Hoffmam Zaira B, Zanphorlin Letícia M, Cota Junio, Diogo José A, Almeida Gabriela B, Damásio André R L, Squina Fabio, Murakami Mario T, Ruller Roberto
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil.
Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil.
N Biotechnol. 2016 Jun 25;33(4):467-72. doi: 10.1016/j.nbt.2016.02.006. Epub 2016 Feb 23.
Xylanases catalyze the hydrolysis of β-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was used to supplement the commercial cocktail Accellerase® 1500 and improved the reducing sugar release by 17% from pretreated sugarcane bagasse. These results indicate that CBM6 can be used as a molecular tool to enhance the catalytic performance of endo-xylanases (GH11) and provide a new strategy for the development of optimized biocatalysts for biotechnological applications.
木聚糖酶催化从木聚糖链中水解β-1,4-连接的木糖基部分，木聚糖是植物细胞壁中最丰富的半纤维素多糖之一。这些酶可以以单一催化结构域的形式存在，也可以以模块化蛋白质的形式存在，该模块化蛋白质由一个或多个连接到催化核心的碳水化合物结合模块（CBM）组成。然而，控制催化结构域与其CBM之间协同效应的分子机制尚未完全了解。因此，本研究的目的是评估属于第6家族的CBM（对木聚糖具有高亲和力）与枯草芽孢杆菌的GH11木聚糖酶（其野生型形式没有CBM）融合的功能效果。野生型酶（BsXyl11）和嵌合蛋白（BsXyl11-CBM6）在大肠杆菌中异源产生，并纯化至同质以进行生化表征。分子融合没有改变对pH和温度的依赖性，但动力学数据显示嵌合酶的催化效率提高了65%。此外，BsXyl11-CBM6嵌合体用于补充商业混合物Accellerase® 1500，并使预处理甘蔗渣的还原糖释放量提高了17%。这些结果表明，CBM6可以用作增强内切木聚糖酶（GH11）催化性能的分子工具，并为开发用于生物技术应用的优化生物催化剂提供了新策略。
