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Purification and characterization of α-Amylase from Miswak Salvadora persica.

Mohamed SA, Almulaiky YQ, Ahmed YM, Al-Bar OA, Ibrahim IH - BMC Complement Altern Med (2014)

Bottom Line: The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases.PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases.The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia. saleh38@hotmail.com.

ABSTRACT

Background: The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste.

Method: The purification method included chromatography of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE.

Results: Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

Conclusions: From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

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Effect of temperature on the thermal stability of miswak α-amylases A1, A4a, A4b, A5a and A5b. The reaction mixture contained in 0.5 ml: 50 mM Tris-HCl buffer, pH 7.5 and suitable amount of enzyme. The reaction mixture was preincubated at various temperatures for 30 min prior to substrate addition, followed by cooling in an ice bath. The enzyme activity was measured using the standard assay method as previously described. Activity at zero time was taken as 100% activity. Each point represents the average of two experiments.
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Figure 6: Effect of temperature on the thermal stability of miswak α-amylases A1, A4a, A4b, A5a and A5b. The reaction mixture contained in 0.5 ml: 50 mM Tris-HCl buffer, pH 7.5 and suitable amount of enzyme. The reaction mixture was preincubated at various temperatures for 30 min prior to substrate addition, followed by cooling in an ice bath. The enzyme activity was measured using the standard assay method as previously described. Activity at zero time was taken as 100% activity. Each point represents the average of two experiments.

Mentions: α-Amylases A1, A4a, A4b, A5a and A5b had different temperature optima ranged from 40 to 60°C (Figure 5). These values resembled that of finger millet α-amylases α -1(b) and α -3 (45°C) and α -2 (50°C) [33]. Broad temperature optima (40-50°C) were detected in wheat α-amylase [29]. A temperature optimum at 37°C was reported for α-amylases from rice [38] and P. erosus tuber [32]. The miswk α-amylases were thermal stable up to 40-60°C (Figure 6). Similarly, α-amylase from wheat was stable up to 60°C [29]. On the contrary, wheat cv. Sakha 69 α-amylases AI and AII were thermally stable with half-maximal activity at 60 and 50°C, respectively [34].


Purification and characterization of α-Amylase from Miswak Salvadora persica.

Mohamed SA, Almulaiky YQ, Ahmed YM, Al-Bar OA, Ibrahim IH - BMC Complement Altern Med (2014)

Effect of temperature on the thermal stability of miswak α-amylases A1, A4a, A4b, A5a and A5b. The reaction mixture contained in 0.5 ml: 50 mM Tris-HCl buffer, pH 7.5 and suitable amount of enzyme. The reaction mixture was preincubated at various temperatures for 30 min prior to substrate addition, followed by cooling in an ice bath. The enzyme activity was measured using the standard assay method as previously described. Activity at zero time was taken as 100% activity. Each point represents the average of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3974154&req=5

Figure 6: Effect of temperature on the thermal stability of miswak α-amylases A1, A4a, A4b, A5a and A5b. The reaction mixture contained in 0.5 ml: 50 mM Tris-HCl buffer, pH 7.5 and suitable amount of enzyme. The reaction mixture was preincubated at various temperatures for 30 min prior to substrate addition, followed by cooling in an ice bath. The enzyme activity was measured using the standard assay method as previously described. Activity at zero time was taken as 100% activity. Each point represents the average of two experiments.
Mentions: α-Amylases A1, A4a, A4b, A5a and A5b had different temperature optima ranged from 40 to 60°C (Figure 5). These values resembled that of finger millet α-amylases α -1(b) and α -3 (45°C) and α -2 (50°C) [33]. Broad temperature optima (40-50°C) were detected in wheat α-amylase [29]. A temperature optimum at 37°C was reported for α-amylases from rice [38] and P. erosus tuber [32]. The miswk α-amylases were thermal stable up to 40-60°C (Figure 6). Similarly, α-amylase from wheat was stable up to 60°C [29]. On the contrary, wheat cv. Sakha 69 α-amylases AI and AII were thermally stable with half-maximal activity at 60 and 50°C, respectively [34].

Bottom Line: The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases.PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases.The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia. saleh38@hotmail.com.

ABSTRACT

Background: The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste.

Method: The purification method included chromatography of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE.

Results: Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

Conclusions: From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

Show MeSH
Related in: MedlinePlus