Immobilization of α-Amylase from Acha (Digiteria exilis) on Different Cellulose Fibre Materials
Alpha amylase was obtained from Acha (D. exilis) sprouted
for 96 h and immobilized on palm wood chips, coconut and cotton wood fibers
according to standard procedures. Palm wood chips maintained enzyme activity
up to 25 h and still retained about 7x10-2 mg glucose mL min-1
of residual activity while coconut and cotton wool lost all activity by
12 h. Enzyme half-life improved by 857.0, 21.4 and 28.6 folds using palm
wood chips, coconut and cotton wool fibre as carriers, respectively. Palm
wood clip improved Vmax and catalytic efficiency by 177 and
163%, respectively. The results concludes that palm wood chip can be used
to immobilize Î±-amylase from D. exilis for several cycles
Î±-Amylase has wide applications in the food industry. For instance,
Î±-amylase is employed in the milling and baking industry (Ruberthaler
et al., 1965; Anderson and Watson, 1982) to hydrolyse starch to
smaller carbohydrates, so as to reduce the dough viscosity and increase
sugar levels, prolong freshness, improve softness and crust quality. Similarly,
in the brewery and beverage industries, (Shallenberger, 1990; Yeshajahu,
1991), Î±-amylase is employed in mash thinning, improves runoff of
wort and the general quality of the end product. The sweetener and confectionery
industries (Johnson, 1976; Wardrip, 1971; Alvin et al., 2002) have
used Î±-amylase to control the ratios of different saccharides to
achieve specific product qualities. It is not surprising therefore that
study into various sources and developments of high quality Î±-amylase
for industrial processes have attracted interests from researchers. Sprouting
cereals (Deatherage et al., 1955; Finney el al., 1972) appear
to be one of the popular sources of industrial amylase for some developing
economies. Solomon et al. (1987) developed standard malt and amylase
for the brewery industry from sorghum while Okon and Uwaifo (1985) have
demonstrated the malting potentials of different sorghum varieties. It
has recently been established that Î±-amylase from acha (Digiteria
exilis) is a superior alternative to sorghum Î±-amylase for industrial
processes (Egwim and Oloyede, 2004). Acha is an unpopular cereal that
is indigenous to the West African Coast. It grows with reasonable yields
in areas of low rainfall and poor sandy or iron stone soils. Farmer believes
acha will grow where no other crop will grow (Amonum, 1990). Acha is not
yet a popular staple food, but it grows extensively in the northern and
middle belt of West Africa, where the soil is not very suitable for the
growth of some more popular cereals like maize, sorghum and millet (Okon,
Currently, industrial enzymes are subjected to immobilization because
it offers both technical and economic advantages. Immobilization offers
some operational advantages over free enzyme; such as choice of batch
or continuous process, rapid termination of reactions, controlled engineering
designs (Brahim et al., 2002). By careful selection of the matrix,
it is also possible to vary the nature of the immobilized derivatives
in order to improve enzyme activity and stability and also to enable easier
storage and handling (Mohy et al., 2000). The immobilized enzyme
can also be used for continuous processes in either fixed or fluidized
bed reactors. In this case, it is possible to use higher enzyme dosage
per volume of reactor than in the soluble enzyme process. This contributes
to high reaction rates and consequently, small reactor size (Bassetti
et al., 1997). These technical advantages allow a reduction in
the operational and capital costs in industrial processes (Zanin and Moraes,
There are several carriers or support materials available for enzyme
immobilization. These include prefabricated and naturally occurring carrier
materials (Johnson, 1979; Wingard et al., 1979; Villenouve et
al., 2000; Kim et al., 2005). The use of cellulose derivative
as support materials for enzyme immobilization is wide spread. For instance
glucoamylase has been bound to halogenacetyl cellulose (Maeda and Suzuki,
1972); glucose isomerase and other industrial enzymes have been immobilized
on cellulose acetate fibre (Krenla and Hinko, 1979; Ogunbayo and Bello,
1986) successfully immobilized lactase on palm wood chips for the treatment
of whey while Antonio et al. (2003) have employed coconut fibre
for the immobilization of polyphenol oxidase.
