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    Identification and characterisation of an enzyme from Monoglobus pectinilyticus associated with degradation of pectin from kiwiberry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Lewis, Aymee
    Pectin is a complex polysaccharide and a very important source of dietary soluble fibre, present in a variety of fruit and vegetables. Pectin possesses a wide range of contributions to a healthy diet and a healthy human gut, such as lowering cholesterol, protecting against intestinal inflammation and maintaining digestive health. Yet pectin is unable to be degraded by human gastrointestinal enzymes, arriving in the large intestine mostly intact. Limited research has showed the domination of Gram-negative bacteria which possess a range of secreted extracellular cell-bound and cell-free pectin degrading enzymes. These enzymes attack the pectin backbone and the accessory side chains resulting in the isolation of mono/oligosaccharides for subsequent uptake. The recent novel discovery of Monoglobus pectinilyticus, a Gram-positive bacterium, expanded the narrow knowledge regarding specific pectinolytic degraders. Genomic studies showed this bacterium contains an arsenal of enzymes specifically for pectin degradation. However, several putative pectin-degrading enzymes produced by M. pectinilyticus were unable to be identified against up-to-date databases, thus suggesting that there may be potentially novel classes of CAZyme(s) to be discovered. The proposed study will attempt to identify such enzyme(s) using New Zealand cultivar kiwiberries as the source of pectin. The initial aim of this study was to investigate the enzyme(s) involved in β-1,4-galactan by growing M. pectinilyticus with a mixture of pectin substrates (0.5% citrus pectin, 0.5% kiwiberry pectin and 0.5% potato galactan), in hope to identify such enzyme(s). Unfortunately, no β-galactosidase/β-galactanase was found. However a potentially novel binding enzyme, protein 0050 was isolated, cloned and expressed, which may be involved in a pectin degrading version of a cellulosome complex. Further research into isolating a variety of secreted proteins activated in presence of galactan during M. pectinilyticus cultivation is needed to understand the complexity of pectin degradation.
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    Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls
    (Wiley, 3/09/2013) Lee KJD; Cornuault VRG; Manfield IW; Ralet MC; Knox JP
    Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG-I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG-I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG-I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.
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    LM6-M: A high avidity rat monoclonal antibody to pectic α-1,5-L-arabinan
    (10/07/2017) Cornuault VRG; Buffetto F; Marcus SE; Crepeau MJ; Guillon F; Ralet MC; Knox JP
    1,5-arabinan is an abundant structural feature of side chains of pectic rhamnogalacturonan-I which is a matrix constituent of plant cell walls. The study of arabinan in cells and tissues is driven by putative roles for this polysaccharide in the generation of cell wall and organ mechanical properties. The biological function(s) of arabinan is still uncertain and high quality molecular tools are required to detect its occurrence and monitor its dynamics. Here we report a new rat monoclonal antibody, LM6-M, similar in specificity to the published rat monoclonal antibody LM6 (Willats et al. (1998) Carbohydrate Research 308: 149-152). LM6-M is of the IgM immunoglobulin class and has a higher avidity for α-1-5-L-arabinan than LM6. LM6-M displays high sensitivity in its detection of arabinan in in-vitro assays such as ELISA and epitope detection chromatography and in in-situ analyses.
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    Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls
    (Springer Verlag, 25/07/2015) Cornuault VRG; Buffetto F; Rydahl MG; Marcus SE; Torode TA; Xue J; Crepeau MJ; Faria-Blanc N; Willats WGT; Dupree P; Ralet MC; Knox JP
    Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.