Detection and identification of extracellular proteases in Trichoderma reesei
thesisposted on 2022-03-28, 20:25 authored by Hana Bali
"A major factor affecting the yield of heterologous protein production from filamentous fungi is the degradation of the target protein by proteases produced by the host organism. One approach to reduce this degradation by proteases is the identification of the proteases produced from the fungi followed by inactivation of the gene(s) encoding the harmful proteases. This project involved characterisation of the protease profile of three strains of the filamentous fungus Trichoderma reesei: QM6a (wild type), Rut-C30 (transformation host) and CVt (producing a heterologous Venus protein). Firstly, zymography was used to allow comparison of extracellular proteases produced from these three strains. Then, liquid assays using specific fluorescent substrates, and mass spectrometric analysis were used to identify the extracellular protease types secreted by the Rut-C30 and CVt strains. Both strains of T. reesei examined were found to produce extracellular proteases that were active across neutral to acidic pH range. Liquid protease activity assays using specific substrates and inhibitors for each protease type showed that there was a resemblance in the protease types which were secreted by T. reesei (Rut-C30 and CVt) strains. Aspartic proteases, chymotrypsin-like proteases and subtilisin-like proteases activities were detected by this method. However, there was higher aspartic protease activity in Rut-C30 supernatants whereas there was higher chymotrypsin-like protease and subtilisin-like protease activities in CVt supernatants. In addition, mass spectrometric analysis (MALDI TOF/TOF MS/MS and LC ESI MS/MS) showed both similarities and differences in the types of proteases between the two strains. Aspartic protease, chymotrypsin-like protease and subtilisin-like protease were identified from both strains. However, three proteases (carboxypeptidase, metallopeptidases and serine protease) were detected from the CVt strain only. This project paves the way for molecular engineering of protease deficient production hosts" -- Abstract.