Production and purification if fibrinolitic lumbokinase enzyme in filamentous fungus Aspergillus oryzae

In this study, it is aimed to produce and purify the lumbrokinase enzyme in high yield in Aspergillus oryzae. Since this enzyme has a fibrinolytic effect, it is used in the treatment of many diseases. The homologous production source for lumbrokinase is Lumbricus bimastus (283 aa). Since homologous production is limited, time-consuming and expensive, researchers have turned to heterologous studies of this enzyme. However, despite the efforts to be produced in different expression systems, sufficient and desired yield could not be produced. A. oryzae is capable of producing large amounts of hydrolytic enzymes such as amylolytic and proteolytic enzymes. Thanks to this feature, A. oryzae constitutes an important source for homologous and heterologous enzyme production. A. oryzae was chosen as the expression system in this study due to its strong protein secretion, rapid growth, easy culture and optimizability. The lumbrokinase enzyme tagged 6X His-tag to the C-terminus and codon-optimized designed to Aspergillus oryzae will be synthesized by ligation into the pUC19 plasmid vector. The incoming vector will be transformed into Escherichia coli TOP10 cells first and then into A. oryzae by protoplast method. Next, the colony will be selected by seeding on CD Agar plates and the selected colonies will be seeded on DPY medium for more specific colonies and protein releases. Controls will be provided by SDS PAGE and expression will be initiated by selecting a colony. IMAC (Immobilized Metal Affinity Chromatography) method will be used for purification of proteins.