Esterases from finger millet malt and their effect on the functionality of cereal non-starch polysaccharides.

Cereal plant cell walls are complex mixture of polysaccharides consisting of
arabinoxylans, (1→3)/(1→4)-β-D-glucans, arabino-galactoproteins and
glucomannans, in addition to the high amounts of lignocellulosic complexes.
Considerable progress was made for the past few decades on the functional aspects of
cereal arabinoxylans due to their potential influence on water holding capacity and
water balance, protein foam stabilization, viscosity, gelling and rheological properties.
Cereal arabinoxylans are β-1, 3/1, 4 linked D-xylose polymers with a large variation
on the nature and position of the substituents in the side chain. Minor substituents
such as acetyl (attached at O-2/O-3 of the xylan backbone), as well as feruloyl groups
(esterified to 5'-OH group of arabinosyl residues in the side chain), are also present in
the side chains of arabinoxylans. The structure of these arabinoxylans varies not only
from source to source, but also with in the same source. Arabinoxylans have been
characterized from various sources such as wheat, barley, maize, rye and ragi with
respect to their structural and functional relationship. Nutritionally, arabinoxylans are
known to alleviate various disease symptoms such as diabetes, atherosclerosis and
colon cancer.
Malting of cereals/millets is an important biotechnological process which
helps in the induction of various hydrolytic enzymes such as α/β-amylases,
α-glucosidases, pullulanases, xylanases and esterases, which in turn, controls the
various physiological and biochemical processes in the grain. The information
pertaining to both starch and major cell wall degrading enzymes such as xylanases
and xylosidases from cereal malts is quite exhaustive. However, the same is not true
with respect to the minor cell wall degrading enzymes such as O-acetyl esterases and
feruloyl esterases.
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Esterases are important class of hydrolytic enzymes that cleave the ester
linkages i.e. acetic acid esterases (EC 3.1.1.6) cleaves the acetyl groups from the
xylose residues of arabinoxylans while cinnamic acid esterases (EC 3.1.1.73)
hydrolyze the cinnamic acid derivatives such as ferulic acid and coumaric acids which
in turn, may modulate the functionality of cereal arabinoxylans.
Finger millet (Eleusine coracana), also known as ragi, is an indigenous minor
millet, rich in calcium and dietary fiber. It is extensively consumed in south India, and
is used in both native and processed (malted) forms. Till date, the effect of O-acetyl
groups and feruloyl groups on the functionality of cereal water-soluble non-starch
polysaccharides has not been addressed. Hence, the present study is undertaken as
there were no reports on isolation, purification and characterization of finger millet
malt esterases i.e. acetic and ferulic acid esterases with respect to their (a) kinetic
properties, and (b) role in modulating the functionality of water-soluble non-starch
polysaccharides from cereals with respect to their viscosity, foam stabilization and
gelling characteristics, which are the most important aspects of polysaccharide
functionality. Accordingly, following objectives are envisaged in the present
investigation.
1. Standardization of the experimental protocols with respect to the isolation of
esterases from finger millet malt, with respect to ionic strength, pH, number of
extractions and temperature. Preparation of synthetic substrate(s) for ferulic acid
esterases (Feruloyl glycerol).
2. Purification of esterases using fractional precipitation, ion exchange and gel
filtration chromatography. Ascertaining apparent homogeneity by native
PAGE/SDS-PAGE. Determination of kinetic parameters of esterases i.e. pH,
temperature optima and stability.
3. Studying the functional characteristics such as viscosity and foam stabilization of
water-soluble polysaccharides of cereals in the presence of enriched enzyme
isolates of esterases.
4. Role of esterases with respect to the mechanism of gelling properties of
polysaccharides.