BMB 464 - Enzyme Purification Paper

BMB 464 - Enzyme Purification Paper - Abstract...

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Abstract Alpha-galactosidase catalyzes the hydrolysis of simple and complex sugars containing alpha -1,4 galactosidic linkages and alpha-1,6 linked terminal galactosyl groups. Due to its widespread presence and functionality in animals, plants and microorganisms, alpha-galactosidase is very useful commercially and medically. Alpha- galactosidase was purified from a culture of brewer’s yeast, Saccharomyces pastorianus , through performing a batch purification from an ion-exchange resin (DEAE-sephadex), a NaCl gradient elution from a DEAE sephadex column, dialysis, and gel filtration, which also aided in determining the molecular weight of the enzyme. The initial specific activity of the enzyme was 0.127 units/mg (μmol/min/mg) and the final specific activity after purification was 1.22 units/mg showing that the enzyme was purified by a factor of 8.89. Analysis of the purified α- galactosidase was performed through sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and native gel electrophoresis to estimate molecular weight of the enzyme, which was found to be 71.98 kDa. By exposing the enzyme to various temperatures and different pH levels, an optimal temperature, 55 o C, and pH, 5, for alpha-galactosidase enzymatic activity were found. The enzyme was also subjected to various concentrations of the substrate p- nitrophenyl –α –D-galactoside (pNPG) and various inhibitors in order to analyze their effects on α- galactosidase reaction rate. Introduction Alpha-galactosidase catalyzes the hydrolysis of simple and complex oligosaccharides and polysaccharides containing alpha-1,4 galactosidic linkages and alpha-1,6 linked terminal galactosyl groups. Since these linkages are widely distributed among microorganisms, plants, and mammals, such as humans, this enzyme is of great biochemical importance and commercial value. α-galactosidase plays an important role in the production of beet sugar since it acts as the enzyme that catalyzes the hydrolysis of raffinose, which interferes with the crystallization of sucrose from sugar beet molasses. Removal of the galactose from the raffinose promotes the forming of the beet sugar (crystallization). (Khire, et al. 1999). Studies of α-galactosidase have also shown that it is also of great medical importance since its absence leads to Fabry’s disease, an inherited, “X-linked,” neurological disorder. This enzymatic defect leads to the systematic accumulation of glycosphingolipids in endothelium cells, vascular smooth muscle cells, myocardium, fibroblasts, perineurium, and other cells in multiple organs. The only specific therapy available for Fabry’s disease is intravenous infusion of recombinant human alpha- galactosidase. (Pleasure, 2005 & Mohanraj, et al, 2002).
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α -galactosidase is also used in the hydrolysis of raffinose and stachyrose, which are present in foods such as beans and soybean milk. These sugars are indigestible to humans and, in turn, they can cause intestinal discomfort and flatulence. For this reason,
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BMB 464 - Enzyme Purification Paper - Abstract...

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