Paenibacillus sp.K1 α-半乳糖苷酶酶学性质

Characterization of the alpha-galactosidase of Paenibacillus sp. K1

利用CODEHOP PCR和Anchor-ligated PCR方法从类芽孢杆菌Paenibacillus sp.K1中克隆得到一个α-半乳糖苷酶基因aga P1,大小为2 190 bp,同源性分析显示,该基因与其他α-半乳糖苷酶基因的序列相似低,是一个新的α-半乳糖苷酶基因。将aga P1在大肠杆菌Origami B（DE3）中表达并纯化获得Aga P1,酶学性质分析显示：以p NPG为底物时,Aga P1最适反应温度为40℃,最适p H 6.5-10,Km值为0.75 mmol/L,最大反应速率Vmax为1.96μmol·min^-1·mg^-1。同时Fe^2＋、Mg^2＋、Ca^2＋、K^＋和甘油能使α-半乳糖苷酶酶活提高1-3倍,而Cu^2＋、Zn^2＋、Fe3＋和还原型谷胱甘肽则抑制该酶的活性。SDS-PAGE检测Aga P1蛋白大小约为80 ku,与理论预测值基本一致;Native-PAGE分析表明正常条件下Aga P1蛋白以二聚体或六聚体形式存在。以上结果显示,Paenibacillus sp.K1产生的α-半乳糖苷酶为一个新的低温α-半乳糖苷酶。

A novel alpha-galactosidase encoded by aga P1 gene was amplilfied in the genome of Paenibacillus sp. K1 by CODEHOP PCR and Anchor-ligated PCR techniques. It was 2 190 bp in size and composed of 729 amino acid residues with 82. 7 ku of predicted molecular weight,and pI 5. 16. The AgaP 1 was expressed in E. coli Origami B（ DE3） and purified with His-tag chromatography column. The enzymic properties of alpha-galactosidase AgaP 1 was analysized and showed optimum temperature of 40 ℃,optimal pH of 6. 5-10,Km0. 75 mmol / L and Vmax1. 96 μmol·min- 1·mg- 1.The AgaP 1 activity could be improved one to three folds by Fe^2 ＋,Mg^2 ＋,Ca^2 ＋,K＋and glycerol,whereas Cu^2 ＋,Zn^2 ＋,Fe^3 ＋and glutathione could completely inhibit the AgaP 1 activity. SDS-PAGE revealed a single band of 80 ku,consistant with the predicted size,while two bands of 150 ku and 440 ku was observed on the native-PAGE gel,corresponding to two and six oligomers of AgaP 1. All of these results suggested that the AgaP 1 produced by Paenibacillus sp. K1 was a novel alpha-galactosidase with the highest enzymic activity at lowtemperature.