Department of Microbiology, University of Georgia, Athens, Georgia, USA
Appl Environ Microbiol. 2020 Jun 2;86(12). doi: 10.1128/AEM.02275-19.
27C64, a Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities, was isolated previously from an insect hindgut. Previous work suggested that this organism's pectin deconstruction system differs from known systems in that its sole pectin methylesterase is cytoplasmic, not extracellular. In this work, we have characterized the specific roles of key extracellular pectinases involved in homogalacturonan deconstruction, including four pectate lyases and one pectin lyase. We show that one newly characterized pectate lyase, PelC, has a novel substrate specificity, with a lower for highly methylated pectins than for polygalacturonic acid. PelC works synergistically with PelB, a high-turnover exo-pectate lyase that releases Δ4,5-unsaturated trigalacturonate as its major product. It is likely that PelC frees internal stretches of demethylated homogalacturonan which PelB can degrade. We also show that the sole pectin lyase has a high value and rapidly depolymerizes methylated substrates. Three cytoplasmic GH105 hydrolases were screened for the ability to remove terminal unsaturated galacturonic acid residues from oligogalacturonide products produced by the action of extracellular lyases, and we found that two are active on demethylated oligogalacturonides. This work confirms that efficient homogalacturonan deconstruction in 27C65 does not require extracellular pectin methylesterase activity. Three of the extracellular lyases studied in this work are also thermostable, function well over a broad pH range, and have significant industrial potential. Pectin is an important structural polysaccharide found in most plant cell walls. In the environment, pectin degradation is part of the decomposition process that turns over dead plant material and is important to organisms that feed on plants. Industrially, pectinases are used to improve the quality of fruit juices and can also be used to process coffee cherries or tea leaves. These enzymes may also prove useful in reducing the environmental impact of paper and cotton manufacturing. This work is significant because it focuses on a Gram-positive bacterium that is evolutionarily distinct from other well-studied pectin-degrading organisms and differs from known systems in key ways. Most importantly, a simplified extracellular deconstruction process in this organism is able to break down pectins without first removing the methyl groups that inhibit other systems. Moreover, some of the enzymes described here have the potential to improve industrial processes that rely on pectin deconstruction.
27C64 是一种革兰氏阳性细菌，具有多种植物细胞壁多糖解构能力，先前从昆虫后肠中分离得到。以前的工作表明，该生物体的果胶解构系统与已知系统不同，因为其唯一的果胶甲酯酶是细胞质的，而不是细胞外的。在这项工作中，我们描述了参与半乳糖醛酸聚糖解构的关键细胞外果胶酶的特定作用，包括四种果胶裂解酶和一种果胶裂解酶。我们表明，一种新表征的果胶裂解酶 PelC 具有新颖的底物特异性，对高度甲基化的果胶的亲和力比对聚半乳糖醛酸低。PelC 与 PelB 协同作用，PelB 是一种高周转率的外切果胶裂解酶，其主要产物是 Δ4,5-不饱和三半乳糖醛酸。很可能 PelC 使内部延伸的去甲基化同半乳糖醛酸自由，PelB 可以降解这些同半乳糖醛酸。我们还表明，唯一的果胶裂解酶具有高 值，并迅速解聚甲基化的底物。筛选了三种细胞质 GH105 水解酶，以确定它们是否能够从细胞外裂解酶作用下产生的低聚半乳糖醛酸产物中去除末端不饱和半乳糖醛酸残基，我们发现其中两种对半甲基化的低聚半乳糖醛酸有活性。这项工作证实，在 27C65 中有效进行同半乳糖醛酸解构不需要细胞外果胶甲酯酶活性。在这项工作中研究的三种细胞外裂解酶也是耐热的，在很宽的 pH 范围内都能很好地发挥作用，并且具有重要的工业潜力。果胶是大多数植物细胞壁中发现的一种重要结构多糖。在环境中，果胶降解是分解过程的一部分，该过程使死亡的植物材料周转，并对以植物为食的生物很重要。在工业上，果胶酶用于改善果汁的质量，也可用于加工咖啡樱桃或茶叶。这些酶在减少造纸和棉花制造对环境的影响方面也可能证明是有用的。这项工作意义重大，因为它专注于一种革兰氏阳性细菌，这种细菌在进化上与其他研究充分的果胶降解生物不同，并且在关键方面与已知系统不同。最重要的是，该生物体简化的细胞外解构过程能够在不首先去除抑制其他系统的甲基基团的情况下分解果胶。此外，这里描述的一些酶有可能改进依赖果胶解构的工业过程。
