Identification of an exo-ß-1,3-d-galactanase from Fusarium oxysporum and the synergistic effect with related enzymes on degradation of type II arabinogalactan

Abstract

An exo-ß-1,3-D-galactanase (Fo/1,3Gal) was purified from the culture filtrate of Fusarium oxysporum 12S. A cDNA encoding Fo/1,3Gal was isolated by in vitro cloning. Module sequence analysis revealed a "GH43_6" domain and a "CBM35_galactosidase-like" domain in Fo/1,3Gal. The recombinant enzyme (rFo/1,3Gal) expressed in Pichia pastoris degraded ß-1,3-galactan and ß-1,3-galactobiose (Gal2), and released only galactose (Gal). In contrast, the enzyme did not hydrolyze p-nitrophenyl ß-D-galactopyranoside, ß-1,4-Gal2, or ß-1,6-Gal2. The enzyme also showed low activity towards native type II arabinogalactans such as larchwood arabinogalactan (LWAG) and gum arabic. Using LWAG as substrate, rFo/1,3Gal released Gal, ß-1,6-Gal2, ß-1,6-galactotriose (Gal3), and ß-1,6-Gal3 substituted with a single arabinofuranose residue accompanied with unidentified oligosaccharides, indicating that the enzyme can by-pass the branching points of ß-1,3-galactan backbones. A time course analysis of products released by rFo/1,3Gal on LWAG revealed that ß-1,6-Gal2 is the main side chain in LWAG and that the activity of rFo/1,3Gal was decreased when degrees of polymerization of side chains increase. rFo/1,3Gal worked synergistically with three other recombinant F. oxysporum enzymes (ß-1,6-galactanase, ß-L-arabinopyranosidase, and α-L-arabinofuranosidase) that degrade side chains, on the degradation of LWAG. However, the synergism was much lower than anticipated, probably because LWAG have longer side chains than the three enzymes used are able to remove or ß-1,3-galactan main chain is interrupted with glycosidic linkages that are different from the ß-1,3-galactosyl linkage. Affinity gel electrophoresis revealed that rFo/1,3Gal specifically bound to ß-1,3-galactan.

Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan

“…To date, all characterized exo-␤-1,3-galactanases have greater activity toward ␤-1,3-galactan than ␤-1,3/␤-1,6-galactan from Prototheca zopfii, LWAG, gum arabic, and radish roots (1,(3)(4)(5)(6)(7)21). BLLJ_1840 has low sequence identities (27% to 28%) with other exo-␤-1,3-galactanases.…”

“…In particular, exo-␤-1,3-galactanase hydrolyzes the ␤-1,3-galactan backbone, bypassing ␤-1,6-galactan side chains, and consequently releases galactose, ␤-1,6-galactooligosaccharides, and their derivatives (1,2). Exo-␤-1,3-galactanases, which belong to glycoside hydrolase family 43 (GH43), have been cloned and characterized from several sources, including bacteria and fungi (1,(3)(4)(5)(6)(7).…”

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family

“…Previously, we isolated, cloned, and characterized various GA-degrading enzymes from Fusarium oxysporum 12S, a phytopathogenic fungus that can grow using GA as the sole carbon source ( 14 , 15 , 16 , 17 , 18 ). In addition to these enzymes, we recently observed that Rha was released from GA by the culture supernatant of this fungus.…”

Biochemical and structural characterization of a novel 4‐O‐α‐l‐rhamnosyl‐β‐d‐glucuronidase from Fusarium oxysporum

“…To elucidate the detailed structure and modify the physical properties of GA, we obtained various AG‐II‐degrading enzymes such as β‐1,6‐galactanase [15], β‐ l ‐arabinopyranosidase [16], exo‐β‐1,3‐galactanase [17], α‐ d ‐galactopyranosidase [11], and α‐ l ‐arabinofuranosidase (unpublished result) from F. oxysporum 12S. When GA was degraded with an F. oxysporum 12S culture supernatant, an oligosaccharide was detected that differed from the reaction products obtained by the above enzymes.…”