Biosynthesis of a thermostable gellan lyase by newly isolated and characterized strain of Geobacillus stearothermophilus 98

The thermophilic strain able to degrade gellan was isolated from Bulgarian hot spring. According to its morphological and biochemical properties and by partial sequencing of its 16S rDNA, it was classified as Geobacillus stearothermophilus. It grew in a synthetic medium with gellan as the only carbon source with a specific growth rate of 0.69 h⁻¹ and generation time of 60 min. The strain produced thermostable gellan lyase extracellularly during exponential phase. Its synthesis was inducible; the enzyme was not registered in culture liquid without gellan. The enzyme activity was increased tenfold in conditions of continuous cultivation compared to data from batch fermentations and enzyme productivity was almost sixfold higher. The enzyme showed optimal activity at 75°C in a very large pH area 4–8.5. This enzyme is the first reported thermostable gellan lyase, its residual activity was 100% after 24 h incubation at 60°C and its half-life was 60 min at 70°C.     

Characterization of alpha-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan.

A bacterium, Bacillus sp. GL1, depolymerizes a heteropolysaccharide (gellan) to a tetrasaccharide (unsaturated glucuronyl-glucosyl-rhamnosyl-glucose) by extracellular gellan lyase. The resultant tetrasaccharide was degraded to the constituent monosaccharides by subsequent reactions of unsaturated glucuronyl hydrolase, beta-d-glucosidase, and alpha-l-rhamnosidase. alpha-l-Rhamnosidase was substantially induced in the bacterial cells when grown in a medium containing gellan as a carbon source. The purified enzyme from the cells was a monomer with a molecular mass of about 100 kDa and was most active at pH 7.0 and 50 degrees C. The enzyme acted on the gellan-degrading product (rhamnosyl-glucose) formed after successive reactions catalyzed by gellan lyase, unsaturated-glucuronyl hydrolase and beta-d-glucosidase, and released rhamnose from the disaccharide. Therefore, the alpha-l-rhamnosidase is found to be responsible as the final enzyme for the complete depolymerization of gellan.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

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.     

Sphinganase, a new endoglycanase that cleaves specific members of the gellan family of polysaccharides.

A sporeforming gram-positive aerobic bacterium was isolated from soil and shown to secrete an endoglycanase that cleaves the tetrasaccharide backbone structure of specific members within the gellan family of related bacterial exopolysaccharides. We refer to these polysaccharides as sphingans. The structures of the sphingans differ by the type and position of side groups that are attached to the backbone. The new enzyme named sphinganase degrades welan, gellan, deacylated gellan, and polysaccharides S-88, S-7, and S-198. However, the enzyme does not attack rhamsan or polysaccharide NW11. Methods for growing the bacteria, isolating the enzyme, and assaying sphinganase activity are presented, and uses for the enzyme are proposed.     

Purification and properties of an enzyme capable of degrading the polysaccharide of the cyanobacterium,Nostoc commune

A novel Nostoc commune-polysaccharide (NPS)-degrading enzyme with a molecular mass of 128.5 kDa was purified from Paenibacillus glycanilyticus DS-1. The optimum pH and temperature of the enzyme activity were 5.5 and 35 degrees C, respectively. The enzyme completely degraded NPS to oligosaccharides, ranging from tetra to hexasaccharides and could degrade the xylan weakly whereas xanthan, gellan, cellulose, curdlan and p-nitrophenyl-beta-D-xylopyranoside were not degraded. Homology analysis of the N-terminal amino acid sequence of the NPS-degrading enzyme against the PIR and SWISS-PROT databases indicated that the sequence was not homologous to any other polysaccharide-degrading enzyme.     

The reinforcement of gellan gel network by the immersion into salt solution

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.     

Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP- and UDP-glucose pyrophosphorylase activities

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 microM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 microM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).     

Production and rheological characteristics of the microbial polysaccharide gellan

B. MANNA, A. GAMBHIR AND P. GHOSH. 1996. Sphingomonas paucimobilis was used for the synthesis of the microbial polysaccharide gellan. In a 60 h fermentation, polysaccharide yield and productivity obtained were 0.45 g gellan per g glucose consumed and 0.21 g I⁻¹ h⁻¹ respectively. The broth showed pseudoplastic behaviour with yield stress. The requirement of the solvent propanol to precipitate gellan from the broth depended on the volume of the broth rather than on gellan concentration. The addition of salt to the broth reduced the propanol requirement. Attempts to separate cells from the highly viscous broth by microfiltration were not successful. Ultrafiltration reduced the propanol requirement but appreciable membrane fouling and loss of gellan was observed.     

The low temperature,rapid dissolution of gellan away from root cultures

Summary A buffer system consisting of 50 mM Tris-HCl-TRIZMA base plus 10 mM EDTA was used to rapidly dissolve gellan gels used for maintaining transformed carrot root cultures. The optimum conditions of pH 7.5 in the presence of 10 mM EDTA for dissolving gellan were first worked out on a model test system containing 0.4% gellan, 0.025% MgSO₄·7H₂O, and blue dye. The conditions were then tested on gellan gels (0.2% gellan plus nutrients) containing carrot roots. This gel dissolution system was rapid (18 to 20 min), did not require heating, and could also be efficiently performed at 4 °C. Furthermore, the buffer system used for gel dissolution is a standard one used for plant cell fractionation studies.     

