INRA, UMR 1163 BCF, 13288 Marseille, France.
Microb Cell Fact. 2011 Dec 6;10:103. doi: 10.1186/1475-2859-10-103.
The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome.
PpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five.
The present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.
在毕赤酵母 GS115 基因组中鉴定出一种编码具有五个不同家族 1 碳水化合物结合模块（CBM1）的非典型多模块糖苷水解酶家族 45 内切葡聚糖酶的基因，命名为 PpCel45A。
成功地在毕赤酵母 X33 中高产量（高达每升培养物 2 克）表达了 PpCel45A（全长开放阅读框）以及三个衍生构建体，包括（i）带有近侧 CBM1 的催化模块，（ii）仅催化模块，和（iii）没有催化模块的五个 CBM1 模块。尽管含有催化模块的构建体对一系列葡聚糖表现出相似的活性，但它们的生化特性比较显示出显著的差异。我们观察到 PpCel45A 的高耐热性（80°C 时半衰期为 6 小时），随着 CBM 和糖基化接头的去除而降低。然而，与存在一个 CBM 相比，一个 CBM 的存在显著增强了对结晶纤维素的结合和结晶纤维素和纤维六糖的水解。
本研究揭示了来自酵母的第一个特征明确的内切 β-1,4 葡聚糖酶的特定特征，其热稳定性有望在与木质纤维素生物质糖化相关的生物技术应用中得到应用，如整合生物加工。
