Degradation Of Exopolysaccharide Research Articles

Degradation of Exopolysaccharides from Lactic Acid Bacteria by Thermal, Chemical, Enzymatic and Ultrasound Stresses.

During isolation, exopolysaccharides (EPS) from lactic acid bacteria are subject of thermal, chemical, enzymatic or ultrasound stress of different intensity that may affect macromolecular properties, for instance molecular mass or (intrinsic) viscosity. These parameters are, however, crucial, as they are associated with the technofunctional potential of EPS replacing commercial thickeners in nonfermented products. The aim of this study was to systematically examine treatments EPS are usually exposed to during isolation and to investigate the underlying degradation mechanisms. Solutions (1.0 g/L) of EPS from Streptococcus thermophilus, isolated as gently as possible, and commercial dextran were analyzed for molecular mass distributions as representative measure of molecule alterations. Generally, acid, excessive heat and ultrasonication, intensified by simultaneous application, showed EPS degradation effects. Thus, recommendations are given for isolation protocols. Ultrasonic degradation at 114 W/cm² fitted into the random chain scission model and followed third- (S. thermophilus EPS) or second-order kinetics (dextran). The degradation rate constant reflects the sensitivity to external stresses and was DGCC7710 EPS > DGCC7919 EPS > dextran > ST143 EPS. Due to their exceptional structural heterogeneity, the differences could not be linked to individual features. The resulting molecular mass showed good correlation (r² = 0.99) with dynamic viscosity.

Photocatalytic reduction and anti-bacterial activity of biosynthesized silver nanoparticles against multi drug resistant Staphylococcus saprophyticus BDUMS 5 (MN310601).

In this study, silver nanoparticles (Ag NPs) was eco-friendly synthesized using purified flavonoid rich content of Morinda citrifolia (M. citrifolia) extract. The synthesized Ag NPs was exhibited at 420nm in UV-spectrometer, and surface morphology with available chemical composition, shape and size of the Ag NPs were confirmed by X-ray diffraction (XRD) variation, scanning electron microscope (SEM) with energy dispersive X-ray spectroscopy (EDX) and transmission electron microscope (TEM). In addition, the excellent phytochemicals and anti-oxidant activity of the Ag NPs were confirmed by total anti-oxidant and DPPH free radical scavenging assays. Further, the concentration dependent inhibition of synthesized Ag NPs against biofilm forming Staphylococcus aureus (S. aureus) was confirmed by minimum inhibition concentration (MIC). The growth cells were arrested in the log phase of the culture and detected by flow cytometry analysis. In addition, the bacterial viability, exopolysaccharide degradation, intracellular membrane damage, matured biofilm inhibition, architectural damage and morphological alteration were confirmed by confocal laser scanning electron microscope (CLSM) and SEM. Furthermore, the synthesized Ag NPs reacted with methylene blue (MB) dye molecules has 100% degradation at an irradiation time of 140min. Conclusively, the eco-friendly synthesized Ag NPs has excellent anti-oxidant, anti-bacterial through intracellular membrane damage, cell cycle arrest and methylene blue dye removal.

Structure and tailspike glycosidase machinery of ORF212 from E. coli O157:H7 phage CBA120 (TSP3)

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a β-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.

Genomic and Biochemical Characterization of Acinetobacter Podophage Petty Reveals a Novel Lysis Mechanism and Tail-Associated Depolymerase Activity.

The increased prevalence of drug-resistant, nosocomial Acinetobacter infections, particularly from pathogenic members of the Acinetobacter calcoaceticus-baumannii complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterized phage Petty, a novel podophage that infects multidrug-resistant Acinetobacter nosocomialis and Acinetobacter baumannii Genome analysis reveals that phage Petty is a 40,431-bp ϕKMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single-subunit endolysin, but it lacks canonical spanins to disrupt the outer membrane. Analysis of other ϕKMV-like genomes revealed that spaninless lysis cassettes are a feature of phages infecting Acinetobacter within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). The product of gene 39, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4-kDa protein gene product 39 (gp39) was cloned and expressed, and its activity against Acinetobacter EPS in solution was determined. The enzyme degraded purified EPS from its host strain A. nosocomialis AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant Acinetobacter strains.IMPORTANCE Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for Acinetobacter infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data need to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this report, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported to be able to infect both A. baumannii and A. nosocomialis The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome and confirm that, unlike the rounding of lambda lysogens lacking functional spanin genes, A. nosocomialis cells infected with phage Petty lyse by bursting. This suggests that phages like Petty employ a different mechanism to disrupt the outer membrane of Acinetobacter hosts during lysis.

Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL−1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL−1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60°C and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

Identification of EPS-degrading activity within the tail spikes of the novel Pseudomonas putida phage AF

We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida. Its 42.6kb genome is related to the “epsilon15-like viruses” and the “BPP-1-like viruses”, a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida-infecting phage φ15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation.

Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens

The extracellular α-amylase enzyme from Bacillus subtilis S8-18 of marine origin was proved as an antibiofilm agent against methicillin-resistant Staphylococcus aureus (MRSA), a clinical strain isolated from pharyngitis patient, Vibrio cholerae also a clinical isolate from cholera patient and Pseudomonas aeruginosa ATCC10145. The spectrophotometric and microscopic investigations revealed the potential of α-amylase to inhibit biofilm formation in these pathogens. At its BIC level, the crude enzyme caused 51.81-73.07% of biofilm inhibition. Beyond the inhibition, the enzyme was also effective in degradation of preformed mature biofilm by disrupting the exopolysaccharide (EPS), an essential component in biofilm architecture. Furthermore, the enzyme purified to its homogeneity by chromatographic techniques was also effective in biofilm inhibition (43.83-61.68%) as well as in degradation of EPS. A commercial α-amylase enzyme from B. subtilis was also used for comparative purpose. Besides, the effect of various enzymes and temperature on the antibiofilm activity of amylase enzymes was also investigated. This study, for the first time, proved that α-amylase enzyme alone can be used to inhibit/disrupt the biofilms of V. cholerae and MRSA strains and beholds much promise in clinical applications.

The T7-Related Pseudomonas putida Phage φ15 Displays Virion-Associated Biofilm Degradation Properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

Composition of bacterial community related to degrading the exopolysaccharide from the cyanobacterium <i>Microcystis aeruginosa</i>

从铜绿微囊藻培养液中提取胞外多糖,化学分析显示其为含8.3%蛋白质的酸性杂多糖.以胞外多糖为碳源接种自然水体的菌群进行富集培养,分析胞外多糖的微生物降解过程及降解菌的组成.结果表明,接种太湖水华期微生物富集菌群后,多糖立即开始被降解,大约在18d后多糖降解过程显著减慢,37d后仍有一部分多糖未能被降解.这些结果说明在自然界中微囊藻胞外多糖是可以被微生物降解的.比较不同水体的菌群对多糖的降解能力后显示,降解菌群只存在于微囊藻水华暴发的阶段.变性梯度凝胶电泳(DGGE)结果显示在整个降解过程中,降解菌群的组成未发生显著变化.对DGGE条带中的DNA片段进行序列分析和系统发育分析表明降解菌群包含以下几类:鞘氨醇单胞菌属(Sphingomonas)3个,褐螺菌属(Phaeospirillum)1个,假单胞菌属(Pseudomonas)1个,红环菌科(Rhodocyclaceae)1个,Hylemonella(无译名)1个,分枝杆菌属(Mycobacterium)1个.;The acidic heteropolysaccharide released by a water-bloom cyanobacterium Microcystis aeruginosa FACHB-912, which contained 8.3% protein, was used as the carbon source in stable enrichment cultures inoculated with natural aquatic microbial communities. The process of microbial breakdown of the exopolysaccharide and composition of the microbial community related to degrading exopolysaccharide were investigated. After inoculation of aquatic microbial community from Lake Taihu, enrichment cultures were obtained. In the enrichment culture, the exopolysaccharide was degraded immediately. The breakdown of the exopolysaccharide slowed markedly after about 18d, left a nondegraded fraction after 37d. The above data, together with comparison experiments on degradation capability of bacterial communities from various aquatic environments, indicated that the exopolysaccharide from M. Aeruginosa FACHB-912 could indeed be degraded by microorganisms coexisting only with the water bloom in nature. Denaturing gradient gel electrophoresis (DGGE) analysis showed composition of the microbial community did not change significantly during exopolysaccharide degradation. Phylogenetic analysis on DNA fragments, which were excised from DGGE gels, placed three degraders in Sphingomonas, one in Phaeospirillum, one in Pseudomonas, one in Rhodocyclaceae, one in Hylemonella, and one in Mycobacterium.

Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Novel bactericidal surface: Catechin‐loaded surface‐erodible polymer prevents biofilm formation

We developed a novel bactericidal surface based on a catechin-loaded surface-erodible polymer. (-)-Epigallocatechin-3-gallate (EGCg), which is the main constituent of tea catechins, showed a dose-dependent inhibitory effect on Escherichia coli biofilm formation and a dose-dependent enhanced destructive effect on biofilm. EGCg-immobilized surfaces were prepared by photopolymerization of liquid biodegradable polyesters. The releasing rate was enhanced with an increase in surface-erosion rate of photocured polymers. Polymers with high releasing capacity dose-dependently reduced biofilm formation on the surfaces. The confocal laser scanning microscopic and scanning electron microscopic observations revealed that EGCg induced biofilm-destructing activities, which include bacterial membrane damage, degradation of exopolysaccharides, and detachment of colonized cells. From these results, potential advantages of the clinical use of catechin-loaded polymer-coated implants or catheters are discussed in terms of a reduced occurrence of biomaterial-centered infections without substantial toxicity or adverse effects.

Effects of supplementation of succinic acid on the production and molecular weight distribution of exopolysaccharides by Antrodia camphorata in batch cultures

AbstractThe effects of organic acid supplementation on both yields and molecular weight distributions of exopolysaccharide (EPS) of Antrodia camphorata were investigated in shaker flasks and air‐lift bioreactors. In the shaker flask study, five out of six organic acid‐supplemented cultures showed negative effects on cell growth, the exception being pyruvic acid‐supplemented culture; lower number average molecular weights (Mn) of EPS were obtained in all the supplemented cultures. EPS production was enhanced by 31% due to the addition of succinic acid. Optimum product yield was obtained between 2.0 and 3.0 g dm−3 succinic acid; however, the specific production of EPS increased monotonically as succinic acid concentration was increased from 0 to 5 g dm−3. Enhancement of EPS yield by 28% and a higher Mn of EPS (around 310 kDa) due to the addition of succinic acid were also demonstrated in an air‐lift bioreactor. In addition, a novel fermentation process resistant to EPS degradation is proposed, based on the inhibition of β‐glucanase activity by the supplementation with succinic acid. Copyright © 2004 Society of Chemical Industry

Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations

To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production. No significant increase of the EPS yields or activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP-glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed. The EPS monomer composition remained unchanged for all experiments. Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation. It was demonstrated that EPS degradation took place enzymatically. Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo-activity. This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH. These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation.

Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation.

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.

Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase.

Biofilm bacteria Enterobacter agglomerans 53b and Serratia marcescens Serr were isolated from a food processing factory. A bacteriophage (SF153b), which could infect and lyse strain 53b, was isolated from sewage. This has been shown to possess a polysaccharide depolymerase enzyme specific for the exopolysaccharide (EPS) of strain 53b. Using batch culture and chemostat-linked Modified Robbins Device systems it was observed that SF153b could degrade the EPS of a mono-species biofilm (strain 53b) and infect the cells. The disruption of the biofilm by phage was a combination of EPS degradation by the depolymerase and infection and subsequent cell lysis by the phage. Strain Serr biofilms were not susceptible to the phage and the biofilm EPS was not degraded by the phage glycanase, with the result that the biofilm was unaffected by the addition of SF153b phage. Scanning electron microscopy confirmed that specific phage could extensively degrade susceptible biofilms and continue to infect biofilm bacteria whilst EPS degradation was occurring.

Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type I exporter and have a novel heptapeptide repeat motif.

The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca2+-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.