levels. The highest improvement in the retained caseinolytic activity was observed under alkaline pH. A small improvement in the retained caseinolytic activity was also observed under acidic pH levels. This research demonstrated the importance of rational design strategies in the selection of the mutation site which might influence the functional resilience of serratiopeptidase under different pH levels.