Purification and characterization of α-galactosidase from Aspergillus niger v. Tiegh RM48
An extracellular α-galactosidase from Aspergillus niger v. Tiegh RM48 was purified to apparent homogeneity by ammonium sulfate (80% saturation) precipitation, Phenyl Sepharose CL-4B hydrophobic column chromatography and Sephadex G-100 filtration chromatography. The specific activity of the enzyme wa...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2009-03-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2009.02.005 |
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| Summary: | An extracellular α-galactosidase from Aspergillus niger v. Tiegh RM48 was purified to apparent homogeneity by ammonium sulfate (80% saturation) precipitation, Phenyl Sepharose CL-4B hydrophobic column chromatography and Sephadex G-100 filtration chromatography. The specific activity of the enzyme was increased approximately 103-fold, from 26.29 IU·mg<sup>-1</sup> protein to 2722.3 IU. mg<sup>-1</sup> protein. The molecular mass of the purified enzyme as determined by SDS-PAGE was 108 kDa. The optimum pH and temperature of this α-galactosidase were pH 4.5 and 60 ℃, respectively. The enzyme was stable between pH 3.0 and pH 6.0, however, its activity was inhibited by all tested metal ions. α-Galactosidase activity was strongly inhibited by Cu<sup>2+</sup>, Fe<sup>2+</sup> and moderately inhibited by Li<sup>2+</sup>, Zn<sup>2+</sup>, Mn<sup>2+</sup>, K<sup>+</sup>, Na<sup>+</sup>, Ca<sup>2+</sup>, Mg<sup>2+</sup> and Co<sup>2+</sup>. Kinetic studies of the α-galactosidase showed that the K<sub>m</sub> and the V<sub>max</sub> for 4-nitrophenyl-α-D-galactopyranoside (pNPG) were 7.37 mmol·L<sup>-1</sup> and 5618 IU· mg<sup>-1</sup>·min<sup>-1</sup>, respectively. The N-terminal amino acids sequence of the α-galactosidase protein was determined as DEKAYSPCYY, no evidence showed the apparent sequence similarity between this enzyme and any other α-galactosidase listed in the database. |
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| ISSN: | 1008-9209 2097-5155 |