Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

Abstract

We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

Structural and biochemical properties of lichenase from Clostridium thermocellum

“…2A) like Ca, Mg and Mn salts [6]. Similar results were also reported for a β-1,3-xylanase belonging to GH26 family [11]. However, higher concentrations of Cu 2+ (>25 mM) resulted in a sharp fall in lichenase activity and the activity dropped to 0 % at 50 mM.…”

Glycosynthase‐Mediated Assembly of Xylanase Substrates and Inhibitors

“…The di‐ and trisaccharide 2‐fluoro‐xylosides 30 and 31 were incapable of inactivating Bcx or Cex. However, these molecules might prove to be mechanism‐based inhibitors of the GH family 26 xylanases 31. 32 Likewise, iminosugars 28 and 29 might also serve as inhibitor candidates for this less‐common class of xylanases.…”

Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides

“…strain XY-214 [34] and from the Vibrio sp. Strain AX-4 [52]. The endo β-(1-3) xylanase from the marine bacterium Alcaligenes sp.…”