Molecular Characterization of the ��-Galactosidase SCO0284 from Streptomyces coelicolor A3(2), a Family 27 Glycosyl Hydrolase
| Autor: | Jae-Seon Park, Soon-Kwang Hong, Uyangaa Temuujin |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Streptomyces coelicolor
010501 environmental sciences 01 natural sciences Applied Microbiology and Biotechnology Mass Spectrometry Substrate Specificity Stachyose chemistry.chemical_compound Bacterial Proteins Hydrolase Glycoside hydrolase Glycosyl Raffinose Melibiose 0105 earth and related environmental sciences Alpha-galactosidase biology 010405 organic chemistry Temperature General Medicine biology.organism_classification Recombinant Proteins 0104 chemical sciences Kinetics Biochemistry chemistry alpha-Galactosidase biology.protein Streptomyces lividans Chromatography Thin Layer Biotechnology |
| Zdroj: | Journal of Microbiology and Biotechnology. 26:1650-1656 |
| ISSN: | 1738-8872 1017-7825 |
| Popis: | The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40°C and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters Vmax and Km for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose. |
| Databáze: | OpenAIRE |
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