| a-galactosidase (EC 3.2.1.22) is a kind of hydrolytic enzyme which can cleaves a-l-6-linked D-galactosyl residues from oligosaccharide and polysaccharide. It have extensive application to food and feed industry, transformation of blood type, the remedy of Fabry, and organ transplantation. Especially in the feed industry, a-galactosidase is an effective method to removal the galactose in soybean meal. We purified this enzyme from the fruiting body of Pleurotus citrinopileatus, Pleurotus djamor and Agaricus bisporus and studied its enzymology properties and degradation effect of oligosaccharide.An acidic a-galactosidase designated as PCGI was isolated from the fresh fruiting bodies of Pleurotus citrinopileatus with 264-fold purification and a specific activity of 7.92 units/mg by ion exchange chromatography and gel filtration chromatography. PCGI is a heterodimeric protein with a molecular mass 57 kDa, consisting of a 33 kDa and a 27 kDa subunit. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 50℃, respectively. Besides, it displayed remarkable resistance to acid protease, neutral protease, a-chymotrypsin, and trypsin. It was strongly inhibited by SDS, Cd2+, Cu2+, Hg2+, Al3+, Fe3+and Ag+ions. Diethypyrocarbonate (DEPC) doubled the activity of PCGI whereas N-bromosuccinimide (NBS) drastically decreased it, indicating tryptophan and histidine residues is necessary groups of the enzyme activity. Futhermore, the results of TCL and substrate specificity showed that it completely degraded raffinose and sthachyose.An acidic a-galactosidase designated as PDGI was purified to homogeneity from the fruiting bodies of Pleurotus djamor with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60 kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 50℃, respectively. Besides, it displayed broad pH stability and resistance to acid protease. It was strongly inhibited by K+, Cd2+, Cu2+, Hg+, Al3+, Fe3+ and Ag+ ions. The chemical modification reagent diethypyrocarbonate (DEPC), 2,3-butanedione (DIC) and trinitrophenol (TNBS) increased the activity of PDGI 1.5-fold whereas N-bromosuccinimide (NBS) and parachloro-mercuri-benzoate (PCMB) drastically suppressed its activity. PDGI displayed activity toward substrates such as stachyose, raffinose, melibiose, locust bean gum, guar gum,2-nitrolphenyl P-D-galactopyranoside. respectively. Furthermore, it completely degraded raffinose and sthachyose.An acidic a-galactosidase designated as ABGI was purified to homogeneity from the fruiting bodies of Agaricus bisporus with 3079-fold purification and a specific activity of 193.12 units/mg by means of ion exchange chromatography and gel filtration chromatography. ABGI is a monomeric protein exhibiting a molecular mass of 43 kDa in SDS-PAGE and gel filtration. Blast search of internal peptide sequences suggested that ABGI is a member of GH family 27. The optimum pH and temperature of the enzyme with pNPGal as substrate were 4.0 and 60℃, respectively. The enzyme remained stable over a pH range of 2.0-9.0. Besides, it displayed resistance to neutral protease and α-chymotrypsin. It was strongly inhibited by Cu2+, Hg2+, Fe3+, Ag+ and SDS. The chemical modification reagent diethypyrocarbonate (DEPC),2,3-butanedione (DIC), carbodiimide (EDC) and trinitrophenol (TNBS) slightly increased the activity of ABGI whereas N-bromosuccinimide (NBS), dithiothreitol (DTT) and parachloro-mercuri-benzoate (PCMB) drastically decreased it. Moreover, it could degraded pNPG, raffinose, sthachyose, guar gum, locust bean gum and melibiose, and the degrade rate was 100%,148.3%,41.5%,30.1%,20.0% and 4.4%, respectively.The above results show that PCGI, PDGI, ABGI have good pH stability and resistance of protease. On one hand it broaden the source of better alpha-galactosidase, on the other hand it can remove the RFOs and relieve the flatulence induced by the RPOs. In addition, we can use ABGI to degradate and of guar gum to improve its commercial value. Hence, PCGI, PDGI, ABGI is a promising candidate for elimination of raffinose oligosaccharides (RPOs) in biotechnological applications. |
PDF Full Text Request