Identification of a trypsin-like serine protease from Trichoderma reesei QM9414
(2007) In Enzyme and Microbial Technology 40(5). p.1087-1094- Abstract
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/667878
- author
- Dienes, Dora ; Börjesson, Johan LU ; Hagglund, Per ; Tjerneld, Folke LU ; Liden, Gunnar ; Reczey, Kati and Stålbrand, Henrik LU
- organization
- publishing date
- 2007
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- serine protease, Trichoderma reesei, Cel7B (endoglucanase I), trypsin
- in
- Enzyme and Microbial Technology
- volume
- 40
- issue
- 5
- pages
- 1087 - 1094
- publisher
- Elsevier
- external identifiers
-
- wos:000245519000014
- scopus:33847617488
- ISSN
- 0141-0229
- DOI
- 10.1016/j.enzmictec.2006.08.013
- language
- English
- LU publication?
- yes
- id
- bab08020-b5f2-44f8-a0d1-1ef1b920dea4 (old id 667878)
- date added to LUP
- 2016-04-01 11:57:26
- date last changed
- 2022-04-13 03:52:25
@article{bab08020-b5f2-44f8-a0d1-1ef1b920dea4,
abstract = {{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.}},
author = {{Dienes, Dora and Börjesson, Johan and Hagglund, Per and Tjerneld, Folke and Liden, Gunnar and Reczey, Kati and Stålbrand, Henrik}},
issn = {{0141-0229}},
keywords = {{serine protease; Trichoderma reesei; Cel7B (endoglucanase I); trypsin}},
language = {{eng}},
number = {{5}},
pages = {{1087--1094}},
publisher = {{Elsevier}},
series = {{Enzyme and Microbial Technology}},
title = {{Identification of a trypsin-like serine protease from Trichoderma reesei QM9414}},
url = {{http://dx.doi.org/10.1016/j.enzmictec.2006.08.013}},
doi = {{10.1016/j.enzmictec.2006.08.013}},
volume = {{40}},
year = {{2007}},
}