A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Abstract

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.Xanthan is an extracellular polysaccharide produced by Xanthomonas campestris that is widely applied as a thickener of aqueous solutions and as a stabilizer of emulsions, foams, and particulate suspensions (12). Xanthan consists of pentasaccharide repeating units: the ␤-1,4-glucan backbone is replaced on alternate glucosyl residues with a trisaccharide side chain consisting of ␣-mannose, ␤-glucuronic acid, and ␤-mannose (see Fig. 1). The inner mannosyl residues are usually acetylated, whereas about 30% of the terminal mannosyl residues are pyruvated.Also, xanthans carrying truncated side chains, produced by mutants of X. campestris, have been described (3,18). Studies of these variant xanthans showed that truncation of the side chain affects the rheological properties of xanthan. The absence of the terminal side chain mannosyl residue results in weaker viscosifier than xanthan (10), whereas the absence of both the terminal mannosyl and the glucuronyl residue results in a viscosifier superior to that of xanthan (7). These truncated side chain xanthans are interesting polysaccharides, both from a scientific and a practical point of view. They are, however, produced at low yields, especially the polytrimer (19). Furthermore, the chain length of the backbone may differ from native xanthan, hampering correct comparison of xanthans with different side chains. Enzymatic modification of the side chains would be a preferable method to obtain tailor-made xanthans, as the effect of a step-by-step removal of side chain residues can be studied, leaving the backbone unaffected.Mixed or pure bacterial cultures that grow on xanthan generally produce a mix of xanthan-degrading enzymes (4,6,8,14,17). One of these degradative enzymes that is potentially useful for xanthan modification is xanthan lyase. Xanthan lyase removes the terminal mannosyl residue via ␤ elimination, yielding a free mannose and a tetrasaccharide repeating unit (Fig. 1). Both nonspecific (16) and pyruvated mannose-specific xanthan lyases (1, 6, 14) have been described. The result...

Prebiotic Content of Bread Prepared with Flour from Immature Wheat Grain and Selected Dextran-Producing Lactic Acid Bacteria

“…Moreover, the larger amount of FOS and the sourdough technology stimulated the biosynthesis of dextran by selected LAB strains employed as starters. Recently, it was demonstrated that EPSs have a prebiotic effect on the human intestinal microbiota (14,16,(48)(49)(50). Furthermore, Olano-Martin et al (17) have shown by in vitro studies that dextran is a good substrate for butyric acid production by intestinal microorganisms.…”

Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

“…strain GL1 is quite different from that in P. alginolyticus strain XL-1, since the posttranslational processing observed in Bacillus sp. strain GL1 has not occurred in P. alginolyticus strain XL-1 (34).…”

“…After submission of this article, results on the xanthan lyase gene (xalA) of Paenibacillus alginolyticus strain XL-1 were reported by Ruijssenaars et al (34). The xalA gene codes for a polypeptide consisting of 936 amino acid residues with a molecular weight of 100,823, including a signal sequence of 36 amino acid residues.…”

Production and Purification of a Novel Xanthan Lyase from a Xanthan-DegradingMicrobacteriumsp. Strain XT11

“…The variant xanthan with the truncated side chain could be produced by X. campestris mutants [ 7 , 10 ], whereas its production level is far from what is required for practical use [ 11 ]. Therefore, because of the molecular design of xanthan, the use of relevant enzymes seems to be a preferable and promising way to obtain a tailor-made xanthan with specific and desired properties [ 12 ]. Differing from other polysaccharide lyases acting on the polysaccharide backbone, xanthan lyase could cleave the linkage between the terminal mannosyl and the glucuronyl residues on the side chain by a β -elimination reaction, introducing a double bond between C4 and C5 of the uronosyl residue and subsequently might be exploited for further chemical modification [ 13 ].…”