The cereals are main food and considered as sources of energy, protein, vitamins
and minerals for poor people in Sudan and some other African countries. Amylases
are useful in abroad range of industrial application which includes food fermentation,
textile and paper industries. α-amylases ubiquitous in nature have been
isolated, purified and characterized from a number of animal, plant, fungal
as well as bacterial sources (Kumar et al., 2009).
The diastatic power (DP) refers to the total saccarifying power activity, which
is considered as contribution of α and β-amylases as well as α-glucosidases
which convert maltose to glucose (Bureng and Worgan,1982).
The thermostability of the DP enzymes is critical in determining fermentable
sugar yield during mashing, where the mash temperature profile is a balance
between the temperature required for starch gelatinization to enable efficient
hydrolysis and the rate of thermal inactivation of the DP enzymes (Evan
et al., 2003). The α and β-amylases of sorghum malt have
been purified and their characteristics were studied by Botes
et al. (1967a,b), Mundy
(1982) and Okon and Uwaifo (1984). Zhang
et al. (2006) showed that the variation in β-amylase activity
was mainly attributable to the environment, although the effect of cultivar
was also highly significant. Etokakpan and Palmer (1990)
calculated activities of α-amylase and the DP from a single extract of
sorghum malt using mercuric chloride to inhibit the SH function.
Sudanese sorghum have higher values of amylolytic activity compared to that
of Nigerian and Australian sorghum malt as reported by Aniche
and Palmer (1990). Etim and Etoakpan (1992) reported
that diastatic activity of three sweet potato varieties was principally due
to β-amylase. According to Omemu et al. (2005),
the crude amylase preparation of A. niger AMO7 had temperature and pH
optima activities at 60°C and 4.0 pH, respectively. Muralikrishna
and Nirmala (2005) reported that α-amylases are more stable compared
to β-amylases. Egwin and Oloyede (2006) found that
the maximum α-amylase yield was attained from 72-120 h of sprouting cereals
and the optimum temperature for α-amylase activity for sorghum malt was
70°C. Maize malt characterization showed that α and β-amylase
had optimal pH between 4-6.5, optimal temperature 50 and 90°C, respectively
(Biazus et al., 2009). Similarly Nagai
et al. (2009) reported optimum pH of α-amylase as 6.0-7.0 and
optimum temperature 40°C and the enzyme was extremely inactivated at pH
higher than 7.0 or lower than 5.0. The heat inactivation occurred at 40°C.
Mohamed et al. (2009) reported that the activity
of α-amylases increased from day 0 to 6 of germination of Triticum estivum
a local Saudi Arabia wheat variety (Balady). Evans and Monday
(2009) found that the rate of α-amylase secretion in sprouting maize,
acha, rice and sorghum for 180 h is significantly high (p≤0.05) with growth
Different amylases give rise to oligosaccharides with specific lengths of glucose
as major products, for this reasons different types of amylases, with unique
properties are isolated and characterized for various application in the starch
industry (Egwin and Oloyede, 2006). Kumar
et al. (2009) found that α-amylase isolated from sorghum is
reversible, unfolded by chemical denaturants at pH 7.0 in 150 mM Hepes containing
13.6 mM calcium and mM DTT.
The objectives of this research are to study the effects of malting and peptone addition on development of amylolytic activities in Sorghum bicolor, Purification of α and β-amylases isolated from Sorghum bicolor malt and characterization of purified α and β-amylases such as storage stability, the optimum temperature and pH.
MATERIALS AND METHODS
This study was applied in Khartoum University, Faculty of Science, Department of Botany, from 2005-2008.
Sorghum malt: Sorghum bicolor cv. (Feterita) grains were purchased from local market. Five hundred grams of sorghum was cleaned and freed from other materials and soaked in water overnight. The grains were spreaded on clean sack made from jute in plastic trays and covered by another jute sack and sprayed with water twice per day at temperature 28±2°C from 1-5 days. Germinated grains were left to dry by the air then ground using an electric grinder, which was operated intermittently so as to avoid heating. The powders were kept in glass bottles for chemical analyses. Three replicates for each experiment were used.
Measurement of diastatic power (DP) of sorghum grains and malt: Five
gram of dry, fine powdered sample were taken in a conical flask containing 100
mL distilled water or 100 mL of 2% peptone solution. The diastatic activity
during the period of germination of sorghum grains was determined by the method
of EBC (1987).
Purification and characterization of α-amylase isolated from sorghum
Extraction of α-amylase: The purification and characterization
of α-amylase isolated from sorghum malt was done according to the method
of Botes et al. (1967a). In order to extract the enzyme, eighty gm of
sorghum malt were suspended in 300 ml of cooled distilled water using cooled
warring blender. The suspension was stirred continuously over night at low temperature
(4°C) and then centrifuged at 14,000 g for 20 min.
