Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds

Abstract

Carboxypeptidase A(CPA) has a great potential application in the food and pharmaceutical industry due to its capability to hydrolyze ochratoxin A(OTA) and remove the bitterness of peptide. However, CPA is a mesophilic enzyme that cannot adequately exert its catalytic activity at elevated temperatures, which seriously restricts its industrial application. In this study, the rational design of disulfide bonds was introduced to improve the thermostability of CPA. The highly flexible regions of CPA were predicted through the HotSpot Wizard program and molecular dynamics (MD) simulations. Then, DbD and MODIP online servers were conducted to predict potential residue pairs for introducing disulfide bonds in CPA. After the conservativeness analysis of the PSSM matrix and the structural analysis of the MD simulation, two mutants with potentially enhanced thermostability were screened. Results showed that these mutants D93C/F96C and K153C/S251C compared to the wild-type(WT) exhibited increase by 10 and 10 °C in Topt, 3.4 and 2.7 min in t1/2 at 65 °C, in addition to rise of 8.5 and 11.4 °C in T5015, respectively. Furthermore, the molecular mechanism responsible for thermostability was investigated from the perspective of advanced structure and molecular interactions. The enhanced thermostability of both mutants was not only associated with the more stable secondary structure and the introduction of disulfide bonds but also related to the changes in hydrogen bonds and the redistribution of surface charges in mutant regions. This study showed for the first time that the rational design of disulfide bonds is an effective strategy to enhance the thermostability of CPA, providing in this way a broader industrial application.

Keywords

• disulfide bonds
• thermostability
• carboxypeptidase a
• rational design