celB, a gene coding for a bifunctional cellulase from the extreme thermophile 'Caldocellum saccharolyticum'

Abstract

'Caldocellum saccharolyticum' is an obligatory anaerobic thermophilic bacterium. A gene from this organism, designated celB, has been cloned in Escherichia coli as part of a bacteriophage λ gene library. This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. The sequence of celB has homology with both exo- and endoglucanases described by others. It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes). Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain. There are internal transcriptional and translational start sites within the gene. The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E. coli. Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000. A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-β-D-cellobioside were also produced. These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product.