Secreted expression of prolyl endopeptidase from Aspergillus fumigatus in Pichia pastoris and characterization of the recombinant enzyme

Abstract:

[Objective] The study aims to heterologously express Aspergillus fumigatus prolyl endopeptidase cDNA and to characterize the recombinant enzyme. [Methods] The cDNA from A. fumigatus CICIM F0044 was obtained by reverse transcription using the total RNA as the template. The PEP gene that encodes the mature prolyl endopeptidase was amplified using polymerase chain reaction with the cDNA as the template. The recombinant expression vector pPIC9K-PEP was constructed by inserting the PEP gene into pPIC9K, which was then transformed into Pichia pastoris GS115 by electroporation. The resulting recombinant enzyme was purified and characterized. [Results] A maximum yield of 647.3 U/L enzyme activity was obtained from the recombinant yeast. The molecular weight of the purified recombinant enzyme was approximately 63 kD. The optimal reaction temperature of the recombinant enzyme was 65 °C. The enzyme is highly thermostable, retaining 90% of enzyme activity after 8 h of exposure to temperatures 55 °C. The recombinant enzyme exhibited an optimal reaction pH of 5.5 and its activity was highly stable from pH 3.0 to 9.0. No decrease in enzyme activity was observed after 10 days of exposure to pH ranging from 6.0 to 8.0 at 37 °C. [Conclusion] The A. fumigatus prolyl endopeptidase cDNA was expressed in P. pastoris. The activity of the recombinant enzyme was stable, which indicates that the recombinant yeast has potential value in industrial applications.