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 Photobacterium 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 1H 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.

中文翻译：

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两种新型藻酸盐裂合酶的生化特性和协同作用。FC615

巨藻和微藻作为第三代生物燃料的原料，在成本、技术和经济等方面具有竞争优势。褐藻中最重要的化合物是海藻酸盐，内溶性和外溶性海藻酸盐裂解酶的协同作用在将海藻酸盐转化为生物燃料的糖化过程中起着至关重要的作用。然而，很少有关于内溶和外溶藻酸盐裂合酶的协同作用的研究，尤其是来自同一菌株的那些。在这项研究中，从海洋细菌 Photobacterium sp 中鉴定出内溶藻酸裂解酶 AlyPB1 和外溶藻酸裂解酶 AlyPB2。FC615。这两种酶表现出完全不同和新颖的酶特性，而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，当AlyPB1和AlyPB2一起作用于海藻酸盐时，海藻酸盐多糖向不饱和单糖的转化率显着提高了约7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，特别是由于它们的 M-/G-偏好和底物尺寸依赖性。此外，首次开发了一种将1H NMR光谱和酶消化与外切酶AlyPB2相结合的海藻寡糖测序新方法，该方法比基于一维和二维的传统方法简单得多。核磁共振波谱。使用这种策略，由 AlyPB1 产生的最终四糖和五糖产物级分的序列很容易确定：四糖级分含有两种结构，ΔGMM 和 ΔMMM，摩尔比为 1:3.2，五糖级分含有四种结构，ΔMMMM、ΔMGMM、ΔGMMM和 ΔGGMM，摩尔比约为 1:1.5:3.5:5.25。这两种新型海藻酸裂解酶的鉴定不仅为寡糖制备提供了极好的候选工具型酶，而且为研究海藻酸在生物燃料生产中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海藻酸盐的结构和功能研究提供非常有用的方法。ΔGMM 和 ΔMMM，摩尔比为 1:3.2，五糖级分包含四种结构，ΔMMMM、ΔMGMM、ΔGMMM 和 ΔGGMM，摩尔比约为 1:1.5:3.5:5.25。这两种新型海藻酸裂解酶的鉴定不仅为寡糖制备提供了极好的候选工具型酶，而且为研究海藻酸在生物燃料生产中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海藻酸盐的结构和功能研究提供非常有用的方法。ΔGMM 和 ΔMMM，摩尔比为 1:3.2，五糖级分包含四种结构，ΔMMMM、ΔMGMM、ΔGMMM 和 ΔGGMM，摩尔比约为 1:1.5:3.5:5.25。这两种新型海藻酸裂解酶的鉴定不仅为寡糖制备提供了极好的候选工具型酶，而且为研究海藻酸在生物燃料生产中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海藻酸盐的结构和功能研究提供非常有用的方法。这两种新型海藻酸裂解酶的鉴定不仅为寡糖制备提供了极好的候选工具型酶，而且为研究海藻酸在生物燃料生产中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海藻酸盐的结构和功能研究提供非常有用的方法。这两种新型海藻酸裂解酶的鉴定不仅为寡糖制备提供了极好的候选工具型酶，而且为研究海藻酸在生物燃料生产中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海藻酸盐的结构和功能研究提供非常有用的方法。