The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides

Abstract

A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylosereleasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a K m of 1.64 ؎ 0.03 mM and a k cat value of 118.8 s ؊1 . Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (␣/␣) 6 barrel fold where the loops connecting the ␣-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a ؊2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts. IMPORTANCEA GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents. P lant biomass is the most abundant source of organic carbon on the planet; therefore, it has become one of the most powerful and sustainable alternatives to petroleum as a platform for generation of biofuels, chemicals, and solvents (1). Nevertheless, plant cell walls are recalcitrant to biological depolymerization, as the extensive interactions between polysaccharides, and between polysaccharides and lignin, restrict access to the battery of degrading enzymes that break down these composite structures (2, 3). Celluloses and hemicelluloses are the most abundant polysaccharides in plants; thus, their separation and degradation are crucial for biomass valorization. Xylan is the major component of hemicelluloses, and it is composed of a backbone of ␤-D-xylopyranosyl residues that can be variably acetylated an...

Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus

“…However, unlike GH30-7 Rex, GH8 Rex exhibits poor activity on polymeric xylan and is designated an exo-oligoxylanase (9). Analyses of kinetic properties of GH8 Rex indicate that Xyl 3 is the most suitable substrate for this enzyme (9,11,33). The K m values of GH8 Rex (BH2105) isolated from Bacillus halodurans increase with increased degree of polymerization in XOS; this results in decreased k cat /K m values.…”

“…␤-Xylosidase releases xylose from the nonreducing ends of XOSs; thus, ␤-xylosidase does not hydrolyze the xylose residue in the reducing end of branched XOS (7,8). Conversely, certain bacterial GH8 xylanases are characterized as reducing-end xylose-releasing exo-oligoxylanases (Rex) (EC 3.2.1.156) (9)(10)(11). GH8 Rex liberates xylose from the reducing ends of XOSs via an inverting catalytic mechanism (9).…”

“…GH8 Rex liberates xylose from the reducing ends of XOSs via an inverting catalytic mechanism (9). This can potentially increase the yield of xylose in hydrolysis of pretreated lignocellulose because Rex readily hydrolyzes branched XOSs that are resistant to enzymatic hydrolysis by endoxylanases and ␤-xylosidases (11).…”

Targeted metatranscriptomics of compost-derived consortia reveals a GH11 exerting an unusual exo-1,4-β-xylanase activity

“…To our knowledge, the present study describes the first example of an exo-xylanase from the GH11 family. compost21_GH11 has high activity on insoluble polymeric xylan, in contrast to GH8 exo-oligoxylanases that show preference for soluble xylooligosaccharides [ 61 , 62 ].…”

Antioxidant activity of xylooligosaccharides produced from glucuronoxylan by Xyn10A and Xyn30D xylanases and eucalyptus autohydrolysates

“…2). The mass spectrum showed the presence of molecular ions of linear and substituted oligomers identified as sodium and potassium adducts (Valenzuela, Lopez, Biely, Sanz-aparicio, & Pastor, 2016).…”