Title
Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense
Author
Gusakov, A.V.
Sinitsyn, A.P.
Salanovich, T.N.
Bukhtojarov, F.E.
Markov, A.V.
Ustinov, B.B.
Zeijl, C.V.
Punt, P.
Burlingame, R.
TNO Kwaliteit van Leven
Publication year
2005
Abstract
Two forms of cellobiohydrolase I (CBH I, Cel7A) were purified from the culture ultrafiltrate of a mutant strain of the fungus Chrysosporium lucknowense, an industrial producer of cellulases and hemicellulases. The enzymes had different molecular masses (52 and 65 kDa, SDS-PAGE data) but the same pI (4.5). Peptide sequencing showed that a single gene encodes both proteins. Both enzymes displayed maximum activity at pH 5.0-5.5; they had similar specific activities against soluble substrates. However, the 65 kDa CBH I was much more efficient in hydrolysis of Avicel and cotton cellulose, and its adsorption ability on Avicel was notably higher in comparison to the 52 kDa enzyme. Using the in-gel tryptic digestion followed by MALDI-TOF mass-spectrometry, it was shown that the 52 kDa enzyme represents the core catalytic module of the intact 65 kDa CBH I without a cellulose-binding module and major part of glycosylated linker. Both enzymes were stable at 50°C for 24 h. At higher temperature, the 65 kDa enzyme showed better thermostability: it retained >90% of activity after 7 h at 60°C and 50% of activity after 3 h at 65°C. The intact CBH I is also notably more thermostable than the Trichoderma reesei CBH I (by 6°C, the data of differential scanning microcalorimetry study). The cbh1 gene was cloned and then the amino acid sequence of Cel7A was deduced from the gene sequence. The enzyme had high degree of similarity (up to 74%) to family 7 cellobiohydrolases and lower degree of similarity (up to 41%) to family 7 endoglucanases. © 2004 Elsevier Inc. All rights reserved.
Subject
Biology
Biotechnology
Cellobiohydrolase
Cellulase
Chrysosporium lucknowense
Purification
Trichoderma reesei
Adsorption
Cellulose
Data acquisition
Differential scanning calorimetry
Fungi
Hydrolysis
Mass spectrometry
Cellobiohydrolase I (CBH I, CeI7A)
Cellulases
Chrysosporium lucknowense
Thermostability
Enzymes
cellulose 1,4 beta cellobiosidase
adsorption
amino acid sequence
article
catalysis
Chrysosporium
Chrysosporium lucknowense
controlled study
differential scanning calorimetry
enzyme activity
glycosylation
high temperature
hydrolysis
matrix assisted laser desorption ionization time of flight mass spectrometry
molecular cloning
molecular dynamics
nonhuman
nucleotide sequence
purification
thermostability
Adsorption
Calorimetry
Cellulose
Data Processing
Enzymes
Fungi
Hydrolysis
Chrysosporium
Chrysosporium lucknowense
Fungi
Gossypium hirsutum
Hypocrea jecorina
Trichoderma
To reference this document use:
http://resolver.tudelft.nl/uuid:b45f59f2-eafb-4ff1-896b-cff2bac04bf5
DOI
https://doi.org/10.1016/j.enzmictec.2004.03.025
TNO identifier
238304
ISSN
0141-0229
Source
Enzyme and Microbial Technology, 36 (1), 57-69
Document type
article