Peptidase-lipase bifunctional enzyme expressed in pichia pastoris

The protein engineering is a shortcut to natural evolution, one of the possible paths is the creation of hybrid enzymes or also called chimeras. These hybrids are the combination of two or more protein sequences, in order to improve or generate new functionalities. The peptidases and lipases are hydrolases capable of hydrolyze various substrates and have combined applications in different fields, as in the food, detergent, leather industries and bioremediation. End-to-end technique was employed to fuse the hydrolases sequences, the main advantage of its use is the independence of protein conformations and structures when compared to other techniques, making the process simpler. The chimera sequence was composed of hydrolases from Fusarium oxysporum. The peptidase was based on the work of Di Pietro et al (1998), which presented a subtilase, and the lipase sequence was based on the similarity with Thermomyces lanuginosus lipase. The position of each enzyme in the chimera was decided based on the in silico analysis of similar structures, where a His-tag was attached to the N-terminal region of the peptidase followed by a linker composed of five amino acids (GGAGG), and then the lipase sequence. The chimera gene was synthetized by GenScript® in pPiczαA expression vector for Pichia pastoris yeast. Please click Additional Files below to see the full abstract