IFP Energies Nouvelles, 1 - 4 avenue du Bois-Préau, 92852 Rueil-Malmaison, France.
Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, 163 avenue de Luminy, 13288 Aix Marseille Université, Marseille, France; INRAE, USC1408 Architecture et Fonction des Macromolécules Biologiques (AFMB), 163 avenue de Luminy, 13288 Marseille, France; Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.
Biochim Biophys Acta Gen Subj. 2021 May;1865(5):129848. doi: 10.1016/j.bbagen.2021.129848. Epub 2021 Jan 15.
Environmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes.
In the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized.
A phylogenetic analysis of the catalytic domain of pro- and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome.
The characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity.
The identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.
环境细菌表达广泛的糖苷水解酶（GH）多样性。从宏基因组来源筛选和表征 GH 可以深入了解未培养原核生物对生物质的降解策略。
在本报告中，我们筛选了堆肥宏基因组中的木质纤维素活性，并鉴定了编码属于 GH9 家族的 6 种酶的基因（GH9a-f）。其中三种酶（GH9b、GH9d 和 GH9e）成功表达并进行了表征。
对原核生物和真核生物 GH9 酶的催化结构域进行系统发育分析表明，存在两个主要亚群。细菌 GH9 具有多种多样的模块化结构，那些含有 N 端 Ig 样结构域的酶，如 GH9b 和 GH9d，与其余酶分离。我们纯化和表征了这两个亚群的 GH9 内切葡聚糖酶，并研究了它们的稳定性、底物特异性和产物谱。GH9e 表现出独特的水解模式，释放出比纤维二糖长的寡糖比例更高。所有酶都表现出连续的行为和对结晶纤维素的协同作用。在同一宏基因组中鉴定的 GH9d 和 GH48 酶之间也存在协同作用。
所鉴定的 GH9 酶具有不同的模块化结构和不同的底物和产物谱。纤维素结合结构域的存在被证明对于结合和消化不溶性纤维素底物是必要的，但对于连续性不是必需的。
从堆肥宏基因组中鉴定出六种 GH9 酶，以及三种特征化成员的功能多样性，突出了该酶家族在细菌生物质解构中的重要性。
