Investigating novel transglycanase activities within the plant kingdom
Integral to the physiological and biochemical properties of the plant, the primary cell wall (PCW) is of great economical interest. Transglycanases are a class of cell-wall remodelling enzymes hypothesised to be involved – among other functions – in cellular elongation and strengthening of the PCW. At present only four transglycanases have been convincingly characterised but the potential existence of many more is likely. To detect potential novel transglycanase activity, broad spectrum fluorescent and radioactive assays were conducted using a variety of potential donor and acceptor substrates. Enzyme extracts were sourced from a range of plants that represented the majority of the plant kingdom. Beansprout extracts reproducibly displayed significant incorportation of radioactivity and fluorescence when incubated with an α-arabinan or β- galactan donor and labelled xyloglucan oligosaccharide (XGO) acceptor. However, further analysis indicated the presence of xyloglucan contamination in donor polysaccharides and thus the activity observed was xyloglucan endotransglucosylase (XET). It has been hypothesised in the literature that linkages formed between the hemicellulosic and pectic matrices may be due to the activity of a transglycanase. This study has found no evidence to support this. In addition, during identification of the gene responsible for mixed-linkage β- (1,3),(1,4)-glucan : xyloglucan endotransglycosylase (MXE) activity – observed in Equisetum – a heterologous Pichia pastoris expression system was developed allowing the synthesis of a novel recombinant hetero-transglycanase (HTG) conferring predominant MXE activity and of five previously unstudied recombinant XET-active xyloglucan endotransglycosylase/ hydrolases (XTHs).