University of Khartoum

Characterization of Amylases from Seedlings of three Sorghum (Sorghum bicolor) Cultivars and three Sorghum Lines

Characterization of Amylases from Seedlings of three Sorghum (Sorghum bicolor) Cultivars and three Sorghum Lines

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Title: Characterization of Amylases from Seedlings of three Sorghum (Sorghum bicolor) Cultivars and three Sorghum Lines
Author: Abbas, Huyam Abbas Hsabelrasoul
Abstract: The amylase family of enzymes is of great significance due to its wide area of potential application and is among the most important enzymes in present-daybiotechnology.Amylase(1,4-α-D-glucan-4 glucanohydrolase; EC 3.2.1.1) from grain sources have dominated applications in industrial sectors. Generally, grains are known to contain a varying quantity of both α- and β-amylases. The aim of this research was therefore to undertake a comparative study on properties of amylases from seedlings of three sorghum cultivars (Wadahmed, Wadakar and Tabat) and three newly developed sorghum lines (L06DWN, L105 and L102), with a view to gain some insight into the suitability of these grains for commercial amylases production. Grains of each cultivar or line were sown separately in local kitan sack for four days then the growth rate and the radical length were measured. The enzymes were extracted in a phosphate buffer solution (0.1 M, pH 7.0) using a refrigerated centrifuge. Amylolytic activity, total soluble protein, pH and temperature optima, effect of element ions, substrate specificity, kineticproperties and storage stability were determined in each enzyme preparation ,all experimentaltreatments were carried out in triplicates and the mean value plus the standard deviation were recorded throughout. Colorimetric as well as spectrophotometric methods were used for characterizing the extracted enzymes. A broad variation was obtained among the sorghum cultivars and lines in germination rate and radical length. The three newly developed sorghum lines gave a very low germination percentage (50%) as compared with the three sorghum cultivars (90 –98%). The highest radical length (5.03cm) was recorded for Wadakar cultivar, while the lowest radicle length (3.33cm) was detected in L102. There were significant differences among the studied germinated grains in their amylolytic activities and had markedly high enzymatic activity (96.34- 106.34 Unit), except L105 (44.27 Unit). The highest level of total soluble protein(0.957 mg/ml) was detected in enzyme preparation of L06DWNwhile the lowest value (0.441mg/ml) was recorded in enzyme preparation of L105. The enzymes were active over wide range of temperature (20 – 80 °C) and pH (2 – 10) values. The study established that the optimum temperature for the reaction was 40 °C, while the optimum pH was 7.0. There was a gradual loss of enzymatic activity on either side of the optimum temperature and optimum pH. Element ions either increased or had no effect on the total activity of the enzyme. Calcium, copper and iron ions markedly increased the enzymatic activity of all extracts.The enzymes had high specificity towards the Merck’s soluble starch and low specificity towards raw sorghum starch as substrates. The extracted enzymes from L06DWM showed the lowest value of Km (0.19 mM) and high catalytic power (Kcat = 1.9 min-1) as compared with that of the other sorghum lines and cultivars. These values revealed that the amylases from L06DWM had a relatively high affinity for starch and they were fully saturated with substrate. During the storage of the enzymes at room temperature, 4°C and -20°C for 30 days, their activities decreased with time. The entire extracted enzymes were fully active (100%) up to 20 days when stored at -20°C while retained 70 – 78% of their original activity after 30 days of incubation. In conclusion, there were significant differences among the studied sorghum grains in their enzymatic properties and activity which could possibly give direction for the selection of sorghum cultivars or lines containing these enzymes for specific food utilization. The high amount of protein content and high catalytic power as well as low km value of amylases from L06DWM might have influenced the choice of this line over the other sorghum cultivars and lines for commercial amylase production.
Description: 81 Pages
URI: http://khartoumspace.uofk.edu/handle/123456789/18404
Date: 2016-01-11


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