Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
Biochimie. 2009 Nov-Dec;91(11-12):1434-42. doi: 10.1016/j.biochi.2009.07.018. Epub 2009 Aug 14.
Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES-GroEL chaperones. Recombinant LJAG31 was an alpha-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k(cat)/K(m)) toward nigerose, maltulose, and kojibiose than other natural substrates having an alpha-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial alpha-1,3-glucosidase to be characterized with a high k(cat)/K(m) value for nigerose [alpha-d-Glcp-(1 --> 3)-d-Glcp]. Transglucosylation of 4-nitrophenyl alpha-d-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl alpha-nigeroside and 4-nitrophenyl alpha-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) alpha-1,3-glucosidase of glycoside hydrolase family 31.
最近完成了对约翰逊乳杆菌 NCC533 的基因组分析。其注释基因 lj0569 编码具有糖苷水解酶家族 31 保守结构域的蛋白质。其在约翰逊乳杆菌 NBRC13952 中的同源基因 ljag31 被克隆并使用大肠杆菌表达系统表达，由于形成包涵体，导致重组 LJAG31 蛋白产量低。通过在培养基中添加高浓度 NaCl、可能通过苯甲醇诱导内源性伴侣蛋白、以及过表达 GroES-GroEL 伴侣蛋白，可提高可溶性重组 LJAG31 的产量。重组 LJAG31 是一种具有广泛底物特异性的α-葡萄糖苷酶，对均相和异相底物均具有活性。与具有非还原端α-葡萄糖苷键的其他天然底物相比，该酶对黑曲霉二糖、麦芽三糖和异麦芽三糖具有更高的特异性（kcat/Km），这表明这些糖可能是有助于约翰逊乳杆菌生长的益生元候选物。据我们所知，LJAG31 是第一个具有高 kcat/Km 值（针对黑曲霉二糖[α-d-Glcp-（1 --> 3）-d-Glcp]）的细菌α-1,3-葡萄糖苷酶。4-硝基苯基α-d-吡喃葡萄糖苷的转葡糖苷作用产生了两种 4-硝基苯基二糖（4-硝基苯基α-黑曲霉二糖苷和 4-硝基苯基α-异麦芽二糖苷）。LJAG31 的这些水解和转葡糖苷性质与糖苷水解酶家族 31 的霉菌（Acremonium implicatum）α-1,3-葡萄糖苷酶不同。
