Starch occurs as highly organized structures and has unique thermal properties
and functionality that have permitted its wide use in food products and industrial
application (Ratnayake and Jackson, 2009). Bioconversion
of starch may be accomplished by acid treatment or enzymatic hydrolysis, of
which the latter is preferred as it offers a number of advantages including
improved yields and favorable economics (Satyanarayana et
al., 2004). Moreover, recent discoveries on the use of micro organisms
as sources of industrially relevant enzymes have led to an increased interest
in the application of microbial enzymes in various industrial processes (Alva
et al., 2007).
Today, food brewing and pharmaceutical industries depend solely on various
extra cellular microbial enzymes (Abu et al., 2005)
and enzymatic hydrolysis of starch can be used to obtain various valuable hydrolyzates
with different compositions (Baks et al., 2008).
Starch is degraded by amylases, of which isoamylase (glycogen-6-glucanohydrolase,
E.C.22.214.171.124) hydrolyses 1, 6- α-D-glycosidic linkages of glycogen, amylopectin
and α and β limit dextrins, producing linear malto oligosaccharides
(Fang et al., 1994). Isoamylase is used primarily
in the production of food ingredients from starch like glucose, maltose, trehalose
and cyclodextrins (Olemposka-Beer, 2007).
Conventionally, conversion of starch to glucose uses cooked or pre gelatinized
substrate and thus is energy intensive and expensive process. With the view
of reducing the energy consumption, currently there is considerable research
on raw starch degrading enzymes (Sun et al., 2008),
which eventually curtail the cost of sugar production in food industries. Only
few micro organisms have been reported to possess the ability to produce raw
starch degrading amylase (Abu et al., 2005) and
almost no published report is available on raw starch debranching fungal isoamylase.
In the present study, saccharification of various native starches, in their uncooked form, by the extra cellular isoamylase synthesized by Rhizopus oryzae was reported with elucidation of various factors influencing the extent of saccharification.
MATERIALS AND METHODS
Enzyme source: The present study was carried out during early summer to early winter (March to December, 2009) of this year. A strain of Rhizopus oryzae PR7 MTCC 9642, isolated from Eastern India was grown in basal medium composed of (g L-1): peptone 0.9; (NH4)2HPO4 0.4; KCl 0.1; MgSO4.7H2O 0.1 and starch 0.25 (pH: 8) at 28°C for 72 h. The culture broth was filtered, the filtrate was centrifuged at 5,000 g for 5 min and the supernatant was used as enzyme source.
Enzyme assay: Isoamylase activity was measured by incubating the assay
mixture (1 mL) containing an equal volume of enzyme and 1% (w/v) Oyster glycogen
in 0.1 (M) phosphate buffer (pH-5) at 55°C for 5 min. The reducing sugar
released was measured by the dinitrosalicylic acid method (Bernfeld,
1955) taking glucose as standard. Blanks were prepared with inactivated
enzymes. One unit of isoamylase was defined as that amount of enzyme that liberated
1 μmole of glucose/mL/min of reaction (Ara et al.,
Saccharification of native raw starches: The native starches namely arrow root (rhizome of Maranta arundinacea), oat (Avena sativa), tamarind kernel, tapioca (Manihot esculenta), millet, arum and pulse dust were collected from market dumps and domestic effluents, washed thoroughly with water, air dried, pulverized and sieved to 40 mesh particle size, before using as substrate for saccharification.
Dry starch granules (10 mg) were incubated with 1 mL of isoamylase diluted
with 0.1 (M) phosphate buffer (pH: 5) in a screw capped tube for various time
periods at 55°C. The resultant supernatant following centrifugation at 2000
g for 2 min was analyzed by DNSA method (Bernfeld, 1955)
using glucose as standard.
The percentage of saccharification was calculated (Baig
et al., 2004) as:
Effect of various factors on saccharification: To determine the effect
of incubation time, saccharification was carried out for various time periods
at optimum conditions. The role of pH on saccharification was determined by
varying the pH of the assay mixture from 4 to 7. The effects of increasing substrate
concentration were estimated by changing the substrate concentrations keeping
other factors unchanged. To check the effects of chemical pretreatment, starch
residues were treated with 10 mM of metal ions, thiol compounds and thiol inhibitors
for 10 min before using them as substrate of saccharification mixture. The effect
of cooking or gelatinization was checked by heating the starch suspension at
100°C for 10 min before enzyme treatment.
The enzyme was able to hydrolyse many types of starch molecules (Fig.
