Maruta Akiho, Yamane Mirei, Matsubara Midori, Suzuki Shiho, Nakazawa Masami, Ueda Mitsuhiro, Sakamoto Tatsuji
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan; International Polysaccharide Engineering Inc., Center for R&D of Bioresources, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan.
Enzyme Microb Technol. 2017 Aug;103:25-33. doi: 10.1016/j.enzmictec.2017.04.006. Epub 2017 Apr 29.
We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-d-galactopyranosidase (GPase) and β-l-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-l-arabinofuranosidase released four times more arabinose than α-l-arabinofuranosidase acting alone. This suggested that a large number of α-l-arabinofuranosyl residues is capped by α-galactosyl residues. H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-d-Galp-(1→3)-α-l-Araf-(1→] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.
我们之前报道过尖孢镰刀菌12S能产生两种具有α - d - 吡喃半乳糖苷酶（GPase）和β - l - 吡喃阿拉伯糖苷酶（APase）活性的双功能蛋白，即FoAP1和FoAP2。本文的目的是从尖孢镰刀菌12S的培养上清液中纯化出第三种GPase，即FoGP1，对其进行表征，并确定其对阿拉伯胶的作用方式。克隆了编码FoGP1的cDNA，并在大肠杆菌中对该蛋白进行了过量表达。模块序列分析显示FoGP1中存在一个GH27结构域。重组酶（rFoGP1）的GPase/APase活性比为330，这与FoAP1（1.7）和FoAP2（0.2）有很大不同。在所测试的天然底物中，rFoGP1对阿拉伯胶的活性最高。与其他已充分表征的GPases不同，rFoGP1从棉子糖等α - 半乳糖基寡糖中释放出少量半乳糖，且对高度被α - 半乳糖基侧链取代的半乳甘露聚糖无活性。这表明FoGP1是一种不同寻常的GPase类型。rFoGP1从阿拉伯胶中释放出了30%的总半乳糖，这表明阿拉伯胶侧链的非还原端存在大量α - 半乳糖基残基。rFoGP1和α - l - 阿拉伯呋喃糖苷酶共同作用释放出的阿拉伯糖比单独的α - l - 阿拉伯呋喃糖苷酶多四倍。这表明大量的α - l - 阿拉伯呋喃糖基残基被α - 半乳糖基残基封端。核磁共振氢谱实验表明，rFoGP1水解了阿拉伯胶中[α - d - Galp - (1→3) - α - l - Araf - (1→)]侧链结构内的α - 1,3 - 半乳糖苷键。总之，rFoGP1对α - 1,3 - 半乳糖苷键具有高活性，但对α - 1,6 - 半乳糖苷键活性可忽略不计或无活性。这种新型的FoGP1可能用于改变阿拉伯胶的物理性质，阿拉伯胶是一种在工业上用作乳化稳定剂和涂层剂的重要多糖。
