Lu Danrong, Zhang Qingdong, Wang Shumin, Guan Jingwen, Jiao Runmiao, Han Naihan, Han Wenjun, Li Fuchuan
National Glycoengineering Research Center and Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, 72 Binhai Rd, Qingdao, 266200 People's Republic of China.
Biotechnol Biofuels. 2019 Nov 4;12:260. doi: 10.1186/s13068-019-1600-y. eCollection 2019.
Macroalgae and microalgae, as feedstocks for third-generation biofuel, possess competitive strengths in terms of cost, technology and economics. The most important compound in brown macroalgae is alginate, and the synergistic effect of endolytic and exolytic alginate lyases plays a crucial role in the saccharification process of transforming alginate into biofuel. However, there are few studies on the synergistic effect of endolytic and exolytic alginate lyases, especially those from the same bacterial strain.
In this study, the endolytic alginate lyase AlyPB1 and exolytic alginate lyase AlyPB2 were identified from the marine bacterium sp. FC615. These two enzymes showed quite different and novel enzymatic properties whereas behaved a strong synergistic effect on the saccharification of alginate. Compared to that when AlyPB2 was used alone, the conversion rate of alginate polysaccharides to unsaturated monosaccharides when AlyPB1 and AlyPB2 acted on alginate together was dramatically increased approximately sevenfold. Furthermore, we found that AlyPB1 and AlyPB2 acted the synergistic effect basing on the complementarity of their substrate degradation patterns, particularly due to their M-/G-preference and substrate-size dependence. In addition, a novel method for sequencing alginate oligosaccharides was developed for the first time by combining the H NMR spectroscopy and the enzymatic digestion with the exo-lyase AlyPB2, and this method is much simpler than traditional methods based on one- and two-dimensional NMR spectroscopy. Using this strategy, the sequences of the final tetrasaccharide and pentasaccharide product fractions produced by AlyPB1 were easily determined: the tetrasaccharide fractions contained two structures, ΔGMM and ΔMMM, at a molar ratio of 1:3.2, and the pentasaccharide fractions contained four structures, ΔMMMM, ΔMGMM, ΔGMMM, and ΔGGMM, at a molar ratio of ~ 1:1.5:3.5:5.25.
The identification of these two novel alginate lyases provides not only excellent candidate tool-type enzymes for oligosaccharide preparation but also a good model for studying the synergistic digestion and saccharification of alginate in biofuel production. The novel method for oligosaccharide sequencing described in this study will offer a very useful approach for structural and functional studies on alginate.
大型藻类和微藻作为第三代生物燃料的原料，在成本、技术和经济方面具有竞争优势。褐藻中最重要的化合物是藻酸盐，内切型和外切型藻酸盐裂解酶的协同作用在将藻酸盐转化为生物燃料的糖化过程中起着关键作用。然而，关于内切型和外切型藻酸盐裂解酶协同作用的研究较少，尤其是来自同一菌株的酶。
在本研究中，从海洋细菌 sp. FC615 中鉴定出内切型藻酸盐裂解酶 AlyPB1 和外切型藻酸盐裂解酶 AlyPB2。这两种酶表现出截然不同且新颖的酶学性质，同时对藻酸盐的糖化表现出强烈的协同作用。与单独使用 AlyPB2 时相比，AlyPB1 和 AlyPB2 共同作用于藻酸盐时，藻酸盐多糖向不饱和单糖的转化率显著提高了约七倍。此外，我们发现 AlyPB1 和 AlyPB2 基于其底物降解模式的互补性发挥协同作用，特别是由于它们对 M-/G-的偏好和底物大小依赖性。此外，首次开发了一种将 1H NMR 光谱与外切酶 AlyPB2 的酶切相结合的藻酸盐寡糖测序新方法，该方法比基于一维和二维 NMR 光谱的传统方法要简单得多。使用该策略，很容易确定 AlyPB1 产生的最终四糖和五糖产物馏分的序列：四糖馏分包含两种结构，ΔGMM 和 ΔMMM，摩尔比为 1:3.2，五糖馏分包含四种结构，ΔMMMM、ΔMGMM、ΔGMMM 和 ΔGGMM，摩尔比约为 1:1.5:3.5:5.25。
这两种新型藻酸盐裂解酶的鉴定不仅为寡糖制备提供了优秀的候选工具型酶，也为研究生物燃料生产中藻酸盐的协同消化和糖化提供了良好的模型。本研究中描述的寡糖测序新方法将为藻酸盐的结构和功能研究提供非常有用的途径。
