Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan

Abstract

A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a ␤-1,6-galactanase that preferentially debranches ␤-1,6-galactobiose from the substrate.Type II arabinogalactans in the Western larch consist of a ␤-1,3-galactan backbone with many side chains at position 6 (15). About half of the main chain galactopyranose residues are replaced by ␤-1,6-galactopyranobiose, and about a quarter are replaced by single galactopyranose units. The rest of the side chains contain three or more glycosidic residues. Arabinofuranose and arabinopyranose residues exist at the nonreducing ends of both the ␤-1,3-galactan main chains and the side chains.Fungi and plants make several type II arabinogalactan-degrading enzymes. These include two endo-␤-1,6-galactanases, one acting on grape arabinogalactans from Aspergillus niger (2) and one acting on radish arabinogalactans from Trichoderma viride (13), a ␤-1,6-galactanase that acts on acid-treated larch wood arabinogalactan (LWAG) from A. niger (10), ␤-galactosidases from radish seeds (19) and spinach (5) that act on ␤-1,3 and ␤-1,6 galactosyl sequences of arabinogalactans but not pectic ␤-1,4 galactan, and two exo-␤-1,3-galactanases (EC 3.2.1.145) from Irpex lacteus (22) and A. niger (14) that cleave the backbone of type II arabinogalactans in an exo-like manner. The latter-named enzymes are able to bypass the branching points of galactan backbones and to release the intact side chains of type II arabinogalactans and thus are very useful for the structural analysis of polysaccharides.To analyze the fine structures of polysaccharides enzymatically, recombinant enzymes are preferred, as they exclude the confounding influence of contaminating carbohydrases. However, only four genes encoding type II arabinogalactan-degrading enzymes have previously been characterized: two exo-␤-1,3-galactanases from Phanerochaete chrysosporium (7) and Clostridium thermocellum (6), an endo-␤-1,6-galactanase from T. viride (9), and a ␤-galactosidase from radishes (8). In this paper, we describe the isolation and characterization of a ␤-1,6-galactanase, termed FoGal1, that is produced by Fusarium oxysporum 12S. In addition, we report the nucleotide sequence of the gene that encodes FoGal1 (Fogal1) and describe the activity of the recombinant enzyme expressed in Escherichia coli.The F. oxysporum 12S strain was isolated from a rotten sugar beet as part of a large-scale screening program (unpublished data). The microorganism is deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (accession no. FERM P-20380; http://unit.aist.go.jp/ipod/cie/index.html). To purify the FoGal1, the F. oxysporum 12S strain was cultivated in 1 liter of LWAG medium consisting of 0.2% NH 4 NO 3 , 0.1% K 2 HPO 4 , 0.05% MgSO 4 · 7H...

Tropism and Pathogenicity of Rickettsiae

“…Thus, programmed necrosis might occur when AeAl2 cells are infected with SFGR. Contrary to our results, a previous report found that some non-pathogenic SFGR species, R. montanensis , and R. peacockii , were able to grow in two mosquito cell lines (the A. albopictus cell line Aa23 and the Anopheles gambiae cell line Sua5B; Sakamoto and Azad, 2007). The reason for this discrepancy is poorly understood; however, the abovementioned growth was only detected by FISH and PCR, rather than in an infective assay.…”

Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

“…When arabinan was isolated from black liquor and used as a substrate, arabinose was the major product. The enzyme substrate specificity is quite similar to that for the enzyme from F. oxysporum (FoGal1) (23). When FoGal1 hydrolyzed larch arabinogalactan, predominantly ␤-1,6-galactobiose and small amounts of galactose and arabinose were produced.…”

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

“…GH5_16 is another example of a monospecific subfamily of secreted enzymes where only a single fully sequenced biochemically characterized β-1,6-galactanase (EC 3.2.1.164) is currently known [32], although partial N-terminal sequence of an Aspergillus enzyme, closely related to sequences of other members of the subfamily, yields the same activity [33]. Several other enzymes with β-1,6-galactanase activity have been moved from GH5 to GH30_5 [23], but subfamily GH5_16 clearly remains within family GH5.…”

“…Several other enzymes with β-1,6-galactanase activity have been moved from GH5 to GH30_5 [23], but subfamily GH5_16 clearly remains within family GH5. The known β-1,6-galactanase (EC 3.2.1.164) is involved in larch wood arabinogalactan degradation [32]. …”