Heterologous expression, characterization, and optimization of production of alpha-galactosidase from aspergillus fumigatus in aspergillus sojae

Gürkök, Sümeyra
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.


Bromofluorocarbene addition to 6-phenylbicyclo[3.2.0]hept-6-ene: characterization and formation mechanism of the products
Balcı, Metin; Algı, Fatih (ARKAT USA, Inc., 2006-4-5)
Bromofluorocarbene addition to 6-phenylbicyclo[3.2.0]hept-6-ene provided fluoro-indanes such as 4,6-difluoro-5-phenylindane, 5,6-difluoro-4-phenylindane, 5,7-difluoro-4-phenylindane, 4-bromo-6-fluoro-5-phenylindane and 5-bromo-6-fluoro-4-phenylindane. The characterization the formation mechanism of the products are discussed.
Optimizing OLED efficacy of 2,7-diconjugated 9,9-dialkylfluorenes by variation of periphery substitution and conjugation length
RATHNAYAKE, Hemali P.; Çırpan, Ali; DELEN, Zeynep; LAHTI, Paul M.; KARASZ, Frank E. (2007-01-05)
The photoluminescence (PL) and electroluminescence (EL) of four 2,7-bis(phenylethenyl)fluorenes (OFPVs) and two 2,7-diphenylfluorenes (OFPhs) are compared to evaluate effects of nonconjugating peripheral substitution and conjugation length on their EL emissions. The OFPVs exhibit very similar PL spectra with 460-480 nm emission maxima but show large variation in the organic light-emitting diode (OLED) efficacy: from a material that does not give persistent emission in test OLEDs (9,9-diheptyl substitution o...
Immobilization of glucoamylase onto activated pHEMA/EGDMA microspheres: properties and application to a packed-bed reactor
Arica, MY; Alaeddinoglu, NG; Hasırcı, Vasıf Nejat (1998-02-15)
Glucoamylase was covalently immobilized onto pHEMA/EGDMA microspheres of two different sizes: 50-100 mu m and 100-200 mu m in diameter. The activity of the enzyme on smaller microspheres was found to be almost twice that of the larger microspheres. A higher enzyme lending was observed on small microspheres (0.64 mg g(-1) support) as compared to large spheres (0.40 mg g(-1) support). The K-m of glucoamylase was significantly increased (approximately five times) upon immobilization, indicating decreased affin...
Özkar, Saim (Elsevier BV, 1985-01-01)
The photochemical reaction of tricarbonyl-η6-1,3,5-cycloheptatriene-chromium(0) (I) with tricyclo[,7]undeca-3,5-diene (II) in n-pentane at 248 K yields the [4 + 6]-cycloadduct tricarbonyl-η6-pentacyclo[,15.02,8.03,7]octadeca-11,13,16-triene-chromium(0) (III). Detachment of the pentacyclic triene ligand from chromium can be achieved with trimethylphosphite. The constitutions of complex III and of the pentacyclic hydrocarbon IV were determined by spectroscopic means.
Tailoring optoelectronic properties of thieno[3,2-b]thiophene comprising homopolymers via electron acceptor moieties: thienopyrrolodione, 2,1,3-benzoselenadiazole, isoindigo
Göker, Seza; Sarigül, Hatice; Toppare, Levent Kamil (Elsevier BV, 2020-09-15)
In this work, thieno[3,2-b]thiophene based homopolymers, namely poly(5,6-bis(octyloxy)-4,7-bis(thieno[3,2-b] thiophen-2-yl)benzo[c][1,2,5]selenadiazole; PBSeThTh), poly(5-(2-ethylhexyl)-1,3-bis(thieno[3,2-b]thiophen-2-yl)4H-thieno[3,4-c]pyrrole-4,6(5H)-dione; PTPDThTh) and poly((E)-6,6 '-bis(thieno[3,2-b]thiophen-2-yl)-1,1 ' diundecyl-[3,3 '-biindolinylidene]-2,2 '-dione; PIIDThTh), were obtained potentiodynamically to tailor optoelectronic properties via altering electron acceptor moieties which were 5,6-b...
Citation Formats
S. Gürkök, “Heterologous expression, characterization, and optimization of production of alpha-galactosidase from aspergillus fumigatus in aspergillus sojae,” Ph.D. - Doctoral Program, Middle East Technical University, 2012.