endo-1,3-β-D-Glucanase (barley)

The aim of this study was to investigate the ability to coat liposomes with β-glucans from Pleurotus eryngii, in order to obtain a suitable formulation for pharmaceutical purposes. After alkali extraction of the fruiting bodies of P. eryngii and solvent fractionation of the raw polysaccharide material, four polysaccharide fractions named PEA2-SS, PEA2-SP, PEA3-SS and PEA3-SP were obtained. Chemical structure analyses by methanolysis, methylation and NMR indicated that all fractions contained two types of β-glucans: one 3-linked with some branching at O-6 and one 6-linked with branches occurring at O-3. The molar mass ranged from 7-36 kDa. Fraction PEA2-SP was most suitable for the coating process according to the resulting charge, hydrodynamic diameter (size) and the PDI of this formulation. This was attributed to the higher Mw displayed by this fraction. After enzymatic degradation, the ability of PEA2-SP to coat the liposomes was lost, indicating the requirement of a minimum polymer molar mass, which is suggested to be around 30 kDa. Furthermore, the presence of a positive charge on the liposome surface seemed to be essential. The novel β-glucans coated liposomes were able to activate dectin-1b, a receptor on macrophages, and may have potential as a drug delivery platform.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defence responses, which include increased activity of β-1,3-glucanase and peroxidase (POD). There is a lack of information regarding β-1,3-glucanase and POD synergistic effects on the plant cell wall modification and characterisation during wheat-RWA infestation. This study aimed to characterise the physicochemical properties of the cell wall-bound POD and β-1,3-glucanase during RWA-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were planted in a glasshouse to a seedling stage before being infested with RWASA2 for 14 days. The findings showed a significant increase in β-1,3-glucanase and POD activities in the infested Tugela-Dn5 and Tugela-Dn1 cultivars over the 14 days. However, in the Tugela enzymes were repressed. In addition, it was shown for the first time that β-1,3 − 1,4-glucanase activity specific toward mixed-linked glucan was significant in the resistant cultivar over 14 days. β-1,3-glucanase, β-1,3 − 1,4-glucanase and POD displayed optimum at pH 5. β-1,3-glucanase and POD displayed temperature optimum at 40 and 50°C, respectively. However, β-1,3 − 1,4-glucanase had temperature optimum at 25°C. β-1,3-glucanase and POD had a thermo-stability at 37°C followed by about 80% relative activity at 70°C, but β-1,3 − 1,4-glucanase displayed thermostability at 25°C and retained more than 75% at 70°C, confirming that β-1,3 − 1,4-glucanase and β-1,3-glucanase induced in the resistant cultivars cell wall were two different enzymes. Mechanism of actions and oligosaccharide displayed that β-1,3-glucanase was highly active against β-1,3-glucan and required a triose and higher oligosaccharide to be active. Our findings demonstrated cell-wall bound POD and β-1,3-glucanase activities significantly increased in wheat after RWASA2 infestation, revealing they acted synergistically to reinforce the cell wall to deter RWASA2 feeding in resistant cultivars.

Background: The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. Results: Here we examined the efficiency and reproducibility of 12 recombinant endo-β(1,3)-D-glucanases for specifically degrading the cell wall β(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, β(1,3)-D-glucan and β(1,6)-D-glucan. We found that the GH16 endo-β(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-β(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant β(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. Conclusions: The discovery of a reliable and efficient recombinant endo-β(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)-D-glucan. This new commercial endo-β(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.

Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barabrum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.