Phosphorylation of alginate: synthesis, characterization, and evaluation of in vitro mineralization capacity

Phosphorylation of alginate was achieved using a heterogeneous urea/phosphate reaction. The degree and stereoselectivity of phosphorylation as well as the effects on the physical properties of the polysaccharide were investigated by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, inductively coupled plasma optical-emission spectroscopy (ICP-OES), and size exclusion chromatography (SEC). Multidimensional NMR studies of the phosporylated alginate revealed that phosphorylation of the M residues occurred predominantly at the C3 (equatorial) carbon of the polysaccharide ring. In addition, a more comprehensive assignment of the (1)H NMR spectrum of alginate, compared with those previously reported in the literature, is provided here. Hydrogel materials were formed from ionically cross-linked blends of phosphorylated alginate and alginate. These blended hydrogels showed an enhanced resistance to degradation by chelating agents compared with cross-linked alginate hydrogels and a reduction in their mineralization potential.     

Temperature-dependent variation in the synthesis of streptococcal group-specific carbohydrate. II. Biosynthetic studies in group A and variant strains.

The biosynthesis of the cell wall polysaccharide and peptidoglycan of group A and A-486-Var streptococci was studied with N-acetyl-[14C]glucosamine, UDP-N-acetyl-[14C]glucosamine, and [14C]glucose. The incorporation of N-acetyl-[14C]-glucosamine into the cell wall four times greater in the A-486-Var cells than in the group A cells. However, the percentage of the total label incorporated into the cell wall polysaccharide at 37 degrees C by the A-486-Var strain was 12%, compared with 66% for the group A cells. When the A-486-Var was grown at 22 degrees C, the proportion of the label incorporated into the cell wall polysaccharide increased to 41%. At 37 degrees C, N-acetyl-[14C]glucosamine was incorporated preferentially into the peptidoglycan of the A-486-Var; almost three times as much of the label was incorporated into the peptidoglycan at 37 degrees C as was incorporated at 22 degrees C. Studies with protoplast membranes of these organisms showed similar differences, with a fourfold greater uptake of UDP-N-acetyl-[14C]glucosamine by the A-486-Var membranes at both incubation temperatures. These studies suggest that a defect in the incorporation of N-acetylglucosamine into the side chain of the polysaccharide is present in the A-486-Var strain at a step following the synthesis of UDP-N-acetylglucosamine. This defect, which may involve the UDP-N-acetylglucosamine transferase, is temperature dependent in the A-486-Var strain.     

Bioresponsive systems based on polygalacturonate containing hydrogels

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 μM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 μM and 57 μM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.     

Bioresponsive systems based on crosslinked polysaccharide hydrogels

Detection of bacterial and fungal contamination is of extreme importance in the fields of medical products or food packaging. Here a diagnostic tool based on pectinase and cellulase triggered release of a dye from a crosslinked polysaccharide matrix was developed. The hydrogel-based bioresponsive matrix consisted of carboxymethylcellulose (CMC) as a substrate for cellulases and polygalacturonate (PGA) as substrate for pectinases. To improve the stability of the hydrogels, methacrylic groups were inserted as crosslinking molecules. For polymerisation, two different methods were used, namely UV and thermal crosslinking. Controlled release triggered by extracellular enzymes of potentially pathogenic/contaminating microorganisms was investigated by the incorporation of Alizarin into the hydrogels. UV polymerised hydrogels turned out to be more suitable than thermal crosslinked polymers. Integration of such polymer based bioresponsive systems in medical surfaces or package systems could therefore act as an in situ monitoring system for detecting the presence of bacteria or fungi like e.g. Aspergillus species.     

Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.     

Screening for novel laccase-producing microbes

AIMS: To discover novel laccases potential for industrial applications. METHODS AND RESULTS: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C. CONCLUSIONS: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478. SIGNIFICANCE AND IMPACT OF THE STUDY: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.     

Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration

An injectable, biodegradable and glucose-responsive hydrogel derived from natural polysaccharide derivatives was synthesized to deliver adipogenic factor of insulin in vitro for adipose tissue engineering. The biodegradable hydrogel based N-succinyl-chitosan (SCS) and aldehyde hyaluronic acid (AHA) with covalently conjugated glucose oxidase and catalase. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of SCS and AHA, respectively. The morphologies and compressive modulus of the freeze-dried hydrogels demonstrated that the incorporated insulin and enzymes results in the formation of a tighter network structure in composite hydrogels. The immobilized enzymes triggered conversion of glucose reduces the pH value of the microenvironment, and results in hydrolysis and increasing swelling of the network basing on Schiff-base cross-linking. The pH inside the hydrogel, kept in PBS solution at pH 7.4 and 37°C, linearly dropped from 7.40 to 7.17 during 4 h of initial period, then slowly increased to 7.36 after 24 h. Correspondingly, the swelling ratio increased from 20.8 to 28.6 at 37°C in PBS with 500 mg/dL glucose. In PBS buffer with 500 mg/dL glucose, about 10.8% of insulin was released from the hydrogel after 8 h of incubation while upon observation. The results demonstrated that the adipogenic factor of insulin would be released from this biodegradable hydrogel device into the local microenvironment in a controlled fashion by the swelling of hydrogel network. These preliminary studies indicate that the biodegradable and glucose-responsive hydrogel may have potential uses in adipose tissue engineering applications.     

Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration

An injectable, biodegradable and glucose-responsive hydrogel derived from natural polysaccharide derivatives was synthesized to deliver adipogenic factor of insulin in vitro for adipose tissue engineering. The biodegradable hydrogel based N-succinyl-chitosan (SCS) and aldehyde hyaluronic acid (AHA) with covalently conjugated glucose oxidase and catalase. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of SCS and AHA, respectively. The morphologies and compressive modulus of the freeze-dried hydrogels demonstrated that the incorporated insulin and enzymes results in the formation of a tighter network structure in composite hydrogels. The immobilized enzymes triggered conversion of glucose reduces the pH value of the microenvironment, and results in hydrolysis and increasing swelling of the network basing on Schiff-base cross-linking. The pH inside the hydrogel, kept in PBS solution at pH 7.4 and 37oC, linearly dropped from 7.40 to 7.17 during 4 h of initial period, then slowly increased to 7.36 after 24 h. Correspondingly, the swelling ratio increased from 20.8 to 28.6 at 37oC in PBS with 500 mg/dL glucose. In PBS buffer with 500mg/dL glucose, about 10.8 % of insulin was seen to be released from the hydrogel after 8 h of incubation. The results demonstrated that the adipogenic factor of insulin would be released from this biodegradable hydrogel device into the local microenvironment in a controlled fashion by the swelling of hydrogel network. These preliminary studies indicate that the biodegradable and glucose-responsive hydrogel may have potential uses in adipose tissue engineering applications.     

Specificities of a chemically modified laccase from Trametes hirsuta on soluble and cellulose-bound substrates

Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently attached onto a laccase from Trametes hirsuta. Different molecular weights of the synthetic polymer were tested in terms of adsorption behaviour and retained laccase activity. Covalent attachment of PEG onto the laccase resulted in enhanced enzyme stability while with increasing molecular weight of attached PEG the substrate affinity for the laccase conjugate decreased. The activity of the modified laccases on fibre bound dye was drastically reduced decreasing the adsorption of the enzyme on various fabrics. Compared to the 5 kDa PEG laccase conjugate (K/S value 47.60) the K/S value decreased much more (47.96–46.35) after the treatment of dyed cotton fabrics with native laccase.     

Immobilized laccase for decolourization of Reactive Black 5 dyeing effluent

Reactive Black 5 industrial dyeing effluent was decolourized by free and immobilized laccase. The stability of the enzyme (194 h free and 79 h immobilized) depended on the dyeing liquor composition and the chemical structure of the dye. In the decolourization experiments with immobilized laccase, two phenomenons were observed – decolourization due to adsorption on the support (79%) and dye degradation due to the enzyme action (4%). Dyeing in the enzymatically recycled effluent provided consistency of the colour with both bright and dark dyes.     

Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans

The cell wall of pathogenic fungi such as Cryptococcus neoformans , provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the i SEC6 strain. In addition, a green fluorescent protein–laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in i SEC6 strains. In contrast, i SEC6 strains retained normal growth at 37°C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans .     