The effect of agitation and aeration on the synthesis and molecular weight of gellan in batch cultures of Sphingomonas paucimobilis

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis.Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.     

The gellan gum biosynthetic genes gelC and gelE encode two separate polypeptides homologous to the activator and the kinase domains of tyrosine autokinases

The high-molecular-weight exopolysaccharide gellan is an important commercial gelling agent produced in high yield by the Gram-negative bacterium Sphingomonas elodea ATCC 31461. The cluster of genes required for gellan biosynthesis contains the genes gelC and gelE. These encode for two polypeptides homologous to the activator domain and the kinase domain, respectively, of bacterial autophosphorylating tyrosine kinases involved in polysaccharide chain length determination. The GelC/GelE pair is an exception to the biochemically characterized Gram-negative tyrosine autokinases since it consists of two polypeptides instead of a single one. The deletion of gelC or gelE resulted in the abolishment of gellan in the culture medium confirming their role in gellan biosynthesis. In addition, ATP-binding assays confirmed the predicted ATP-binding ability of GelE. Interestingly, GelE contains an unusual Walker A sequence (ASTGVGCS), where the invariant lysine is replaced by a cysteine. This residue was replaced by alanine or lysine and although both mutant proteins were able to restore gellan production by complementation of the gelE deletion mutant to the production level observed with native GelE, only the mutated GelE where the cysteine was replaced by alanine was demonstrated to bind ATP in vitro. The importance of specific tyrosine residues present in the C-terminal domain of GelE in gellan assembly was also determined. The tyrosine residue at position 198 appears to be essential for the synthesis of high-molecular-weight gellan, although other tyrosine residues may additionally contribute to GelE biological function.     

Photocrosslinkable gellan gum film as an anti-adhesion barrier

The purpose of this study was to develop a gellan gum-based film which could be photocrosslinked for medical applications. Gellan gum was grafted with cinnamate to yield the photo crosslinkable polymer (gellan gum-cin). This material had 14.7% of its d-galacturonic residues reacted with cinnamate groups and displayed maximum absorption at 254nm. Investigation of the photochemical properties showed that the crosslinking efficiency was 82% after 16min of UV irradiation. The anti-adhesion films prepared from gellan gum-cin polymers exhibited high gel contents (88±2%) and suitable mechanical properties. When implanted into rats, the gellan gum-cin film exhibited the most promising anti-adhesion potential in 2 out of 10 rats without forming any tissue adhesion. Furthermore, the gellan gum-cin film could effectively inhibit inflammation in rats based on the results of fluid leukocyte analyses. The gellan gum-cin film thus has potential in clinical applications.     

Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Effects of Potassium Chloride and Sodium Chloride on the Thermal Properties of Gellan Gum Gels

The exothermic and endothermic peaks in cooling and heating curves of differential scanning calorimetry (DSC) for gellan gum gels without and with potassium chloride and sodium chloride were analyzed. The gelling and melting temperatures shifted to higher temperatures with increasing gellan and salt concentration in the concentration range of gellan from 0.3 to 2.0% (w/w). The exothermic and endothermic enthalpy increased with increasing gellan and salt concentrations. Cooling DSC curves showed one exothermic peak for samples with salts and at low gellan concentration. Heating DSC curves showed many peaks for all samples except 0.3% (w/w) gellan gum gels. The sol-gel transition of samples was examined numerically by using a zipper model approach. The introduction of cations increases the number of junction zones or zippers and decreases the rotational freedom of parallel links. This makes the structure of junction zones more heat resistant, and increases the elastic modulus of the gel.     

Rheological properties of concentrated solutions of gellan in an ionic liquid

The rheological properties of solutions of gellan were examined at high concentrations where there is entanglement coupling between gellan chains. An ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) was used as a solvent. Concentrated solutions of gellan in BmimCl were obtained by using a hot-molding technique. The concentration of gellan was varied from 1.9 × 10² to 4.8 × 10² kg m⁻³. The measurement temperature ranged from 50 to 100 °C. The master curve of the angular frequency dispersion of the storage modulus for the gellan solutions showed a rubbery plateau at high angular frequency. The molecular weight between entanglements (M_e) for gellan was obtained from the plateau modulus. From the concentration dependence curve of M_e, M_e for gellan in the molten state was determined to be 2.3 × 10³.     

Effect of pH on the conformation of gellan chains in aqueous systems

The optical rotation and fluorescence anisotropy for gellan aqueous systems were measured at pH 4, 7, and 10 to elucidate the effect of pH on the conformation of gellan chains. The optical rotation study suggests that pH affects the conformation of helical gellan chains and their aggregation behavior but the coil-helix transition temperature. By comparing the chain mobility estimated from the fluorescence anisotropy between different pH conditions, it has been revealed that the degree of expansion of random-coiled gellan chains varies with pH. These results indicate that the effect of pH is explained by the change in the anionic nature of gellan chains rather than in the shielding effect of hydrogen ions surrounding gellan chains as a cation species.     

Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology

A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket-Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket-Burman design. Most suitable medium composition for production of gellan was (g/l): molasses-112.5; tryptone-1; casaminoacid-1; disodium hydrogen orthophosphate-1; manganese chloride-0.947 and the optimum gellan production was 13.814 g/l.     

The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members

Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.