|Ph.D Student||Goldman Dan|
|Subject||Biochemical Characterization of the Glycoside Hydrolase|
Family 68 Levansucrase from Zymomonan Mobilis
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
Glycoside hydrolases (GH) are a widespread group of enzymes hydrolyzing the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety. Zymomonas mobilis levansucrase is a member of glycoside hydrolase family 68, and can catalyze both sucrose hydrolysis, and the polymerization of fructose to levan (β-(2-6)-fructofuranose). We demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pHs above 7.0, the enzyme is mainly a dimer, whereas at pHs below 6.0, the protein forms well-ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly, the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructo-saccharides (degree of polymerization 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of 6 to 8 protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils and to synthesize levan at pH 6.0.
Large quantities of the recombinant enzyme were produced using fed- batch fermentation with defined media of E. coli high-cell-density-culture. To avoid the accumulation of growth-inhibiting acidic by-products of incomplete substrate oxidation (such as acetic acid), a fermentaion strategy involving a pre-determined feeding rate was employed in order to limit the growth on the carbon source and to maintain the dissolved oxygen concentration above 30% . This procedure was adopted to 50 liter scale, resulting in optical cell density of 300 at 600 nm (150 gram-dry-cell-weight per liter) and 32 grams of enzyme per 1 liter of cell culture. Based on the fact that the enzyme can be easily converted from soluble to insoluble forms, depending on the pH, an efficient method to purify the enzyme was developed, resulting with over 95% pure protein and 90% total yield.