Photopolymerization of methacrylated chitosan/PNIPAAm hybrid dual-sensitive hydrogels as carrier for drug delivery

A series of hybrid hydrogels based on glycidyl methacrylated chitosan (CS-GMA) and N-isopropylacrylamide (NIPAAm) were designed and prepared via photopolymerization technology. The hydrogels were characterized by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and optical transmittance. The interior morphology of hydrogels was investigated by scanning electron microscopy (SEM). The swelling experiment results revealed that hybrid hydrogel exhibited combined pH and temperature sensitivities. Acid orange 8 (AO8) and 5-flurouracil (5-Fu) were selected as model drugs for examining their release from hydrogels. The results suggested that hydrogel composition and pH value of buffer solution had great influences on release profiles.     

Enzymatic Biobleaching of Two Recalcitrant Paper Dyes with Horseradish and Soybean Peroxidase

A stilbene dye (Direct Yellow 11) and a methine dye, Basazol 46L, recalcitrant to common chemical bleaches, were treated with horseradish and soybean peroxidases. Both enzymes were effective at chromophore removal. When compared to laccase in combination with a mediator (ABTS), soybean peroxidase was more effective at oxidative dye removal, especially for the methine dye.     

Biodegradation of benzidine based dye Direct Blue-6 by Pseudomonas desmolyticum NCIM 2112

Pseudomonas desmolyticum NCIM 2112 was able to degrade a diazo dye Direct Blue-6 (100 mg l(-1)) completely within 72 h of incubation with 88.95% reduction in COD in static anoxic condition. Induction in the activity of oxidative enzymes (LiP, laccase) and tyrosinase while decolorization in the batch culture represents their role in degradation. Dye also induced the activity of aminopyrine N-demethylase, one of the enzyme of mixed function oxidase system. The biodegradation was monitored by UV-Vis, IR spectroscopy and HPLC. The final products, 4-amino naphthalene and amino naphthalene sulfonic acid were characterized by GC-mass spectroscopy.     

Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization

The process parameters influencing the production of extracellular laccases by Streptomyces psammoticus MTCC 7334 were optimized in submerged fermentation. Coffee pulp and yeast extract were the best substrate and nitrogen source respectively for laccase production by this strain. The optimization studies revealed that the laccase yield was maximum at pH 7.5 and temperature 32 degrees C. Salinity of the medium was also observed to be influencing the enzyme production. An agitation rate of 175 rpm and 15% inoculum were the other optimized conditions for maximum laccase yield (5.9 U/mL). Pyrogallol and para-anisidine proved to be the best inducers for laccase production by this strain and the enzyme yield was enhanced by 50% with these inducers. S. psammoticus was able to decolourize various industrial dyes at different rates and 80% decolourization of Remazol Brilliant Blue R (RBBR) was observed after 10 days of incubation in dye based medium.     

Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.     

Distinct functions of polysaccharide deacetylases in cell shape,neutral polysaccharide synthesis and virulence of Bacillus anthracis

Peptidoglycan deacetylases (PGNG‐dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG‐dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG‐dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG‐dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S‐layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.     

New Possibilities for Biospecific Chromatography

Polyacrylamide hydrogels covalently modified with duck eggwhite ovomucoid were synthesized by radical copolymerization. These hydrogels can protect insulin (physically immobilized therein) against the action of proteolytic enzymes. Biospecific interaction of the polysaccharide component of the ovomucoid with lectins targets the hydrogel particles to the wall of the small intestine.     

Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete <Emphasis Type="Italic">Phoma</Emphasis> sp. UHH 5-1-03

A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer (∼150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography–tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.     

Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli.

Certain D-amino acids, such as D-methionine and D-cystine, were incorporated into cells of Escherichia coli under conditions inhibiting protein and cell wall synthesis. Part of the radioactivity of D-14C-amino acids incorporated into the cells was found in the isolated cell wall peptidoglycan. A covalent linkage between the amino group of the D-amino acids and the peptidoglycan was presumed to be the main cause of the binding of the D-amino acids to peptidoglycan, because the amino group of the D-amino acids in the incorporation product was substituted. Whether the carboxyl terminus was substituted was unknown. The formation of the D-amino acid-peptidoglycan linkage was insensitive to beta-lactam antibiotics such as benzylpenicillin and ampicillin (500 micrograms/ml) and therefore was not due to the reaction of DD-transpeptidation which is involved in the biosynthesis of peptidoglycan. The D-amino acids also strongly inhibited the formation of peptidoglycan-bound lipoprotein in the E. coli cells. The results may suggest the correlation between binding of D-amino acid to peptidoglycan and inhibition of formation of the bound form of lipoprotein.