&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 ° / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12614859/index.pdf |
| Date | 01 October 2012 |
| Creators | Gurkok, Sumeyra |
| Contributors | Ogel, Zumrut Begum |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | Ph.D. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for METU campus |
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