The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

中文翻译：

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黄褐球菌FD-1 v3家族5糖苷水解酶第一内切甘露聚糖酶（RfGH5_7）的分子克隆，表达及生化特征。

黄褐球菌的纤维素酶RfGH51 / 2包含一个N端模块，即具有推定的内切葡聚糖酶活性的5族糖苷水解酶GH5_4，而C端结构域则是一个推定的内甘露聚糖酶（GH5_7）。两个推定的催化模块被具有广泛配体特异性的80族碳水化合物结合模块（CBM80）隔开。克隆假定的甘露聚糖内切酶模块GH5_7（RfGH5_7），在大肠杆菌BL-21（DE3）细胞中表达并纯化。纯化的RfGH5_7的SDS-PAGE分析显示，分子大小约为35 kDa。RfGH5_7的底物特异性分析显示，对刺槐豆半乳甘露聚糖（298.5 U / mg）具有最大活性，其次是魔芋葡甘露聚糖（256.2 U / mg）和角豆半乳甘露聚糖（177.2 U / mg）。RfGH5_7在最佳pH 6.0和60°C的温度下显示出最大活性。RfGH5_7在pH 6.0和9.0之间显示稳定性，并在50°C之前保持热稳定性。10 mM Ca2 +离子使酶活性提高了33％。RfGH5_7的熔融温度为84°C，不受Ca2 +离子或螯合剂的影响。RfGH5_7对刺槐豆半乳甘露聚糖的Vmax为389 U / mg，Km为0.92 mg / mL。TLC分析表明，RfGH5_7将刺槐豆的半乳甘露聚糖水解成甘露糖，甘露二糖，甘露三糖，并且甘露寡糖的聚合度更高，表明其具有内在作用催化机理。这项研究揭示了一种高活性和热稳定的甘露聚糖内切酶，具有相当大的生物技术潜力。RfGH5_7的熔融温度为84°C，不受Ca2 +离子或螯合剂的影响。RfGH5_7对刺槐豆半乳甘露聚糖的Vmax为389 U / mg，Km为0.92 mg / mL。TLC分析表明，RfGH5_7将刺槐豆的半乳甘露聚糖水解成甘露糖，甘露二糖，甘露三糖，并且甘露寡糖的聚合度更高，表明其具有内在作用的催化机理。这项研究揭示了一种高活性和热稳定的甘露聚糖内切酶，具有相当大的生物技术潜力。RfGH5_7的熔融温度为84°C，不受Ca2 +离子或螯合剂的影响。RfGH5_7对刺槐豆半乳甘露聚糖的Vmax为389 U / mg，Km为0.92 mg / mL。TLC分析表明，RfGH5_7将刺槐豆的半乳甘露聚糖水解成甘露糖，甘露二糖，甘露三糖，并且甘露寡糖的聚合度更高，表明其具有内在作用催化机理。这项研究揭示了一种高活性和热稳定的甘露聚糖内切酶，具有相当大的生物技术潜力。