Cloning, Sequencing and Overexpression of a Rhodothermus marinus Gene Encoding a Thermostable Cellulase of Glycosyl Hydrolase Family 12
Solveig HALLDORSDOTTIRa, Eirny Tholl THOROLFSDOTTIRa, Remi SPILLIAERTa, Sigridur THORBJARNARDOTTIRa, Astridur PALSDOTTIRa, Gudmundur O. HREGGVIDSSONb, Jakob K. KRISTJANSSONb, and Gudmundur EGGERTSSON*a
a Laboratory of Molecular Genetics, Institute of Biology, University of Iceland, IS-108 Reykjavik, Iceland
b Department of Biotechnology, Technological Institute of Iceland, Keldnaholt, IS- 112 Reykjavik, Iceland
Rhodothermus marinus is a marine thermophilic eubacterium isolated from alkaline submarine hot springs. It is an obligate aerobe and grows optimally at 65℃, pH7 and 2% NaC1 (1). Its 16S rRNA sequence indicates that it is most closely allied to the Flexibacter-Cytophaga-Bacterioides group.
A gene library from R. marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants 3 were active on carboxymethylcellulose (CMC). 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 weight 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 using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on CMC and lichenan, but not on birch xylan or laminarin. The expressed enzyme had 6 terminal histidine residues and was purified by using a Ni-NTA column. The enzyme had a pH optimum of 7 and highest initial activity at 100℃. The enzyme had a half life of 8 h at 90℃.
A comparison of the amino acid composition of 12 cellulases of class 12 shows that the R. marinus enzyme and 4 enzymes from two Thermatoga species contain a significantly higher percentage of glutamic acid than their mesophilic counterparts.
1. Alfredsson, G.A., Kristjansson, J.K, Hjorleifsdottir, S. and Stetter, K.O. (1988) J. Gen. Microbiol., 134, 299- 306.
2. Andresson, O.S. and Fridjonsson, O.H. (1994) J. Bacteriol., 176, 6165-6169.
