Characterization of purified xylanase from finger millet (Eleusine coracana-Indaf 15) malt

Xylanase (E.C. 3.2.1.8) was purified to apparent
homogeneity from 96 h finger millet (Eleusine
coracana, Indaf-15) malt by a three step purification procedure
via ammonium sulphate fractionation, DEAEcellulose
ion exchange and Sephadex G-75 gel permeation
chromatographies with a recovery of 4.0% and fold puri-
fication of 60. Xylanase, having a molecular weight of
29 ± 2 kDa was found to be monomeric on SDS-PAGE.
pH optimum of the enzyme was found to be in the range of
5.0–5.5. The activation energy was 25 kJmol–1. 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 lmol min–1, respectively. Metal ions such as
Ca2+, Mg2+, Mn2+, Cu2+, Fe2+, Ag2+ and Ni2+ enhanced
xylanase activity at 5 mM concentration. p-chloromercuribenzoate,
citric, oxalic and boric acids inhibited the
enzyme in concentration dependent manner. The mode of
action of xylanase was found to be ‘‘endo’’ as determined
by the analysis of products liberated from larchwood xylan
by ESI-MS and H1NMR. In vitro studies using Bifidobacterium
and Lactobacillus sp. confirmed the prebiotic
activity of the xylo-oligosaccharides.