The search for improved immobilized enzymes for technical and economic
reasons is currently on the increase. The present study is aimed at immobilization
of Î±-amylase obtained from Acha (Digitera exilis), on cellulose
carriers like palm wood, coconut and cotton wool fibres.
MATERIALS AND METHODS
All chemicals used for the present study were of analytical grade
(Analar) being products of British Drug House (BDH) Chemical Limited Role,
England. Acha was obtained from National Cereal Research Institute (NCRI)
Badeggi, Niger state, Nigeria.
Palm wood was cut from the bush around while coconut fibre was collected
from the waste deposits of coconut sellers in the open market, cotton
wool was purchased from the open market.
Preparation and Assay of Crude Amylase Enzyme
Acha was spouted for 96 h and milled in pre chilled 0.05 M citrate
buffer, pH 6.0, the resulting homogenate was centrifuged at 10,000xg for
10 min. The supernatant contain the crude Î±-amylase stored in buffer.
Î±-Amylase activity was assayed by modification of the method described
by Gil-Jin et al. (1997). Aliquot (0.1 mL) of crude enzyme was
pipetted into separate test tube and 0.9 mL of 2% soluble starch was added
and incubated in a shaking water bath at 50Â°C for 30 min. The reaction
was stopped by adding DNSA (Dinitro Salicylic Acid) reagent and boiled
for at least 3 min for colour development. Absorbance was read at 550
nM against reagent blank. Enzyme activity was thereafter was computed
from a standard glucose curve (0.1-1 mM glucose mL-1).
Immobilization of Enzyme
Immobilization carriers used are palm wood (Raphia hooker), coconut
(Cocus nucifera) and cotton wool fibres. Palm wood chip was cut
to approximately 1x1x1 cm from the woody portions of the palm fronds of
Raphia hooker. Coconut fibre was obtained from the fibrous epicarp
of coconut fruit, the coconut fibre was cut to smaller shreds for easy
The fibres were delignified by boiling in hot water several times until
the resulting water was clear. The fibres were air dried and stored. Immobilization
of crude Î±-amylase on the different cellulose fibres followed the
method of Ogunbayo and Bello (1986). Dried palm wood chip(250 pieces)
were soaked in 400 mL of chilled crude Î±-amylase for at least 4 h
while maintaining cold condition. The excess crude enzyme was drained
and the chips were washed and dried for 30 min. Gum arabic (50%) was prepared
and used as binder. The binder solution was poured layer by layer into
the dried chips to have a thorough mixing with the chips. The chips were
then left to dry for at least 5 h. This procedure was repeated for coconut
and cotton wool fibres. The immobilized enzyme was packed into a glass
column reactor (60x6 cm), the reactor was maintained at 45Â°C and 2%
starch solution was run through the packed column. The substrate and product
valves were adjusted to a steady flow rate. The product of starch hydrolysis
was collected and the total reducing sugar was assayed using DNSA reagent
and expressed as enzyme activity.
Enzyme activity in the present study is defined as the amount of enzyme
required to liberate a unit of glucose per minute. (mM glucose min-1)
at reaction condition.
Decay Constant (Îº), Half-Life (T1/2) and Kinetic Parameters
Îº and t1/2 of free and immobilized enzymes were computed
following the method described by Fabricio et al. (2004).
Kinetic parameters were determined by varying the substrate concentration
(0.1-1%) and Vmax and Km were extrapolated from
Hane`s Plot ([s]/vo vs [s])
RESULT AND DISCUSSION
The result of the activity-time profile of Î±-amylase immobilized
an difference cellulose fibre supports is shown in Fig.
