α-L-Arabinofuranosidase (Ustilago maydis)

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3²-α-L-Araf-(1-4)-β-D-xylobiose (A³X), 2³-α-L-Araf-(1-4)-β-D-xylotriose (A²XX), 3³-α-L-Araf-(1-4)-β-D-xylotriose (A³XX), 2²-α-L-Araf-(1-4)-β-D-xylotriose (XA²X), 3²-α-L-Araf (1-4)-β-D-xylotriose (XA³X), 2³-α-L-Araf-(1-4)-β-D-xylotetraose (XA²XX), 3³-α-L-Araf-(1-4)-β-D-xylotetraose (XA³XX), 2³ ,3³-di-α-L-Araf-(1-4)-β-D-xylotriose (A²⁺³XX), 2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA²⁺³XX), 2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA²⁺³XXX) and 3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA³A³XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A^2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

Dietary fiber plays an important role in the prevention of colorectal cancer, and arabinoxylans are an important source of this in grains, with some studies reporting the inhibition of cancer cell growth. However, very few studies have been conducted on this, and most previous studies have used oligosaccharides derived from arabinoxylans of specific molecular weight. The aim of this work is to extract, isolate, and analyze arabinoxylans from two different Argentinian genotypes of wheat (hard and soft) and study if they have the capacity to decrease the cellular viability of a colon cancer line (HCT-116). To determine whether the molecular size influences the inhibition of HCT-116 cell viability, specific hydrolysis was performed with endoxylanase, and the cells were exposed to the hydrolyzed arabinoxylans. The arabinoxylans treatment resulted in HCT-116 cell viability of 74% for the soft genotype and 64% for the hard genotype in comparison to nontreated cells. Hydrolyzed-arabinoxylans result in HCT-116 cell viability of 68% for soft and 36% for hard genotypes (the lowest IC₅₀ values) compared to nontreated cells. More importantly, no decrease after the arabinoxylans treatment was observed in the viability of murine noncancer cells known to rapidly respond to polysaccharide presence. The arabinoxylans from hard wheat showed more disubstituted xylose and α-1,2/α-1,3 linkages than the arabinoxylans from soft wheat, the possible cause for showing the best in vitro biological effect. The results showed other beneficial effects than the prebiotic ones and support the use of enzymatic treatment to increase the biological impacts of arabinoxylans.