Recombinant production and biotechnological applications of the hydrophobic protein DewA by Pichia pastoris

Hydrophobins have great potential in many biotechnological applications due to changing their surface characteristics as they adhere to surfaces. In recent years, although there has been a significant increase in the biotechnological applications of hydrophobins, industrial production has still not been achieved due to yield problems. Therefore, more studies are needed on the recombinant production of hydrophobins. In this thesis, the recombinant production of class I hydrophobin DewA protein belonging to Aspergillus nidulans, which is determined to have high contact angle in the literature, was aimed. For this purpose, firstly, the secretion signal sequence and the stop codon of the DewA gene (UNIPROT:52750) were removed. Then, the restriction enzymes that did not cut the obtained sequence were determined using the bioinformatics program and EcoRI and XbaI enzyme cutting sites were added to the sequence. Then, sequence synthesis was made after codon optimization. As a result, DewA protein was recombinantly produced using P. pastoris X-33 strain under AOX1 promoter by transferring into pPICZ a-A vector. The highest yield was obtained at 1% methanol concentration as 77 mg/L in 96 hours. The molecular weight of the obtained recombinant protein was about 15 kDa. As a result of coating the DewA protein on the glass surface changes the surface characteristics and makes it hydrophobic; On teflon surfaces, it has been proven to change the surface characteristics and make them hydrophilic. Then, the surface stability of the protein was evaluated by applying hot SDS and UV to these surfaces. As a result, the surface-coated DewA protein was resistant to hot SDS application on both glass and teflon surfaces; ın the UV application, it was understood that while the protein was degraded by UV exposure on glass surfaces, it preserved its structure on teflon surfaces. In the thesis study, the DewA protein of A. nidulans was cloned into the pPICZα-A vector and recombinantly produced for the first time in the P. pastoris X-33 strain under the control of the AOX promoter.