Structure of Secondary Cell Wall Polymers in Cell Surfaces of Anaerobes and Interaction between Them and SLH Domains

厌氧菌细胞表面次生细胞壁聚合物的结构及其与SLH结构域的相互作用

• 批准号: 15380060
• 负责人: SAKKA Kazuo
• 金额: $ 9.02万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380060/
• 关键词: Secondary cell wall polymers; Peotidoglycan; SLH Modules; Clostridium josui; Clostridium stercorarium; Clostridium thermocellum; SLHドメイン; 嫌気性細菌; 細胞表層タンパク質; 2次細胞壁; 細胞壁; キシラナーゼ; モジュラー酵素; クロストリジウム

1.Secondary cell wall polymers(SCWP) and peptidoglycans were prepared from 3 clostridia, C.josui, C.stercorarium and C.thermocellum2.SLH module polypeptides from C.josui xylanase Xyn10A, C.stercorarium xylanase Xyn10B and C.thermocellum cell surface protein SdbA were produced in Escherichia coli and purified.3.Interactions between these polypeptides and various cell wall fractions were investigated. As a result, it was found that the SLH module polypeptides from C.josui Xyn10A and C.stercorarium Xyn10B bound to SCWP but that the SLH module polypeptide from C.thermocellum SdbA unexpectedly bound to peptidoglycan.4.Binding assay using peptidoglycan fraction prepared from E.coli confirmed that the SLH module polypeptide from C.thermocellum SdbA had an affinity for peptidoglycan although it is generally believed that SLH modules bind to SCWP. The SLH module polypeptide from C.thermocellum SdbA should be applicable for cell surface engineering in bacteria lacking SCWP.5.SCWP of C.josui, C.stercorarium and C.thermocellum were hydrolyzed with HC1 and constituent amino sugars were qualitatively analyzed. N-Acetylglucosamine and N-acetylmannosamine were detected as constituents for C.stercorarium SCWP and N-acetylgalactosamine as well as these amino sugars were identified for C.josui and C.thermocellum SCWP, suggesting that SCWP of these two bacteria include an unidentified structure.

1.以3种梭状芽孢杆菌、c.j usui、c.c stercorarium和c.c thermocell2为原料制备了次级细胞壁聚合物（SCWP）和肽聚糖。在大肠杆菌中制备并纯化了木聚糖酶Xyn10A、木聚糖酶Xyn10B和热细胞表面蛋白SdbA的SLH模块多肽。研究了这些多肽与不同细胞壁组分之间的相互作用。结果发现，来自c.j usui Xyn10A和c.c stercorarium Xyn10B的SLH模块多肽与SCWP结合，而来自c.c thermocellum SdbA的SLH模块多肽却出乎意料地与肽聚糖结合。利用大肠杆菌制备的肽聚糖片段进行结合实验，证实了来自C.thermocellum SdbA的SLH模块多肽对肽聚糖具有亲和力，尽管一般认为SLH模块与SCWP结合。产自C.thermocellum SdbA的SLH模块多肽可用于缺乏scwp的细菌的细胞表面工程。用HC1水解葡萄球菌、牛耳球菌和热cellum的SCWP，并对其组成氨基糖进行定性分析。在C.stercorarium SCWP和N-acetylmannosamine中检测到N-Acetylglucosamine和N-acetylmannosamine，在C.josui和C.thermocellum SCWP中检测到这些氨基糖，表明这两种细菌的SCWP包含一个未知的结构。

项目成果

R.Araki, T.Kimura, K.Sakka, K.Ohmiya 他: "Essential role of the family-22 carbohydrate-binding modules for β-1,3-1,4-glucanase activity of Clostridium stercorarium Xyun10B"FEBS Lett.. 561. 155-158 (2004)

R.Araki、T.Kimura、K.Sakka、K.Ohmiya 等人：“家族 22 碳水化合物结合模块对粪臭梭菌 Xyun10B 的 β-1,3-1,4-葡聚糖酶活性的重要作用”FEBS莱特。561。155-158（2004）