Identification of a trypsin-like serine protease from Trichoderma reesei QM9414
Författare
Summary, in English
In the present work the genetically modified Trichoderma reesei strain QM9414 was used to produce full-length Ce17B (endoglucanase I, EGI) under the control of the constitutive Aspergillus nidulans gpdA promoter in the presence of glucose. However, the full-length Ce17B enzyme was found to be truncated to lower molecular weight components in the culture broth. Truncation of recombinant proteins produced in fungi may be due to protease activity. In order to identify major sectreted proteases, protease activity was assessed in culture filtrate of the T reesei QM9414 recombinant. Zymogram analysis revealed the presence of proteolytic activity corresponding to one protein, which was subsequently purified by a combination of ion exchange and size exclusion chromatography. The protein has a molecular mass of 25 kDa, and an isoelectric point of 7.3. By matching tryptic peptide fragments analyzed by tandem mass spectrometry to fungal proteins available in databases as well as to expressed sequence tag (EST) sequences, and comparing the coded amino acids to full-length amino acid sequences, the purified protein was found to be homologous to several trypsin-like fungal serine proteases, with the highest homology to the protease P27 from Trichoderma harzianum. The purified protein was further characterized using benzoyl-arginyl-p-nitroanilide (BApNA) as substrate. It was found to have maximum activity at pH 8 and 50 degrees C, with a k(m)-value of 0.3 mM.
Avdelning/ar
Publiceringsår
2007
Språk
Engelska
Sidor
1087-1094
Publikation/Tidskrift/Serie
Enzyme and Microbial Technology
Volym
40
Issue
5
Länkar
Dokumenttyp
Artikel i tidskrift
Förlag
Elsevier
Ämne
- Chemical Engineering
- Biological Sciences
Nyckelord
- serine protease
- Trichoderma reesei
- Cel7B (endoglucanase I)
- trypsin
Status
Published
ISBN/ISSN/Övrigt
- ISSN: 0141-0229