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Detection and identification of extracellular proteases in Trichoderma reesei

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posted on 28.03.2022, 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.

History

Table of Contents

1. Introduction -- 2. Materials and methods -- 3. Results and discussions -- 4. Summary and future prospects.

Notes

"October 2012 Includes bibiographical references A thesis submitted in fulfilment of the requirements for the degree of Master of Philosophy, Department of Chemistry and Biomolecular Sciences, Macquarie University, Australia".

Awarding Institution

Macquarie University

Degree Type

Thesis masters research

Degree

MPhil, Department of Chemistry and Biomolecular Sciences, Macquarie University, Australia

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2013

Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Junior Te'o

Rights

Copyright disclaimer: http://www.copyright.mq.edu.au Copyright Hana Bali 2013.

Language

English

Extent

1 online resource (99 pages) illustrations

Former Identifiers

mq:28212 http://hdl.handle.net/1959.14/267752 2066124