Efficient heterologous expression in Pichia pastoris, immobilization and functional characterization of a scorpion...

Industrial processes have expanded with the ability to clone and express recombinant immobilized enzymes in microorganisms such as Pichia pastoris that have commercially attractive amounts of the appropriate genes. This report describes the overexpression in Pichia pastoris, immobilization, and functional characterization of a secreted phospholipase A₂ from scorpion venom Scorpio maurus: rPLA₂(-5). After 48 h of culture, the recombinant rPLA₂(-5) was secreted into the culture medium and expressed at about 9 mg/L. Comparative analyses of the kinetics and hydrolysis of rPLA₂(-5) monolayers at various surface pressures were conducted with the same form produced in Escherichia coli. As a second part of the study, rPLA₂(-5) overexpressed in Pichia pastoris was immobilized by adsorption on CaCO₃, with about 78 percent of the activity. In comparison to the free enzyme, rPLA₂(-5) was studied for stability. Immobilization improved the thermal stability of rPLA₂(-5) and even the stability at acidic pH. Moreover, we found that the immobilization improved the stability of rPLA₂(-5) towards bile salts, Tween 80, Triton X-100, and SDS, as well as its stability towards many organic solvents. Until now, this is the first study to describe the overexpression and immobilization of a scorpion venom phospholipase A₂ that possesses an interesting stability characteristic that makes it useful for a wide range of biotechnological applications.