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Truncation of the cellulose binding domain improved thermal stability of endo-β-1,4-glucanase from Bacillus subtilis JA18
The C-terminus region of endo-β-glucanase Egl499 from Bacillus subtilis JA18 was suggested to be a putative family 3 cellulose-binding domain (CBD) by computer analysis. To prove this proposal, C-terminus truncation mutant Egl330 was constructed and expressed. Compared with Egl499, Egl330 lost the c...
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Published in: | Bioresource technology 2009, Vol.100 (1), p.345-349 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The C-terminus region of endo-β-glucanase Egl499 from
Bacillus subtilis JA18 was suggested to be a putative family 3 cellulose-binding domain (CBD) by computer analysis. To prove this proposal, C-terminus truncation mutant Egl330 was constructed and expressed. Compared with Egl499, Egl330 lost the cellulose binding capability at 4
°C, confirming the C-terminus region was a CBD. Binding of the CBD to Avicel was inhibited by carboxymethylcellulose (CMC), but not by barley β-glucan and glucose at concentration of 0.1% and 0.5%. Kinetic analysis showed both the turnover rate (
k
cat) and the catalytic efficiency (
k
cat/
K
m) of Egl330 increased for the substrate CMC compared to Egl499. A great improvement in thermal stability was observed in Egl330. The half life of Egl330 at 65
°C increased to three folds that of Egl499, from 10 to 29
min. After treated at 80
°C for 10
min, Egl330 could recover more than 60% of its original activity while Egl499 only recovered 12% activity. UV spectrometry analysis showed Egl330 and Egl499 differed in refolding efficiency after heat treatment. |
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ISSN: | 0960-8524 1873-2976 |
DOI: | 10.1016/j.biortech.2008.06.001 |