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首页> 外文期刊>Applied Microbiology >Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs
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Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

机译：青春期双歧杆菌β-甘露聚糖酶携带甘露聚糖结合和细胞协会母题的表达和表征。

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摘要

The gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k _(cat) was determined to be 444 s~(?1) and K_(m) 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.
机译：克隆了来自青春双歧杆菌（生活在人类肠道中）的β-甘露聚糖酶（EC 3.2.1.78）BaMan26A的基因，并对基因产物进行了表征。发现该酶是模块化的并且包含推定的信号肽。它具有糖苷水解酶家族26的催化模块，预计的免疫球蛋白样模块和家族23的两个推定的碳水化合物结合模块（CBM）。该酶可能是通过分选酶机制（LPXTG模体）或通过C端跨膜螺旋。该基因在大肠杆菌中表达，没有天然信号肽或细胞锚。进行了两种变体：一种包含所有四个模块，称为BaMan26A-101K，另一种在CBM之前被截短，称为BaMan26A-53K。包含CBM的BaMan26A-101K显示对解离常数为0.34μM（8.8 mg / L）的角豆半乳甘露聚糖具有亲和力，而BaMan26A-53K没有结合，表明至少23个推定的CBM为CBM。甘露聚糖绑定。对于BaMan26A-53K，使用角豆半乳甘露聚糖作为底物在最佳pH值为5.3时，k_（cat）被确定为444 s〜（？1），K_（m）为21.3 g /升。两种酶变体都将魔芋葡甘露聚糖以及角豆和瓜尔豆胶半乳甘露聚糖水解为寡糖混合物。发现象牙坚果甘露聚糖的主要产物是甘露三糖。高效阴离子交换色谱表明，甘露二糖和甘露糖的产量较低。甘露二糖不被水解，而甘露三糖的水解速度明显低于较长的寡糖。

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