Enzymatic modification of bacterial exopolysaccharides; xanthan lyase as a tool for structural and functional modification of xanthan

Bacterial extracellular polysaccharides (EPSs) can be applied, e.g., in foods, as a thickener or stabilizer. The functional properties that make a polysaccharide suitable for such applications are largely determined by the primary structure, i.e., the sugar composition, the linkage types between the sugar units, and the presence of side chains and non-sugar substituents. The aim of this research was to obtain EPS-modifying enzymes that could be used as tools both for studying structure-function relationships of (food-grade) EPSs and for the production of tailor-made EPSs with a specific, desired functionality. EPS-degrading microorganisms could serve as a source of such enzymes. To get an idea of the probability of finding EPS-degrading microorganisms, a comparative biodegradability study was carried out on eight EPSs, six of which were produced by lactic acid bacteria (Chapter 2). Human faeces or soil were used as inocula. Xanthan, clavan, and the EPSs of Streptococcus thermophilus strains SFi39 and SFi12 were readily degraded. The four other EPSs, produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59, were not. Xanthan, the most relevant food-grade EPS, was chosen as the target for further studies. For efficient screening of polysaccharide-degrading microorganisms, plate methods are required that discriminate between intact and degraded polysaccharide. Such methods can make use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity. Alternatively, dye-labelled polysaccharides can be applied. Chapter 3 presents a survey of plate methods based on the above principles. A mixed xanthan-degrading culture was obtained from soil by enrichment on xanthan. From this culture, Paenibacillus alginolyticus XL-1 was isolated. This strain degraded 28% of the xanthan molecule and appeared to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including a xanthan lyase. Xanthan lyase removes the terminal mannosyl residue of the trisaccharide xanthan side chain by aβ-eliminative mechanism, resulting in a double bond in the side chain glucuronyl residue. Xanthan lyase is the only polysaccharide lyase that is exo-acting, releasing residues from the outside of a polysaccharide molecule. All other polysaccharide lyases described to date are endo-acting, attacking the polysaccharide backbone. In P. alginolyticus XL-1, xanthan lyase production is induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A 97-kDa xanthan lyase was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C (Chapter 4). The gene encoding the pyruvated mannose-specific xanthan lyase of P . alginolyticus XL-1, designated xalA , was isolated. The xalA gene encodes a 936-amino acid protein, including a 36-amino acid signal sequence. The XalA protein belongs to polysaccharide lyase family 8, which until now only contained chondroitinases and hyaluronate lyases. The part of the xalA gene encoding the 900-amino acid, 96,887-Da mature enzyme was expressed functionally in Escherichia coli . Like the native enzyme, the recombinant enzyme is specific for pyruvated xanthan. Heterologous production of XalA in E. coli increased the volumetric productivity by a factor 30, compared to production by P. alginolyticus . The recombinant xanthan lyase was used as a tool to modify xanthan, which resulted in a dramatic loss of the capacity to form gels with locust bean gum. Besides xanthan lyase, P. alginolyticus XL-1 produces other enzymes that could be useful for xanthan modification, such as a xanthan deacetylase and an enzyme releasing uronic acid, or uronic acid-containing oligosaccharides, from xanthan lyase-modified xanthan. Since these enzymes were produced at very low titers, P. alginolyticus XL-1 is not a suitable production organism for xanthan-modifying enzymes. Strain XL-1 may be very useful, however, as a source of genes for heterologous production of xanthan-modifying enzymes.