Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

S. Halldórsdóttir, E. T. Thórólfsdóttir, R. Spilliaert, M. Johansson, S. H. Thorbjarnardóttir, A. Palsdottir, G. Ó Hreggvidsson, J. K. Kristjánsson, O. Holst, G. Eggertsson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)

Abstract

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxy-methylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C.

Original languageEnglish
Pages (from-to)277-284
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume49
Issue number3
DOIs
Publication statusPublished - 1998

Bibliographical note

Funding Information:
Acknowledgements This work was supported by grants from the Icelandic National Research Council and the EC generic project Biotechnology of Extremophiles ERB BI04 CT96 0488.

Fingerprint

Dive into the research topics of 'Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12'. Together they form a unique fingerprint.

Cite this