Purification and Catalyst Kinetics of α-galactosidase Derived from Lactobacillus salivarius XH4B
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Abstract:
In this study, α-galactosidase was derived from Lactobacillus salivarius XH4B (GeneBank accession number: JX1125456), and purified by super filtration and size exclusive chromatography of Sephedax G200. The results showed that the compounds with molecular weight (MW) more than 100 kDa had enzymatic activity and those washing constituents of 370~400 min showed significant activity. The SDS-PAGE appeared that the MW of the monomer of the enzyme was 70~80 kDa, while the native enzyme was a polymer with a MW exceeded 100 kDa. Compared with the crude enzyme, the purification efficiencies of super filtration and column chromatography were 149.80% and 391.91%, respectively. The optimal catalyst conditions determined by responding surface method were citric buffer pH 5.5, ion strength 0.15 mol/L and reaction temperature 52 ℃. The Km value of the enzyme to pNPG was 0.817. Compared to pure water system, only K+ and Na+ could improve the catalyst activity to 102.50% and 104.18% respectively. EDTA (96.54%), Mg2+ (91.53%), Ca2+ (82.51%), and DTT (79.38%) could reserve most of the activity, while Zn2+, Cu2+, Pb2+, Fe3+ and Vc could only remain 5~7% activity.
