A gene encoding endo-β-(1→6)-galactanase from Trichoderma viride was cloned by reverse transcriptase–PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His6 tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed β-(1→6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse α-l-arabinofuranosidase-treated arabinogalactan protein from radish. It produced β-(1→6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and β-(1→6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-β-(1→6)-galactanase. As far as we know, this is the first time an endo-β-(1→6)-galactanase has been cloned.

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