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Identification of a trypsin-like serine protease from Trichoderma reesei QM9414

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Author

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.

Publishing year

2007

Language

English

Pages

1087-1094

Publication/Series

Enzyme and Microbial Technology

Volume

40

Issue

5

Document type

Journal article

Publisher

Elsevier

Topic

  • Chemical Engineering
  • Biological Sciences

Keywords

  • serine protease
  • Trichoderma reesei
  • Cel7B (endoglucanase I)
  • trypsin

Status

Published

ISBN/ISSN/Other

  • ISSN: 0141-0229