Researcher: Debosmita Sikdar and Ivy Kanungo, M. Tech Biotechnology,
Government College of Engineering and Leather Technology
The linear polysaccharide, β-1, 4-xylan (hemicellulose), is a major component of plant cell walls.
The enzyme xylanase hydrolyses β-1, 4-xylan into xylose, thereby making it useful for preparation
of different value added products having wide industrial applications. Xylose is the second most
abundant sugar present in lignocellulosic biomass after glucose. Xylanases are produced by diverse
genera and species of bacteria, actinomycetes and fungi. However, filamentous fungi are the
principal producer of xylanase. Xylanase plays a major role in micro-organisms thriving on plant
and agricultural sources for the degradation of plant matter into usable nutrients, increasing the
sustainability of biomass. Several efforts are underway to achieve an efficient as well as
commercially viable process development for xylose production and purification. Xylanase can be
explored in various industrial applications such as paper-pulp industries, textile, food and bakery
industries as a greener and eco-friendly approach.
METHODOLOGY AND RESULTS
In this work, a Three Phase Partitioning (TPP) method has been employed for isolation of this
enzyme from Agaricus bisporus (basidiomycete). This is a novel bio separation approach for
isolating industrially important enzymes with the enhancement of catalytic power of the enzyme.
TPP technique is widely adopted for the purpose of commercial downstream processing of
enzymes using agro-residues that too in a cost effective manner. The agro-wastes harbour huge
amount of microbial population over them especially fungal.
• These fungal spores were isolated, cultured and fermented in laboratory, to derive xylanase.
• The isolated enzyme extract wasassayed for calculating xylanase activity.
• The enzyme extract was made to undergo optimisation under variable experimental
conditions such as ammonium sulphate concentration, ratio of culture filtrate to tertiary
butanol (v/v) and pH.
Experimentally it was found that all three variables influence the degree of enzyme production as
well as its activity. Maximum enzyme activity was obtained at 50 % ammonium sulphate
saturation (w/v) when the other conditions were kept constant. In the same way, at 1:2 ratio of
culture filtrate to t-butanol (v/v) and at pH 6 keeping constant the remaining experimental variables
individually, maximum enzyme activity was achieved.
Thus, it can be concluded that the xylanase enzyme isolated using TPP method can be implemented
in paper-pulp and other industries that might pave the path towards a clean, environmentally safe
and sustainable perspective.
• The fungus Agaricus bisporus was made to undergo submerged fermentation to produce
xylanase using Potato Dextrose Agar media and Tamarind Kernel Powder was used as the
• Three phase partitioning uses t-butanol and ammonium sulphate to precipitate xylanase
from aqueous solution at the intermediate phase.
• For optimizing best three-phase partitioning result of endo-xylanase isolation from culture
filtrate, effect of various process parameters such as percent saturation of ammonium
sulphate, crude extract to t-butanol ratio and pH of the culture medium were analyzed.
After carrying out the respective optimisation experiments, it was concluded that the combination
of 50 %( w/v) ammonium sulphate saturation with 1:2 ratios of xylanase to t-butanol (v/v) at ph
6.0 was optimal for attaining the best recovery of xylanase.
The data obtained is conclusive to state that TPP is a scalable and quite efficient as an initial step
of bio separation of xylanase. This enzyme might be effectively used in a number of industrial
applications as a substitute of harsh chemical-based reagents thereby embarking in the pathway of
non-polluting, viable and ecologically sound strategy.