Algal polysaccharides: current status and future prospects

Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of β-1,3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via elastin-like polypeptides (ELPs)-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized β-1,3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after 12 reaction cycles, demonstrating its excellent reusability. The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.

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A novel all-in-one strategy for purification and immobilization of β-1,3-xylanase directly from cell lysate as active and recyclable nanobiocatalyst

Green synthesis of silica-coated magnetic nanocarriers for simultaneous purification-immobilization of β-1,3-xylanase.

Many of the current methods for enzyme purification and immobilization suffer from several drawbacks, such as requiring tedious multistep procedures or long preparation, and being environmentally unfriendly, due to the chemicals and conditions involved. Thus, a simple technique for direct purification and immobilization of target enzymes from cell lysates was proposed. The elastin-like polypeptides (ELPs)-SpyCatcher chimera could mediate the formation of silica carriers within seconds and the target enzymes were then covalently immobilized on silica carriers via SpyCatcher/SpyTag spontaneous reaction. These tailor-made carriers were easily prepared, with precisely controlled morphology and size, as well as none-consuming surface modification needed, which could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification.

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A Simple Technique for Direct Immobilization of Target Enzymes from Cell Lysates Based on the SpyTag/SpyCatcher Spontaneous Reaction.

Enzyme-assisted valorization of lichenan represents a green and sustainable alternative to the conventional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are essential components of state-of-the-art enzyme cocktails for lichenin bioconversion. The LPMOs named SpyTag fused LPMOs (AST) from Chaetomium globosum was functionally expressed in E. coli and exhibited 1.25-fold synergism with lichenase, whereas AST alone produced no detectable reducing sugars. HPLC results further confirm that AST does not alter the endogenous hydrolysis mode of lichenase but rather enhances its hydrolysis efficiency by disrupting the long chain of lichenan and releasing more reducing ends. To the best of our knowledge, this was the first report on the synergistic effect of LPMOs and lichenase, which may have great synergistic potential in the conversion of lichen biomass. Furthermore, a novel strategy for the covalently immobilizing AST and lichenase on silica nanoparticles (SNPs) from the cell lysate in a single step was proposed, which exhibited high activity recovery (82.9%) and high immobilization yield (94.8%). After 12 independent runs, about 67.4 % of the initial activity of the immobilized enzymes was retained. The resulted biocatalyst systems exhibited the green and sustainable strategy in the bioconversion of lichen biomass as well as other diverse polysaccharides.

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The synergism of lytic polysaccharide monooxygenases with lichenase and their co-immobilization on silica nanospheres for green conversion of lichen biomass

Lytic polysaccharide monooxygenases (LPMOs) with synergistic effect on polysaccharide hydrolase represent a revolution in biotechnology, which may accelerate the conversion of biomass to the second-generation biofuels. Discovering more hydrolases that have synergism with LPMOs will considerably expand the knowledge and application of biomass degradation. The LPMOs named CgAA9 were verified to exhibit 1.52-fold synergism when incubated with β-1,3-xylanase at a molar ratio of 3:1. The ion chromatography results proved that CgAA9 did not alter the endogenous hydrolysis mode of β-1,3-xylanase. Meanwhile, to decrease the operational cost of enzymes, a novel strategy for immobilizing LPMOs and β-1,3-xylanases based on the biomimetic silica nanoparticles was developed. It enabled preparation of immobilized enzymes directly from the cell lysate. The immobilization efficiency and activity recovery reached 84.6 and 81.4%. They showed excellent reusability for 12 cycles by retaining 68% of initial activity. The optimum temperature for both free and immobilized biocatalyst were 40 and 37 °C, indicating they were ideal candidates for typical simultaneous saccharification and fermentation (SSF) in ethanol production from algea biomass. This was the first report on the synergy between LPMOs and β-1,3-xylanase, and the strategy for enzyme self-immobilization was simple, timesaving, and efficient, which might have great potentials in algae biomass hydrolysis. • The lytic polysaccharide monooxygenases (LPMOs) from Chaetomium globosum were firstly verified to boost the hydrolysis of β-1,3-xylanases for β-1,3-xylan.
 • A novel strategy for simple preparation of SpyCather-modifed silica nanopartilcles and intelligent immobilization of target enzymes from the cell lysate was proposed.
 • The immobilized LPMOs and β-1,3-xylanases could be reasonable alternatives for typical simultaneous saccharification and fermentation (SSF) in manipulation of algae biomass.

Enzymatic degradation of algal 1,3-xylan: from synergism of lytic polysaccharide monooxygenases with β-1,3-xylanases to their intelligent immobilization on biomimetic silica nanoparticles

Mengqi Liu

Papers

Enzymatic degradation of algal 1,3-xylan: from synergism of lytic polysaccharide monooxygenases with β-1,3-xylanases to their intelligent immobilization on biomimetic silica nanoparticles