Abstract
BackgroundMacroalgae 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Macroalgae and microalgae, as feedstocks for third-generation biofuel, possess competitive strengths in terms of cost, technology and economics
Gel filtration analysis showed that under the given reaction conditions, a small amount of Unsaturated monosaccharide (UDP1) was produced when using AlyPB2 only, while the yield of UDP1 was dramatically increased approximately sevenfold when AlyPB1 and AlyPB2 acted on alginate together (Fig. 8b). These results suggest that the digestion of alginate by AlyPB1 strongly promotes the activity of AlyPB2 to release UDP1 from substrates, which is consistent with the finding that AlyPB2 prefers to digest alginate oligosaccharides rather than polysaccharides
We found that 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 over a short time, demonstrating that the synergistic effect of AlyPB1 and AlyPB2 was quite remarkable
Summary
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. Macroalgae and microalgae are attracting increasing attention and are emerging as alternative and environmentally friendly feedstocks for the production of biofuels, such as bio-ethanol, bio-hydrogen and biooil, due to rising energy demands [1,2,3,4]. The main polysaccharide component of brown macroalgae is alginate, which composes approximately 40% of the dry weight of algal biomass [9]. Alginate is the major component of the brown algal cell wall and has recently attracted interest due to its potential for biofuel, food and pharmaceutical applications [13, 14]
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