Purification and Characterization of a Serine-Like Protease from Solanum dubium

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Elamin,Fatima Musbah Abbas Mohammed
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University of Khartoum
ABSTRACT The main objectives of this investigation are to extract, purify and characterize an enzyme from Solanum dubium (S. dubium) seeds with milk clotting activity and at the same time free of toxic substance (s). The dry and presoaked seeds, ripe and unripe, were extracted with the buffers such as sodium phosphate (pH 7.1), Tris HCl (pH 8.5), Sodium Acetate (pH 5.2) in addition to a distilled water treatment. Significant differences were observed in total protein content and clotting ability of the seed extracts abtained by the use of different buffers and those extrated with distilled water. Buffer extraction and presoaking produced more extractable protein with better clotting ability. The increase in protein content as a result of soaking ripe seeds is further supported by the high performance liquid chromatography (HPLC) amino-acid profiles. The extracted enzymes were filtered and fractionated on Sephadex G-75, anion exchange, and gel filtration chromatography columns. The purified enzyme is characterized by being thermostable (up to70 ºC) with a pH range of 6-7(for 1% casein substrate). The enzyme is inhibited by serine protease inhibitors such as phenylmethanesulfonylfluroide (PMSF) having 35%, 27% residual activity at 1mM and 2mM concentrations respectively and is also inhibited by chymostatin at a residual activity of 51% and at a concentration of 100_6;/L. It is, therefore, concluded that the enzyme is of the chymotrypsin-like serine protease class. Moreover, the enzyme activity was enhanced by the addition of CaCl2, made highly stable in the presence of various metal ions such as KCl, KF, MgSO4, MgCl2, Fe2 (SO4)3, ZnCl2, and AlSO4, ending up in 50% reduction in activity as the result of the addition of CuSO4, HgCl2, ZnSO4 and CoSO4. Gel filtration and SDS-PAGE methods showed that the purified enzyme consists of three bands with molecular masses of 11KD, 35KD and 40KD. The enzyme has Km value of 0.6 mM and Vm value of 66.7 unit/min .mg. Seeds scarification was found to be an important factor for germination and for gibberellic acid effect (GA3), especially at a concentration of 1000ppm, which proved to be an effective dormancy breaker, followed by hot water treatment at 80I0;C for 10 minutes. The growth regulators, strength of basic growth medium and sucrose at different concentrations were studied for their effects on callus induction in seeds. Four concentrations (0.5, 1.0, 1.5, and 2.0 mg/L) from each of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA) and 2, 4-dichlorophenoxy-acetic acid (2, 4-D), alone or in combination with 0.5 mg/L of 6-benzylamino purine (BAP), were used for callus induction. NAA alone or in combination with BAP gave higher callusing percentages (80 to100%). The concentrations used of NAA and BAP also shortened the time for callus development and improved it growth quality. It appeared that low sucrose concentration was more effective in inducing callus growth (88% for 10g/L and 60% for30g/L). The enzyme extracted from seed callus tissue of S. dubium exhibited more or less similar characteristics of the enzyme extracted from seeds; again, suggesting chymotrypsin-like serine protease.The similarities are Present in the effects of different metal salts, characteristics of the three molecular mass bands and Km and Vm values. The only major difference is in the optimum pH range which is higher for callus tissue enzyme (7-8). The extraction of the enzyme from callus tissue is a new finding. The toxicity of the enzyme extracted from S. dubium seed was studied by neutrophils, 3T3cell line, neutrophils viability, oxidative burst test, and MTT test. The results indicated that the enzyme can be safely considered as non-toxic.
174 Pages
Solanum dubium;amino acid;milk;water;enzyme;protein;oxidative; Cytotoxicity