A Pyruvated Mannose-Specific Xanthan Lyase Involved in Xanthan Degradation by Paenibacillus alginolyticus XL-1

The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C.     

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

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.     

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

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.     

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1.

The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55 degrees C.     

Biodegradation of Xanthan Gum by Bacillus sp

Strains tentatively identified as Bacillus sp. were isolated from sewage sludge and soil and shown to elaborate extracellular enzymes that degrade the extracellular polysaccharide (xanthan gum, polysaccharide B-1459) of Xanthomonas campestris NRRL B-1459. Enzyme production by one strain was greatly enhanced when the strain was incubated in a mixed culture. Products of degradation were identified as d-glucuronic acid, d-mannose, pyruvylated mannose, 6-O-acetyl d-mannose, and a (1-->4)-linked glucan. These products correlate with the known structure of the gum. The complexity of the product mixture indicated that the xanthanase was a mixture of carbohydrases. The xanthanase complexes were similar to one another in temperature stability, pH and temperature optima, degree of substrate degradation, and enzymolysis products. Differences in pH stability, salt tolerance, recoverability, and yields of enzyme were observed.     

A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1

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.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

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 degrees 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.     

Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.     

Xanthan lyases--novel enzymes found in various bacterial species

Xanthan lyases, cleaving the terminal beta-mannosidic linkage of the side-chain of the exopolysaccharide xanthan from Xanthomonas campestris, have been obtained from several sources. These include a Bacillus species, a Corynebacterium species and a mixed culture. The lyases were initially associated with endo-beta-glucanases cleaving the main chain of xanthan. Partial purification of the enzymes was achieved and the Bacillus preparation was separated by FPLC into material free of endoglucanase and glycosidase activities. The lyase was active on polysaccharides with and without acetate and pyruvate. The optimal size of the substrate appeared to be in the range of degree of polymerization (DP) 25-35, i.e. 5-7 repeat units of the polysaccharide. No activity was found against xanthan modified by reduction of the carboxyl groups or by the addition of amine or hydroxyethyl groups. The combined action of the lyase and the endoglucanase yielded a series of oligosaccharides, each with a side-chain terminating in an unsaturated uronic acid and containing the molar ratio of D-glucose to D-mannose, 2:1.     

Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459

The nutritional requirements of Xanthomonas campestris NRRL B1459 for optimal xanthan production were studied in a chemically defined medium. Of the carbon sources tested, a 4% sucrose or glucose medium yielded the highest xanthan titers. The further addition of certain organic acids, such as succinate, pyruvate, and α-ketoglutarate, stimulated xanthan production; excess concentrations of these organic acids inhibited xanthan formation. Certain amino acids (e.g., glutamate) and nitrate salts were superior to ammonium salts for xanthan production. Concentrations of these nitrogen sources higher than the optimal levels inhibited xanthan production while stimulating growth. Xanthan production was also sensitive to high concentrations of inorganic phosphate. High xanthan potencies, up to 30 g/kg of broth, were achieved in these shake-flask studies, in which completely defined media were used.     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

黄原胶寡糖生物活性的研究

利用黄原胶降解菌Cellulom onassp.XT11生产的黄原胶降解酶,对黄原胶进行生物降解,生产具有不同粘度/还原末端比的黄原胶寡糖,并研究了黄原胶寡糖在清除羟基自由基、植物防卫反应中激活因子活性和对植物病原菌抑制能力等方面的生物活性,结果表明黄原胶寡糖具有清除羟基自由基能力,并能激活植物防卫系统以抵御病原菌的侵染,同时对野油菜黄单孢菌也具有抑菌活性。     

Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.     

Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation

Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo 32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Xanthan degragation by biofilm in porous media

Biological activity in oil reservoirs can cause significant problems such as souring and plugging. This study focuses on the problem of polymer degradation and permeability reduction due to biofilm formation during polymer injection for improved oil recovery. Polymers are included in injection fluids to increase their viscosity. Results relating biological processes and polymer degradation to fluid‐dynamic conditions in a laboratory model porous medium are presented.

A transparent flow cell with an etched two‐dimensional network of pores served as a model porous medium. A sterile xanthan polymer and natural sea water solution were continuously injected into the porous medium. A bacterial culture capable of xanthan degradation was introduced into the cell by a single injection. Some of the cells from this culture attached to the pore walls forming an immobile bacterial culture, termed biofilm. The development of this biofilm, its xanthan degradation and its effect on permeability were measured.

The effects of injection rate and rate transitions were analyzed. Injection fluid viscosity was reduced by 30% after 5 min flow through the porous medium at the maximum steady state degradation rate observed. Permeability was significantly reduced by the xanthan degrading biofilm, causing an increase in pressure drop through the porous medium of up to 80%. Polymer injection in oil reservoirs may, therefore, have negative effects on oil recovery, unless efficient biofouling control is applied. The methodology presented may serve as a tool in the development of biofouling control measures in porous media.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

Xanthan production in bubble column and air-lift reactors

A bubble column (0.05 m(3)) and an air-lift fermentor (1.2 m(3)) were used for the production of the exocellular microbial polysaccharide xanthan with Xanthomonas campestris in a synthetic medium. Upon oxygen depletion in the liquid, the xanthan production rate dropped sharply and then became a linear function of the oxygen transfer rate. The volumetric mass transfer coefficients for oxygen conformed to the correlation of Suh et al. Using this correlation in combination with the model for xanthan batch fermentation suggested by Peters et al., the xanthan fermentations in the bubble column were well described. The model also correctly predicted the time course of the molecular weight of the polysaccharide even when a complex medium was used. In the air-lift fermentor, however, the xanthan production rate and the xanthan yields with respect to oxygen and glucose were lower than expected at the overall oxygen transfer rate. The poor performance of the air lift was traced back to the lack of any oxygen supply in the downcomer.