一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质

Purification and characterization of a novel α-galactosidase from Penicillium sp.F63 CGMCC1669

摘要:

从丝状真菌中筛选到一株产α-半乳糖苷酶的菌株F63,对该菌株进行了形态观察和18SrDNA序列分析,该菌株属于青霉属。采用硫酸铵沉淀、阴离子交换层析和分子筛层析等方法分离纯化了该菌株的一种α-半乳糖苷酶。经过聚丙烯酰胺凝胶电泳,此酶蛋白的分子量约为82kDa。该α-半乳糖苷酶反应的最适pH为5.0,最适温度为45℃。此α-半乳糖苷酶的热稳定性在40℃以下,pH稳定性为pH5.0-6.0。与已报道的α-半乳糖苷酶的活性都受到Ag⁺的强烈抑制不同的是,该α-半乳糖苷酶受Ag⁺的抑制作用不显著。以pNPG为底物的Km值为1.4mmol/L和V_max=1.556mmol/L.min^-1.mg^-1。该酶可以有效降解蜜二糖、棉子糖和水苏糖,但不能降解末端含α-半乳糖苷键的多糖。通过利用质谱技术对纯化的α-半乳糖苷酶进行鉴定以及内肽的N端测序证明该蛋白为一种新的α-半乳糖苷酶。

Abstract:

An α-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The α-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The α-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45℃. The enzyme is stable between pH5.0 and 6.0 below 40℃. The α-galactosidase activity is slightly inhibited by Ag⁺, which is dissimilar to other α-galactosidases. Kinetic studies of the α-galactosidase showed that the K_m and the V_max for pNPG are 1.4mmol/L and 1.556mmol/L.min^－1.mg^－1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the α-galactosidase is a novel one.