Cloning and heterologous expression of subtilisin SAPN, a serine alkaline protease from Melghiribacillus thermohalophilus Nari2AT in Escherichia coli and Pichia pastoris

The sapN gene, encoding the extracellular subtilisin SAPN, a serine alkaline protease from Melghiribacillus thermohalophilus Nari2AT, was isolated, sequenced, and heterologously expressed in Escherichia coli BL21(DE3) pLysS using pUT57 and pTrc99A vectors and in E. coli BL21-AITM using the GatewayTM pDESTTM 17 vector. Conversely, three cassettes encoding pre-pro-subtilisin (rSAPN/SP-Pro-M), pro-subtilisin (rSAPN/Pro-M), and the mature-subtilisin (rSAPN/M) were hyperexpressed in Pichia pastoris SMD1168 and X33 using the pPICZ?C vector. rSAPNs were purified, characterized, and compared to wild-type SAPN. The deduced amino acid sequences exhibited high similarity with subtilisins from Bacillus strains. The highest sequence identity (96 %) was observed with the Bacillus licheniformis MP1 protease, with a 10-residue difference. Compared to SAPN and untagged rSAPNs, (His)6-tagged enzymes showed the highest activity and stability at alkaline pH and high temperature, the highest hydrolysis degree on crab and shrimp by-products, and the best catalytic efficiency. It was found that His6-rSAPN/SP-Pro-Ms expressed in P. pastoris strains was more active than those produced in E. coli. To initiate structure-function relationships, a 3D-model of the Pro-SAPN was built based on the available structures of common subtilisins. These data constitute a pivotal first step toward the creation of new efficient rSAPNs with enhanced catalytic properties and high potential for biotechnological and industrial uses.

Automated summary

In this study, the sapN gene encoding subtilisin SAPN from Melghiribacillus thermohalophilus Nari2AT was successfully isolated, cloned, and expressed in both P. pastoris and E. coli, with His6-tagged enzymes showing the highest activity and stability. Comparative analysis revealed that these enzymes have great potential for industrial applications due to their catalytic efficiency and hydrolysis degree.