Characterization of Galactosidases from Aspergillus niger: Purification of a Novel α-Galactosidase Activity (original) (raw)
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Enzyme and Microbial Technology, 2001
Enzymes with ␣-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major ␣-galactosidase forms (␣-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. ␣-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. ␣-Gal I is a completely different enzyme that belongs to family 36. ␣-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. ␣-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. ␣-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2-5, whereas ␣-gal I was most stable at pH 5-6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. ␣-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. ␣-Gal I released galactose from locust bean gum only in combination with A. niger -mannosidase. Kinetic experiments showed that ␣-gal I hydrolyzed p-nitrophenyl-␣-D-galactopyranoside and melibiose more efficiently than ␣-gal II-IV. The distinct hydrolytic and biochemical properties of ␣-gal I and ␣-gal II-IV further signifies the difference between ␣-galactosidases of family 27 and 36.
Purification and characterization of an alpha-galactosidase from Aspergillus fumigatus
Brazilian Archives of Biology and Technology, 2005
Aspergillus fumigatus secreted invertase (β-fructofuranosidase) and α-galactosidase enzymatic activities able to hydrolyzing raffinose oligosaccharides (RO). α-Galactosidase was induced by galactose, melibiose and raffinose, but galactose was the most efficient inducer. It was purified by gel filtration and two ion exchange chromatographies and showed Mw of 54.7 kDa. The purified enzyme showed maximal activity against pnitrophenyl-α-D-galactopyranoside (pNPGal) at pH 4.5-5.5 and 55 °C, and retained about 80% of the original activity after incubation for 90 minutes at 50ºC. The K M for pNPGal was 0.3 mM. Melibiose was hydrolyzed by the enzyme but raffinose was very poor substrate.
European Journal of Biochemistry - EUR J BIOCHEM, 2001
Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
Electronic Journal of …, 2009
Aspergillus parasiticus microbial type culture collection (MTCC)-2796, a new source of α-galactosidase is an efficient producer of enzyme in basic medium under submerged fermentation conditions. Maximum αgalactosidase production (156.25 Uml-1) was obtained when the basic medium is supplemented with galactose (0.5% w/v) and raffinose (0.5% w/v) as carbon source and yeast extract as nitrogen source. Enzyme production was also enhanced considerably in the presence of wheat bran (1.0% w/v). Enzyme secretion was strongly inhibited by the presence of Hg 2+ , Cu 2+ , and Co 2+ in the medium and to some extent by Zn 2+ and Ni + , while marginal increase in the enzyme production was observed when Mg 2+ and Mn 2+ were added in the medium. Among amino acids checked (aparagine, cysteine, glutamine, leucine and proline), glutamine (1 mM) was found to be an enhancer for the enzyme production. The temperature and pH range for the production of enzyme were 25ºC to 35ºC and 6.5 to 7.5, respectively with maximum activity (50 Uml-1) at 30ºC and pH 6.5 under static fermentation condition.
Process Biochemistry, 2010
Bacillus stearothermophilus secretes ,-mannanase and oa-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of aL-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified P-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The P-D-mannanase activity exhibited thermostabiity, retaining nearly full activity after incubation for 24 h at 70°C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for V.. and Km of 455 60 U/mg and 1.5 0.3 mg/ml, respectively, at 70°C and pH 6.5.
Electronic Journal of Biotechnology, 2009
Aspergillus parasiticus microbial type culture collection (MTCC)-2796, a new source of α-galactosidase is an efficient producer of enzyme in basic medium under submerged fermentation conditions. Maximum αgalactosidase production (156.25 Uml -1 ) was obtained when the basic medium is supplemented with galactose (0.5% w/v) and raffinose (0.5% w/v) as carbon source and yeast extract as nitrogen source. Enzyme production was also enhanced considerably in the presence of wheat bran (1.0% w/v). Enzyme secretion was strongly inhibited by the presence of Hg 2+ , Cu 2+ , and Co 2+ in the medium and to some extent by Zn 2+ and Ni + , while marginal increase in the enzyme production was observed when Mg 2+ and Mn 2+ were added in the medium. Among amino acids checked (aparagine, cysteine, glutamine, leucine and proline), glutamine (1 mM) was found to be an enhancer for the enzyme production. The temperature and pH range for the production of enzyme were 25ºC to 35ºC and 6.5 to 7.5, respectively with maximum activity (50 Uml -1 ) at 30ºC and pH 6.5 under static fermentation condition.
Characterization of immobilized β-galactosidase from Aspergillus niger
Delta Journal of Science, 2013
β-galactosidase enzyme was isolated from Aspergillus niger, and immobilized in sodium alginate gel. The maximum activity of the free enzyme was obtained at 65 o C, pH 3.5 and its not affected by immobilization. The free enzyme had pH stability range from 3.5 to 6.5 and it was increased by immobilization process especially at acid pH values. The free enzyme retained 90.28, 85.09, 45.49, and 19.2 % of its initial activity after incubation at 30, 40, 50, and 60 o C, for 60 min respectively. Thermal stability was enhanced by immobilization process. The kinetic parameters for soluble and immobilized enzyme were also determined, and immobilization led to decrease in Km value (5.12 mM for free form to 1.48 mM for immobilized form), indicating decreased affinity by the enzyme for its substrate. Vmax was also decreased by immobilization process, and it was reached from 86.66 μmol ONP.min-1 for free enzyme to .38.02 μM ONP.min-1 for immobilized form.
Applied and Environmental Microbiology, 1999
A gene encoding a third ␣-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other ␣-galactosidases revealed that it belongs to a subfamily of ␣-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic ␣-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger -galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.