肠膜明串珠菌α-葡萄糖苷酶基因克隆表达及酶学性质

Cloning and Expression of an α-Glucosidase Gene from Leuconostoc mesenteroides and Biochemical Characterization of the Recombinant Enzyme

肠膜明串珠菌(Leuconostoc mesentcroides ATCC8293)基因组中存在一个编码糖苷酶家族3保守区段的基因(Leum-0289),根据该基因序列设计引物,经过扩增和重组,成功将该基因插入到表达载体pET28a(+)中,实现了该基因的诱导表达。该基因包含2226个碱基,编码742个氨基酸,编码蛋白质理论预测分子质量为75kDa,表达蛋白经SDS-PADE分析,分子质量与理论值基本一致。纯化后的蛋白经酶活测定,发现该酶只能水解α-对硝基苯酚葡萄糖苷(pNPG)底物,无水解甘露糖、蔗糖、纤维二糖、乳糖和龙胆二糖的活性,遂命名为α-葡萄糖苷酶。进一步酶学性质研究表明该酶的最适反应温度和最适反应pH分别为40℃和pH6.5,Fe2+、Ca2+、Mg2+对重组α-葡萄糖苷酶有明显激活作用,而Cu2+、Zn2+对其有一定的抑制作用。

A putative glycosidase family 3 gene （Leum _0289） was amplified from Leuconostoc rnesentcroides ATCC 8293 genomic DNA and sub-cloned into the vector pET28a（ ＋ ） to obtain a recombinant plasmid pET28a-Glu. The gene is composed of 2 226 bp nucleotides which encodes 742 amino acid residues with calculated molecular mass of 75 kDa. The enzyme assay results showed that the protein can not hydrolyze maltose, sucrose, cellobiose, lactose and gentiobiose except p-nitrohenyl-α- glucopyranoside substrate. The protein was then designated α-glucosidase. The optimum temperature and pH was 40℃ and 6.5, respectively. The enzyme is activated by Fe^2 ＋ , Ca^2＋ and Mg^2＋ , but inhibited by Cu^2＋ and Zn^2 ＋.