Purification of sorghum malt α-amylase: Crude α-amylase was prepared by extracting as described above. The clear extract was 40% saturated with solid ammonium sulphate which precipitated the α-amylase. The precipitate was allowed to settle over night, collected by centrifugation, dialyzed to remove the salts and then freeze dried. The supernatant which contained the β-amylase was used for the preparation of β-amylase.
Fifteen grams of the crude α-amylase were dissolved in 1500 mL 0.1M acetate buffer (pH5.9) containing 0.2% calcium acetate and 60 g decolorizing charcoal was added. The mixture was heated at 70°C for 15 min with stirring. The suspension was cooled to 4°C and the charcoal and coagulated protein removed with sold ammonium sulphate, the precipitate which formed collected by centrifugation, dissolved in 0.1 M tris buffer pH 7.0 +0.2% CaAc and dialyzed overnight against the same buffer.
The dialyzed solution from the previous step was applied to 4x12 cm DEAE cellulose column. The column was first eluted with 0.1 M tris buffer pH 7.0+0.2% CaAc and the eluate discarded. This was followed by elution with 0.1M tris buffer pH7.0 + 0.3 Nacl+0.2 CaAc in which the bulk of the activity appeared.
Enzyme assay (α and β-amylase): Soluble starch (Merck, special for diastatic power determination) was used as substrate for α and β-amylase activity. Apparent saccharifying power was estimated from the amount of reducing sugars, calculated as maltose produced from 2 mL of 2% starch solution in 0.05M acetate buffer of pH 4.6, when treated with 0.1 mL of suitable diluted enzyme solution for 10 min at 30°C. The enzyme reaction was stopped by adding 2 mL of 3,5-dinitrosalicylic acid reagent used for the determination of reducing sugars.
Purification and characterization of β-amylase isolated from sorghum malt
Purification steps of sorghum malt β-amylase: The purification of β-amylase as described by Botes et al. (1967b) was followed. The supernatant obtained after precipitation of α-amylase at 40% saturation with ammonium sulphate, contained the β-amylase. The supernatant was precipitated with 70% ammonium sulphate. The precipitate was allowed to settle overnight collected by centrifugation, dialyzed to remove the salts and freeze-dried.
Twenty grams of the crude β-amylase were dissolved at room temperature in 2000 mL 0.1% cysteine (pH 6.0) containing 0.2% EDTA. The pH of the solution was lowered to 3.2 by addition of 5.0 N acetic acid and the solution was kept at this pH for one hour at room temperature. The pH of the solution was then adjusted to 4.6 by the addition of solid sodium acetate.
The solution was cooled to-2°C and fractionated with ethanol at -10 to -12°C. The precipitate obtained at an alcohol concentration between 33.33 and 66.66% v/v contained in a refrigerated centrifuge. The precipitate was dissolved in chilled 0.05 M acetate buffer of pH 5.0 and dialyzed overnight against the same buffer.
The dialyzed solution was applied to a 4x8 cm DEAE-cellulose column. The column was first eluted with 0.05 M acetate buffer of pH 5.0 and the eluate discarded. This was followed by elution with 0.15 M acetate buffer of pH 5.0 which resulted into two partly separated elution peaks. β-amylase activity was associated only with the last peak. Those fractions which contained the enzyme were combined, 90% saturated with solid ammonium sulphate and the precipitate was collected by centrifugation.
Protein determination: Protein was quantified by the method of Bradford
(1976) with bovine serum albumin as standard.
Storage stability of α and β-amylases: Twenty fractions of 0.1 mL from purified enzyme preparations were taken in 20 test tubes. Ten of these tubes were stored in a refrigerator at 4°C, the other were placed in a freezer at -20°C. The enzyme activity were assayed with 7 days interval for both the frozen enzyme preparations and the enzyme stored at 4°C. The decrease in the absorbance values was measured and the percent of remaining activity was calculated.
Effect of temperature and pH on the α and β-amylase activity: The optimum temperature of α and β-amylases was determined using different temperatures for enzymes reaction (30.40, 50, 60, 70 and 80°C). The enzymes activity was assayed for each temperature reaction at pH 4.6 and 5.0 for α and β-amylases, respectively for 10 min. The optimum pH values of α and β-amylases for the reaction mixture was determined using acetate buffer at different pH values (2.0, 4.0, 5.0. 6.0, 7.0, 8.0 and 9.0). The enzymes activity was measured at each pH value at 30°C for 10 min.
RESULTS AND DISCUSSION
Diastatic Power (DP): During the process of germination, it was clear
that DP was started by the onset of germination from the Ist day and continued
until it reached the maximum value at the 4th day of germination as 62.0 IOB.
