Molecular Characterization of the ��-Galactosidase SCO0284 from Streptomyces coelicolor A3(2), a Family 27 Glycosyl Hydrolase

Abstract

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.

Characterization of a Deep-Sea Actinobacterium Strain Uncovers Its Prominent Capability of Utilizing Taurine and Polyvinyl Alcohol

“…The Actinobacteria bacteria ubiquitously distribute in marine sediments ( Solano et al, 2009 ), sea water ( Sheikh et al, 2019 ), marine organic aggregates ( Lam, 2006 ), marine sponges ( Gandhimathi et al, 2009 ; Sheikh et al, 2019 ), and deep-sea gas hydrate reservoirs ( Wang et al, 2014 ). Notably, Actinobacteria have broad application potentials, such as production of large number of secondary metabolites for developing novel antibiotics ( Wang et al, 2014 ; Kamjam et al, 2017 ); production of variety of enzymes including alkaline protease, xylanase, α-galactosidase ( Temuujin et al, 2016 ; Sanjivkumar et al, 2017 ; Thakrar and Singh, 2019 ). In addition, Actinobacteria could degrade and metabolize foreign compounds such as heavy metals, hydrocarbons, pesticides and plastics, and they are also potential candidates for bioremediation ( Rathore et al, 2021 ).…”