Aspergillus Fumigatus Alfa-Galaktosidazının Aspergillus Sojae'de heterolog üretimi, karakterizasyonu ve üretiminin optimizasyonu.

α-Galactosidase is an exo-glycosidase that hydrolyses non-reducing, α-1,6-linked α-galactose units from oligosaccharides, galactomannans, and galactolipids. α-Galactosidase activity has biotechnological, industrial, and medical importance. α-Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, α-galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, α-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 α-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 α-galactosidase production (10.4 U/mL) was achieved. Recombinant α-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 α-D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for α-galactosidase activity were between pH 4–6 and 50–60 °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 12–29%. To analyse the influence of osmotic stress as a means of further inducing α-galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both ‘salt-adapted’ and ‘salt non-adapted’ 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 α-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 α-galactosidase was studied. α-Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNPαGal as monitored by TLC, ESI-MS, and HPLC.Ph.D. - Doctoral Progra