1) of which apart from potato starch (Merck), other types of native starches
showing promising results, were starch granules extracted from arrow root, tamarind
kernel, oat and tapioca.
||Saccharification of various native starches by Isoamylase
of Rhizopus oryzae
||Effect of incubation time on saccharification of native starches
by Isoamylase of Rhizopus oryzae
Therefore, further experiments were carried out with the bioconversion of these
starch residues only.
The maximum amount of bioconversion was accomplished within 60 min of incubation (Fig. 2) which was followed by a slow rate of increase in sugar production, but it took longer time for tamarind kernel and oat starch.
As pH of the saccharification media is one of the important determinants of
bioconversion process,saccharification was tested at various pH, which showed
that except tamarind kernel powder, the best pH for bioconversion was 5.0 (Fig.
||Effect of pH on saccharification of native starches by Isoamylase
of Rhizopus oryzae
||Effect of temperature on saccharification of native starches
by Isoamylase of Rhizopus oryzae
On the other hand, the highest amount of sugar production was found at a temperature
of 55°C (Fig. 4) for all types of starch tested.
Saccharification rate was found to increase with the increase in substrate
concentration (Fig. 5) up to 1% (w/v). But, with further increase
in substrate concentration to 1.5% (w/v), the sugar production dropped in cases
of potato starch (Merck), tamarind kernel powder and oat starch.
|| Effect of substrate treatments on saccharification of starch
|Substrate: 10 mg mL -1
||Effect of starch concentration on saccharification by Isoamylase
of Rhizopus oryzae
As pretreatment of substrate plays an important role in saccharification, the
starch granules were treated both thermally and chemically, which showed a remarkable
increase in sugar production after gelatinization or cooking. An 85-200% increase
in sugar production (Table 1) was found after gelatinization.
Amongst the chemicals, Mn2+ brought about a uniform increase in sugar
production for all the substrates used, whereas thiol compounds like reduced
glutathione (GSH) and dithiothreitol (DTT) enhanced the sugar production up
to a certain extent.
Starches extracted from different sources showed different susceptibility towards
enzymic hydrolysis. These differential rates of saccharification could be explained
by their different granular morphology (Howling, 1989).
For all types of substrates, the sugar production rate was initially high which
gradually reduced after certain time, probably due to substrate and/or enzyme
limitation or as a result of product accumulation and consequent product inhibition
(Ray et al., 1994).
The best pH for saccharification by isoamylase of R. oryzae PR7 was
slightly acidic, similar to the optimum pH of bacterial isoamylases (Castro
et al., 1992; Wu et al., 1994; Fang
et al., 2005). The higher pH required for tamarind kernel powder
suggested the existence of distinct crystalline organization (Colonna
et al., 1992) of the starch granule extracted from it.
The optimum temperature of the isoamylase from the present strain for sugar
production was higher than the temperature optima (30-40°C) reported from
other microbial isoamylases (Ueda and Nanri, 1967; Fang
et al., 1994; Ara et al., 1993; Spencer-Martins,
1982; Olemposka-Beer, 2007), which indicated that
the isoamylase from the present strain may be used conveniently at moderately
high temperature in sugar manufacturing industries.
Although, there was an initial positive correlation between starch concentration
and sugar production, with further increase in substrate concentration there
came a decline in saccharification, probably due to enzyme limitation. The difference
in the level of substrate saturation amongst the native starches tested could
be attributed to the nature of the starch granules, as amylolytic hydrolysis
of native starch granule is governed by the specific surface area, not by the
mass concentration of each granule (Kim et al., 2008).
Gelatinization brought about a remarkable increase in saccharification, as
the kinetics of the reaction at lower temperature were much slower than above
the gelatinization temperature where the starch exists in a molecularly dispersed
sol in the presence of an excess of water (Howling, 1989)
and hydrothermic action on starch molecules enhanced its rate of depolymerization
that made them more accessible to enzyme action.
The enhancement of saccharification by Mn2+ might be achieved by the increased activity of the enzyme used, but thiol compounds probably increased the affinity of the substrate towards the enzyme as indicated by the differential response by different types of starch.
In view of energy cost, effective utilization of natural resources, direct
hydrolysis of starch below the gelatinization temperature is desirable (Kelly
et al., 1995). The sacchrification potential of the isoamylase produced
by the present strain may add economy in sugar production from raw native starches.
The authors wish to thank University Grants Commission, India for financial assistance.