1. Enzymes activity in coconut fibre showed an initial increase in
activity within the first 4 h, activity dropped sharply by the 8 h and
then reduced to zero. The cotton wool maintained enzyme activity for up
to 10 h and dropped sharply to zero by the 12 h, while the palm wood chip
maintained fairly steady enzyme activity up to 15 h and there after activity
reduced gradually to a residual activity of about 53x10-4 mM
glucose min-1 at 25 h. The present observation indicates that
palm wood chip may be the cellulose fibre of choice amongst the three
for immobilizing Î±-amylase from D. exilis.
Enzyme molecules may have leached from coconut and cotton wool fibres
since the fibres are in tread and shred forms. While palm wood chips may
have created a microenvironment within the matrix of the palm wood chip
cut into cubes. Ogunbayo and Bello (1986) have successfully immobilized
lactase on palm wood chips and achieved up to 50% stability while Antonio
et al. (2003) have studied the effectiveness of polyphenol oxidase
naturally immobilized on coconut fibre (Cocus nucifera). The present
observation indicates that palm wood chips are the most suitable carrier
for Î±-amylase immobilization. Indeed, the immobilization confers
stability on the enzyme as portrayed by the extended half-life (Table
||The extended half-life as shown by immobilization on palm wood chips
show its superiority over coconut and cotton wood fibre.
||The half-life of crud Î±-amylase immobilized on palm wood chip
is 40 and 30 times greater than that of coconut and cotton wood fibre,
respectively. This observation is in line with the finding of Bassetthi
et al. (1997) who had earlier reported an increase in invertase
half-life when immobilized.
||Decay constant and half-life of Î±-amylase immobilized on different
cellulose fibre supports
||Activity-time profile of alpha amylase from D. exilis immobilized
on different supports
Fabricio et al. (2004) have shown that the operational stability
of lipase immobilization on chitin is quite favourable. While Adriano
et al. (2005) have shown that immobilization of G acylase improves
operational stability. It seems obvious therefore that one of the main
targets in immobilization is to improve enzyme stability and half-life
for possible reuse. The present study has achieved enzyme stability by
immobilization on palm wood chip, coconut and cotton wool fibres.
||Immobilization on palm wood chips extends Î±-amylase
half-life from 0.14 h (free enzyme) to 120 h, which is about 857 folds
increase, while coconut and cotton wool fibres achieved 21.4 and 28.7
folds extension of enzyme half-life.
||.Varavinit et al. (2002) achieved an extension of Î±-amylase
half-life (10 cycles reuse) by immobilization on cellulose fibre from
bagasse. Torchilin et al. (1978, 1979) have shown that stability
of immobilized enzymes is due to intramolecular linkage conferred
by the carriers and depends on the length of such linkage. This may
explain why palm wool chip is a better carrier in the present study.
The result of varying substrate concentration on the activity of Î±-amylase
immobilized on palm wood chips is shown in Fig. 2. The
result shows a normal Mechealis-Menten`s pattern. This observation suggests
that immobilization has not altered the reactive site of the enzyme.
||Effect of substrate concentration on alpha-amylase activity immobilized
on palm wood chips
||Hans plot to determine Km and Vmax of alpha-amylase from D. exilise
||Kinetic parameters of free and immobilized Î±-amylase from D.
exlis immobilized on palm wood chips.
Hane`s plot is shown in Fig. 3 to obtain the enzyme
kinetic parameters. Table 2 compares the kinetic parameter
of free and immobilized Î±-amylase on palm wood chips. The result
shows that immobilization improves both the Vmax and catalytic
efficiency by 177 and 163%, respectively. This observation suggests that
the interaction between the carrier and the enzyme exposed the enzymes
active site to a better orientation for maximum turn over.
Similarly, Cellulose frbres can be used to immobilize Î±-amylase
from D. exilis for better application. The study there concludes
that immobilization improves stability, enzyme half-life and maximizes
turn over. Palm wool chips (1x1x1 cm) are better carrier for immobilizing
Î±-amylase from D. exilis than coconut and cotton wool fibres.
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