Identification of a GH62 α-l-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum

Abstract

An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a "Glyco_hydro_62" was present at position 28-299 of AXS5. This is a family of α-L-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. (1)H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding D-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

“…In fact, in our previous study, we showed that xylooligosaccharides can reversibly bind in the xylan-binding pocket of CBM13 linked to ␤-1,4-xylanase using decorated xylooligosaccharides (56). Moreover, cleavage of arabinofuranosyl side chains linked to O2 and O3 of single-substituted xylose residues in arabinoxylan by GH62 arabinofuranosidase was previously reported (49).…”

GH62 arabinofuranosidases: Structure, function and applications

“…Early on product profiles indicated that SlAbf62A hydrolyzed AXOS of DP 2 6 (unknown Araf substitution patterns) (Vincent et al, 1997). 1 H NMR analysis showed Penicillium chrysogenum GH62 ABF (PcAbf62A-m2,3) hydrolyzed -L-1,3 faster than -8 L-1,2 linked Araf from singly substituted Xylp in AX (Lange et al, 2006;Sakamoto et al, 2010) and that AnAbf62A-m2,3 released -1,3 three times faster than -1,2 linked Araf from a 1:1 XA 2 XX+XA 3 XX mixture (Wilkens et al, 2016). Using different AXOS substrates ScAbf62A, PfAbf62A-m2,3 and PfAbf62C-m2,3 were demonstrated to hydrolyze singly -1,2 and -1,3 linked…”

First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies

“…To date, all of the biochemically characterized GH62 are arabinoxylan ␣-L-arabinofuranohydrolases, in as much that they specifically cleave either ␣-1,2-or ␣-1,3-L-arabinofuranosidic linkages in arabinoxylans (11). In the case of GH62 ␣-Larabinofuranosidases from Penicillium chrysogenum and Penicillium funiculosum, 1 H NMR and hydrolytic fingerprinting revealed that these enzymes cleave ␣-1,2-or ␣-1,3-bonds that specifically link arabinofuranosyl moieties to single-substituted D-xylosyl residues in arabinoxylan (12)(13)(14).…”