Identification of two functionally different classes of exocellulases
Identification of two functionally different classes of exocellulases
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DOI：
10.1021/bi9520388
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发表时间：
1996-01-16
期刊：
影响因子：
2.9
通讯作者：
Wilson, DB
中科院分区：
文献类型：
--
作者：
Barr, BK;Hsieh, YL;Wilson, DB
在纤维素酶混合物中存在两类协同作用：内切纤维素酶和外切纤维素酶之间的协同作用，以及某些外切纤维素酶之间的协同作用。外切纤维素酶传统上被定义为从纤维素的非还原末端释放纤维二糖，但该定义不足以解释外切/外切协同作用。最近的一些报道表明，一些外切纤维素酶能够从还原端水解纤维素。两种具有不同特异性的外切纤维素酶类的存在可以为exo/exo协同作用提供解释。在本文中，我们报告了三个Thermomonospora fusca（E3，E4和E6）和两个里氏木霉（CBH I和CBH II）外切纤维素酶的底物特异性标记的纤维低聚糖。我们描述了一种新的非放射性技术，用于确定底物特异性，其中离子喷雾质谱法被用来分析的产品的酶促纤维五糖标记O-18在还原端。外切纤维素酶的反应性也进行了研究，在非还原性结束与C-14标记的纤维五糖，和纤维低聚糖减少与硼氢化钠。标记在反应产物中的分布支持存在两种功能类别的外切纤维素酶。一类（含有CBH I、E4和E6）优先从还原端切割纤维寡糖，而另一类（含有E3和CBH II）优先从非还原端切割。外切纤维素酶的这种分类与exo/exo协同作用实验以及已发表的纤维素酶晶体学数据一致。
There are two classes of synergism in cellulase mixtures: synergism between endocellulases and exocellulases, and synergism between certain exocellulases. Exocellulases have been defined traditionally as releasing cellobiose from the nonreducing ends of cellulose, but this definition is inadequate to explain exo/exo synergism. Several recent reports indicate that some exocellulases are capable of hydrolyzing cellulose from the reducing end. The existence of two exocellulase classes with different specificities could provide an explanation for exo/exo synergism. In this paper, we report the substrate specificity of three Thermomonospora fusca (E3, E4, and E6) and two Trichoderma reesei (CBH I and CBH II) exocellulases on labeled cellooligosaccharides. We describe a new nonradioactive technique for determining substrate specificity, in which ion-spray mass spectrometry was used to analyze the products of enzymatic digests of cellopentaose labeled with O-18 at the reducing end. Exocellulase reactivity was also investigated on cellopentaose labeled at the nonreducing end with C-14, and cellooligosaccharides reduced with NaBH4. The distribution of label in the reaction products supports the existence of two functional classes of exocellulases. One class (containing CBH I, E4, and E6) preferentially cleaves cellooligosaccharides from the reducing end, while the other (containing E3 and CBH II) preferentially cleaves from the nonreducing end. This classification of exocellulases is consistent with exo/exo synergism experiments, and with published cellulase crystallographic data.