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 alpha-amylase from miswak. The second goal is to study the storage stability of alpha-amylase in toothpaste.
Method: The purification method included chromatographaphy of miswak alpha-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE.
Results: Five alpha-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. alpha-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak alpha-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 degrees C-60 degrees C. Miswak alpha-amylases were stable up to 40 degrees C-60 degrees C after incubation for 30 min. Ca+2 activated all the miswak alpha-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 alpha-amylases. PMSF, rho-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on alpha-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak alpha-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak alpha-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.
Conclusions: From these findings, alpha-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude alpha-amylase of miswak, which contained the five alpha-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.