BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via ß-1,4-glycosidic bonds.

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via ß-1,4 glycosidic bonds, such as ß-(Man1 â†’ 4Man), ß-(Glc1 â†’ 4Glc) and ß-(Xyl1 â†’ 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3-6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 µmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 µmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome.

Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes, IDSPel16 and IDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed in Escherichia coli and functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ℃, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 °C, maintaining 77.51% residual activity after preincubation at 60 °C for 1 h, whereas IDSPel17 denatured rapidly at 60 °C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with a Vmax of 942.90 ± 68.11, whereas IDSPel17 had a Vmax of only 28.19 ± 2.23 µmol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG2) and unsaturated trigalacturonate (uG3) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2 and unsaturated galacturonic acid (uG1) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo- and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries. KEY POINTS: • Two novel pectate lyase genes, IDSPel16 and IDSPel17, were isolated and characterized from the sheep rumen microbiome. • Both IDSPel16 and IDSPel17 are alkaline pectate lyases, releasing unsaturated digalacturonate and unsaturated trigalacturonate from polygalacturonic acid. • IDSPel16, a bifunctional pectate lyase with endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities, could be a potential candidate for industrial application.

Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota.

ß-Glucanases are a suite of glycoside hydrolases that depolymerize ß-glucan into cellooligosaccharides and/or monosaccharides and have been widely used as feed additives in livestock. In this study, two novel glucanase genes, IDSGluc5-26 and IDSGluc5-37, derived from sheep rumen microbiota, were expressed and functionally characterized. The optimal temperatures/pH of recombinant IDSGLUC5-26 and IDSGLUC5-37 were 50 °C/5.0 and 40 °C/6.0, respectively. Notably, IDSGLUC5-26 showed considerable stability under acidic conditions. Both IDSGLUC5-26 and IDSGLUC5-37 showed the highest activities toward barley ß-glucan, with Vmax values of 89.96 ± 9.19 µmol/min/mg and 459.50 ± 25.02 µmol/min/mg, respectively. Additionally, these two glucanases demonstrated hydrolysis of Icelandic moss lichenan and konjac gum, IDSGLUC5-26 releasing cellobiose (G2; occupying 17.37% of total reducing sugars), cellotriose (G3; 23.97%), and cellotetraose (G4; 30.93%) from barley ß-glucan and Icelandic moss lichenan after 10 min and suggestive of a typical endo-ß-1,4-glucanase (EC.3.2.1.4). In contrast, IDSGLUC5-37 was capable of liberating dominant G3 (64.11% or 67.55%) from barley ß-glucan or Icelandic moss lichenan, suggesting that the enzyme was likely an endo-ß-1,3 - 1,4-glucanases/lichenase (EC3.2.1.73). These findings describe the expression and characterization of two novel glucanase genes from sheep rumen microbiota. The two recombinant enzymes, particularly the acid-stable IDSGLUC5-26, will be of interest for potential application in food-/feed-additive development.

Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization.

Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze-thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization.

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation.

The capacities for thermal and inhibitor tolerance are critical for industrial enzymes and loss of activity is a major challenge in deploying natural enzymes for commercial applications. Protein engineering approaches, such as site-directed mutagenesis and directed evolution, have been devoted to modifying natural enzymes. Recently, a post-translation protein engineering strategy, the SpyTag/SpyCatcher system, was introduced. Here, we have generated a thermo- and ion-tolerant cyclized xylanase (C-TFX) by fusing the SpyTag and SpyCatcher peptides to its N- and C- terminus respectively. Compared with the linear enzyme, C-TFX retained greater residual activity after heating or metal ion exposure. Intrinsic fluorescence and circular dichroism analysis revealed that the isopeptide bond mediated by SpyTag/SpyCatcher cyclization contributed to enhanced thermo- and ion-stability, probably by stabilizing its secondary and conformational structure. In addition, the heat-challenged C-TFX was observed to degrade natural lignocellulosic substrates efficiently. The cyclized xylanase was more stable and resistent to denaturation and aggregation than the linear enzyme. The "superglue" SpyTag/SpyCatcher cyclization system enables the enzyme to maintain its structural conformation, which will be of particular interest in engineering of enzymes for industrial application such as feed additives and functional oligosaccharides production.

Inhibitory effect of phloroglucinol on α-glucosidase: Kinetics and molecular dynamics simulation integration study.

Regulation of α-glucosidase (EC 3.2.1.20) and its inhibitors is of great interest to researchers due to its clinical relevance as a target enzyme for the treatment of α-glucosidase-mediated diseases, such as type 2 diabetes mellitus and Pompe disease. In this study, we conducted a phloroglucinol-induced inhibition kinetics assay and performed computational molecular dynamics (MD) simulations to assess binding manner in α-glucosidase. The results showed that phloroglucinol reversibly inhibited α-glucosidase in a dose-dependent but non-competitive manner (Ki=2.07±0.16mM). Interestingly, the maximum peak wavelength and the hydrophobic surface remained unchanged during the inhibition reaction, with computational MD simulations further revealing that phloroglucinol bound in front of the active site pocket rather than in the α-glucosidase active site. Therefore, we speculate that phloroglucinol-specific inhibition is mild and the inhibitor likely binds to a single binding site near but not in the active site. Our study provided insight into the effects and mechanisms associated with a mild inhibitor of α-glucosidase activity and promotes fundamental research and potential applications of inhibitors for treatment of α-glucosidase-mediated clinical disease.