Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1 6)-galactanase gene

Abstract

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.

A β-l-Arabinopyranosidase from Streptomyces avermitilis Is a Novel Member of Glycoside Hydrolase Family 27

“…2A). The N-terminal catalytic domain (domain I, residues 45-339) has a (␤/␣) 8 barrel, which is observed in many glycoside hydrolases. The second domain (domain II, residues 340 -430) is an eight-stranded anti-parallel ␤-domain containing tandemly repeated Greek key motives, but in imperfect shapes.…”

Endo-β-1,3-galactanase from Winter Mushroom Flammulina velutipes

“…Endo-␤-1,6-galactanase (EC 3.2.1.164) isolated from Trichoderma viride specifically acts on ␤-1,6-galactan side chains (17,18). Based on hydrophobic cluster analysis, this endo-␤-1,6-galactanase has been categorized into GH family 5 (19 -21).…”

Characterization of an Exo-β-1,3-D-galactanase fromStreptomyces avermitilisNBRC14893 Acting on Arabinogalactan-Proteins

“…-1,3-Galactan, -1,3--1,6-galactan from Prototheca zopfii, native and -L-arabinofuranosidasetreated AGPs from radish, -1,3-and -1,6-linked galactooligosaccharides, and arabinan were prepared as described previously. [4][5][6][7][8] Methyl -glycoside of -1,3-linked galactotetraose and -pentaose, and -1,6-linked galactopentaose and -hexaose were a kind gift from Dr. P. Kováč of the NIDDK (National Institutes of Health, Bethesda, MD). -1,3-Galactosyl galactosaminide and -1,3-galactobiose were purchased from Dextra Laboratories (Reading, UK).…”

“…Hence, we focused on enzymes capable of degradation of AGPs. [4][5][6][7][8][9] Exo--1,3-galactanase is an especially powerful tool to analyze AGP since it is able to accommodate branched side chains when the enzyme hydrolyzes the main-chain -1,3-linkage of the carbohydrate moiety of AGPs. In spite of its usefulness, so far, exo--1,3-galactanase has been purified from only two organisms, Irpex lacteus (Il1,3Gal) and Aspergillus niger (An1,3Gal).…”