Saccharification Of Alginate Research Articles

Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615

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.

Saccharification of Brown Macroalgae Using an Arsenal of Recombinant Alginate Lyases: Potential Application in the Biorefinery Process

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

Comparison of different types of pretreatment and enzymatic saccharification of Macrocystis pyrifera for the production of biofuel

In this work, the brown algae Macrocystis pyrifera were pretreated with dilute sulfuric acid, water and three different types of ionic liquids (ILs): 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1,5-diazabicyclo[4.3.0]non-5-ene acetate ([DBNH][OAc]) and 1,8-diazabicyclo-[5.4.0]–undec-7-ene–sulfurdioxide–monoethanolamine (DBU–MEA–SO2–SIL), to disassemble the complex polysaccharide structure. After each pretreatment procedure, enzymatic saccharification was performed to release the monosaccharides. The main building blocks of M. pyrifera were processed by derivatization via acid methanolysis and subjected to gas chromatographic analysis. It was found that the main constituents were alginate (60.6wt.%) and cellulose (22.6wt.%) of total carbohydrate content. The degradation of alginate requires the action of alginate lyase and oligoalginate lyase, which hydrolyze the main chain in a synergistic mechanism releasing uronic acid (unsaturated uronate). Upon saccharification of cellulose, cellulases and β-glucosidase were used allowing the release of glucose. It was found that the best pretreatment strategy for M. pyrifera consisted of a pretreatment with 2vol.% sulfuric acid, followed by saccharification of cellulose with a mixture of cellulases at pH5.2 for 4h at 50°C or by saccharification of alginate with the enzyme lyase/oligoalginate lyase at pH7.5 for 2h at 37°C. The process resulted in a release of 68.4wt.% of glucose (55.74±0.05mg glucose/g algae) whereas in the case of alginate 85.8wt.% of uronic acid (193.7±10.6mg uronic acid/g algae) was released. To the best of our knowledge this is the first time that saccharification of both cellulose and alginate from brown algae is reported.