Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene.
The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no. AB104898. A gene encoding endo-beta-(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 His(6) 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 beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase. As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned.
Kotake
Toshihisa
T
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
Kaneko
Satoshi
S
Kubomoto
Aya
A
Haque
Md Ashraful
MA
Kobayashi
Hideyuki
H
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; C478178:endo-beta-(1-6)-galactanase; D001616:beta-Galactosidase