Then it decreased on the 5th day (Fig. 1). These results agree
with that obtained by Etim and Etoakpan (1992), who
reported that diastatic activity of three potato varieties was principally due
to β-amylase. Also the purification of β-amylase in this study revealed
that the fourth day of germination of sorghum showed the highest amount of enzyme
activity and decreased in the fifth day of germination, these results also confirmed
by Egwin and Oloyede (2006). The results showed that
the Sudanese sorghum malt have higher values of amylolytic activity compared
to that of Nigerian and Australian sorghum malt as reported by Aniche
and Palmer (1990). Figure 1 also showed that there was
no significant variation (p≤0.05) between the extraction procedures followed
either by distilled water or distilled water with 2% peptone.
Purification of α and β-amylase: Table 1
represented the results of the purification steps of α-amylase. Heating
the crude enzyme for 15 min at 70°C increased the specific activity of sorghum
malt from one to five days by 1.64, 2.1, 2.56, 3.16 and 3.51 folds. At the same
time the total protein decreased by 43, 57, 71, 74 and 76% in the same order
as compared by the initial value for sorghum malt. The values of enzyme yield
were 93, 90, 74, 82 and 82% for sorghum malt. It is clear that heat step of
purification is successful step, this result supported the findings of Kumar
et al. (2005) and Egwin and Oloyede (2006).
||Comparative development of Diastatic Power (DP) in Sorghum
||Purification steps of α-amylase isolated from Sorghum
bicolor (Feterita) germinated for five days
||Purification steps of β-amylase isolated from Sorghum
bicolor (Feterita) germinated for five days
The results using DEAE-cellulose chromatography indicated that the purification
folds were 6.63 and 6.94 for 4th and 5th day of germination, respectively and
the yields of enzyme activity were 79 and 76%, respectively. The reduction of
protein was 87 and 88%, respectively, compared to that of initial value.
The specific activity was increased by 9.67, 10.12, 15.10, 23.17 fold at day 1,2,3,4 respectively and then decreased to 19.37 at the 5th day of germination compared to initial value. At the same time the total protein were decreased by 93, 92, 94, 96 and 95%, respectively. The enzyme yields were 66, 78,82, 84and 83%, respectively (Table 2). Botes et al. (1967b) reported that the acid treatment reduced α-amylase while the β-amylase activity remained constant then alcohol fractionation removed the last traces of α-amylase and gave considerable purification of β-amylase. In this study it was clear that high purification fold (35.42) was obtained during the 4th day of germination, where DEAE-cellulose chromatography was used, these might confirm the Botes et al. (1967b) results. The total protein was reduced by 98% and the yield of enzyme activity was 80% compared to initial value. At the same time during the 5th day of germination the specific activity was reduced in all steps of purification treatment.
Storage stability of α and β-amylases: The storage stability
of purified α and β-amylases at -20 and 4°C for 56 days was determined.
It was observed that there was no significant loss in the enzymes activity during
the storage period for 56 days in-20°C for both enzymes isolated from sorghum
malt. The loss of activity at 4°C during the period of 56 days was 19% for
α, whereas the loss of β-amylase activity during storage period at
4°C at days 35, 42, 49 and 56 were 20, 25, 30 and 34% (Fig.
2). Theses results confirm the findings of Muralikrishna
and Nirmala (2005),who observed that α-amylases are more stable compared
The effect of temperature and pH on α and β-amylases: The
influence of temperature on α and β-amylases activity showed that
enzyme activity increased progressively with increase in temperature from 30°C
reaching a maximum at 70°C for α-amylase and 50°C for β-amylase
as shown in (Fig. 3).
||Sorghum bicolor α-and β-amylases stability
at two different storage conditions
||Temperature dependent activity profile of Sorghum bicolor
malt α-and β-amylase
Above 70 and 50°C there was a reduction in α and β-amylases
activity, respectively. These results agree with that reported by Egwin
and Oloyede (2006), they found that the optimum temperature for α-amylase
activity isolated from sorghum malt was 70°C.The results showed that the
activity of α-amylase decreased markedly after being heated for 15 min
at temperature up to 70°C this agree with the findings of Bureng
and Worgan (1982). On the other hand, the β-amylase enzyme was stable
at 50°C for 10 min; otherwise it showed markedly destroyed at temperature
The effect of pH on α and β-amylases activity isolated from sorghum
malt was showed that the enzymes specific activity was reached a maximum value
at pH 5.0 and 5.5, respectively (Fig. 4). These results conversely
with that obtained by (Egwin and Oloyede, 2006). As
the same time the obtained data agree with the finding of Nirmala
and Muralikrishna (2003) who found that the optimum of pH α-amylases
for finger, millet was a range of 5.0-5.5.
||Sorghum bicolor malt α-and β-amylases activity
as a function of pH at 30°C
Mohamed et al. (2009) reported that the optima
of different α-amylases of wheat have broad pH optima range from 5.0 to
7.0. This pH finding of β-amylase agrees with that of Okon
and Uwaifo (1984) who reported that the optimum pH of β-amylase was
The authors like to thank Dr. Sirag Ali Ibrahim for his valuable advice and help during this study.