Purification and properties of a xanthan depolymerase from a heat-stable salt-tolerant bacterial consortium

Ahlgren, Jeffrey A.

doi:10.1007/bf01569906

Cited by 11 publications

(23 citation statements)

References 10 publications

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“…They found no depolymerase activity in their cultures because the b-1,4-linked glucan backbone remained intact. The other type is the xanthan lyase, which eliminatively cleaves the terminal pyruvated b-D-mannosyl-b-D-1, 4-glucuronosyl linkage of the side chain of xanthan [9]. A xanthan lyase was first obtained from a mixed culture [10], and recently purified from Bacillus and Paenibacillus [11,12].…”

Section: Introductionmentioning

confidence: 99%

Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides-induced phytoalexin synthesis in soybean cotyledons

Liu

Huang

Dong

et al. 2005

Process Biochemistry

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Section: Introductionmentioning

confidence: 99%

Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides-induced phytoalexin synthesis in soybean cotyledons

Liu

Huang

Dong

et al. 2005

Process Biochemistry

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“…Although some bacteria and microbial mixed cultures have been reported to assimilate xanthan (2,6,7,8,19,40,41), we first elucidated an enzymatic route for complete depolymerization of xanthan in Bacillus sp. strain GL1 (12,29).…”

mentioning

confidence: 99%

“…A few xanthan lyases from bacteria or mixed culture fluids have been characterized (1,12,33,42), but the molecular cloning of their genes and overproduction of the enzymes have not yet been achieved. As the first step to prepare modified xanthans and analyze the structure-function relationship of the enzyme, in this study we cloned a gene encoding the xanthan lyase of Bacillus sp.…”

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confidence: 99%

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

Hashimoto

Miki

Tsuchiya

et al. 2001

Appl Environ Microbiol

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When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

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“…1). Both nonspecific (16) and pyruvated mannose-specific xanthan lyases (1,6,14) have been described. The resulting polytetrasaccharide differs from the polytetrasaccharide produced by X. campestris mutants: xanthan lyase-modified xanthan bears a 4,5-ene-glucuronic acid instead of a glucuronic acid as a terminal side chain residue.…”

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confidence: 99%

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

Ruijssenaars

Hartmans

Verdoes

2000

Appl Environ Microbiol

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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...

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Purification and properties of a xanthan depolymerase from a heat-stable salt-tolerant bacterial consortium

Cited by 11 publications

References 10 publications

Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides-induced phytoalexin synthesis in soybean cotyledons

Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides-induced phytoalexin synthesis in soybean cotyledons

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

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

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