A novel α-galactosidase from Arthrobacter sp. GN14 isolated from Grus nigricollis feces: gene cloning，heterologous expression and characterization

Abstract:

Abstract:［Objective］Cloning and heterologously expressing the α-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 recombinantα-alactosidase (rAgaAGN14) was purified to electrophoretic homogeneity by Ni2 + -NTA metal chelating affinity chromatography，and then the enzyme characterizations were determined． Amino acids sequences of agaAGN14 (AgaAGN14) and α-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 α-galactosidases in public databases，and＜43% identities with α-galactosidases from gastrointestinal microorganisms.AgaAGN14 was put in a phylogenetic branch sharing the catalytic motifs KWD and SDXXDXXXR，and close to α-galactosidases from soil microorganisms and far from α-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℃，stability and activity (＞50%) at pH 6.0－9.0，and activities of 28%，30% and 80% at 10℃ ，20℃ and 37℃ ，respectively; exhibited Km，Vmax and kcat values of 0.41 mmol/L，18.28 μmol/min/mg and 25.36 s－1，respectively，using pNPG as the substrate at 45℃ and pH 6.5; strongly inhibited by Ag+，Hg2+ and SDS，partial inhibited by K+，Ca2+，Mn2+，Fe3+，Ni2+，Cu2+ and β- mercaptoethanol，and little influenced by Co2+，Pb2+，Zn2+，Mg2+，Na+and EDTA．［Conclusion］ 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 α-galactosidase from Arthrobacter strain were first reported. rAgaAGN14 was a novel α-galactosidase．