Xylanase from Ragi Malt and It’s Use in the Preparation of Xylo-Oligosaccharides from Cereal Brans

Xylanases (EC 3.2.1.8) are the major enzymes which bring about the
depolymerization of heteroxylan, the major hemicellulosic component of plant cell walls.
Xylanases from plant sources still remains unexplored and hence the present study. It
focuses on the characterization of purified xylanase from finger millet (Ragi; Eleusine
coracana, Indaf-15) malt and its utilization in obtaining xylo-oligosaccharides. Xylanase
was purified to apparent homogeneity from 96 h ragi malt by a three step purification
procedure via ammonium sulphate fractionation, DEAE-cellulose ion exchange and
Sephadex G-75 gel filtration chromatographies with a recovery of 4.0 % and fold
purification of 60. Xylanase, having a molecular weight of 29.5 kDa was found to be
monomeric on SDS-PAGE. pH optimum of the enzyme was found to be in the range of
pH 5.0-5.5. Xylanase showed maximum stability at 35°C in a pH range of 5.0-6.0. Km
and Vmax of purified xylanase were found to be 0.2 % and 4.5 Cmoles min-1 respectively.
Metal ions such as Ca2+, Mg2+, Mn2+, Cu2+, Fe2+, Ag2+ and Ni2+ enhanced xylanase
activity at 5 mM concentration. P-chloromercuribenzoate (PCMB), citric, oxalic and
boric acids inhibited the enzyme in concentration dependent manner. Purified ragi
xylanase hydrolyzed wheat and ragi bran soluble polysaccharides (SP) yielding xylooligosaccharides
which were purified on Biogel P-2 column followed by HPLC and their
composition was determined by GLC. The oligosaccharides consisted of arabinose and
xylose. The purified oligosaccharides were characterized by ESI-MS and H1NMR. Wheat
bran SP yielded four oligosaccharides (WO-1, WO-2, WO-3 and WO-4). WO-1 and
WO-2 were identified as arabinose containing xylotetraose and xylotriose respectively
whereas, WO-3 and WO-4 were identified as unsubstituted xylotriose and xylobiose
respectively. Ragi bran SP yielded only one oligosaccharide (RO-1), identified as
xylobiose. The xylo-oligosaccharides liberated from both wheat and ragi brans were
proved to be prebiotic by in vitro fermentation experiments using Bifidobacteria,
Lactobacilli and Pediococci spp. in terms of growth characteristics such as absorbance
(600 nm) and pH of the culture broth and dry cell mass. Acetic acid was the major short
chain fatty acid (SCFA) liberated due to in vitro oligosaccharide fermentation. The thesis
ends with summary and conclusions succeeded by references quoted.