[A novel alpha-galactosidase from Arthrobacter sp. GN14 isolated from Grus nigricollis feces: gene cloning, heterologous expression and characterization].
Клучни зборови
Апстракт
OBJECTIVE
Cloning and heterologously expressing the alpha-galactosidase gene (agaAGN14) from Arthrobacter sp. GN14 isolated from feces of black-neck crane (Grus nigricollis).
METHODS
The full-length agaAGN14 was cloned based on degenerate PCR and GC TAIL-PCR (thermal asymmetric interlaced PCR), ligated into pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. The recombinant alpha-galactosidase (rAgaAGN14) was purified to electrophoretic homogeneity by Ni(2+)-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. Amino acids sequences of agaAGN14 (AgaAGN14) and alpha-galactosidases from Actinobacteria and gastrointestinal microorganisms were aligned and used for constructing a neighbor-joining phylogenetic tree.
RESULTS
The 2109-bp full-length agaAGN14 (66.8% GC content) encodes a 702-residue polypeptide (AgaAGN14; 77.5 kDa). AgaAGN14 showed the highest identity of 53.7% with alpha-galactosidases in public databases, and < 43% identities with alpha-galactosidases from gastrointestinal microorganisms. AgaAGN14 was put in a phylogenetic branch sharing the catalytic motifs KWD and SDXXDXXXR, and close to alpha-galactosidases from soil microorganisms and far from alpha-galactosidases from gastrointestinal microorganisms. The purified rAgaAGN14 efficiently hydrolyzed pNPG, raffinose, melibiose, stachyose, rapeseed meal and cottonseed meal; showed apparent optimal at pH 6.0 and 45 degrees C, stability and activity (> 50%) at pH 6.0-9.0, and activities of 28%, 30% and 80% at 10 degrees C, 20 degrees C and 37 degrees C, respectively; exhibited K(m), V(max) and k(cat) values of 0.41 mmol/L, 18.28 micromol/min/mg and 25.36 s(-1), respectively, using pNPG as the substrate at 45 degrees C and pH 6.5; strongly inhibited by Ag+, Hg2+ and SDS, partial inhibited by K+, Ca2+, Mn2+, Fe3+, Ni2+, Cu2+ and beta-mercaptoethanol, and little influenced by Co2+, Pb2+, Zn2+, Mg2+, Na+ and EDTA.
CONCLUSIONS
The Arthrobacter strain isolated from feces of Grus nigricollis, and the sequence analysis, phylogenetic analysis, heterologous expression and recombinant enzyme's biochemical characterizations of an alpha-galactosidase from Arthrobacter strain were first reported. rAgaAGN14 was a novel alpha-galactosidase.
