A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked ß(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells.

Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan-hsa-miRNA-145 (CS-miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS-miRNA nanocomplexes were produced with a range of (+/-) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS-miRNA complexes offer a promising non-viral platform for breast cancer gene therapy.

A rational approach towards the design of chitosan-based nanoparticles obtained by ionotropic gelation.

Chitosan is a linear aminopolysaccharide that has been widely used for the formation of chitosan-based nanoparticles by ionic gelation with sodium tripolyphosphate (TPP). Often, the experimental design used to obtain these systems does not take into consideration important variables, such as the degree of acetylation (DA) and the molecular weight (Mw) of chitosan. In this work, we studied the formation of chitosan-TPP nanoparticles with chitosan samples of varying DA and Mw (DA0 ∼ 0-47% and Mw ∼ 2.5-282 kDa). We addressed the influence the degree of space occupancy and the degree of crosslinking on the physical properties of chitosan-TPP nanoparticles. Nanoparticles that comprised chitosan of DA ∼ 0-21.7% behaved differently than those made of chitosan of DA ∼ 34.7-47%. We attributed these differences to the polymer conformation and chain flexibility of the distinct chitosans in solution. Moreover, chitosan of high Mw were found to have a stronger preference for incorporating into the formed nanoparticles than do low-Mw ones, as determined by SEC-HPLC. These results open new perspectives to understand the formation of chitosan nanoparticles by the ionic gelation technique.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells.

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 µM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample.

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851 Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation.

Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Förster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provide in vitro proof-of-concept that these approaches can be used to label CS-NPs and colocalize them following their interactions with mammalian cells.

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3.

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0-7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, (1)H NMR of polymer and LC-HILIC-MS(n) after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

Combined HILIC-ELSD/ESI-MS(n) enables the separation, identification and quantification of sugar beet pectin derived oligomers.

The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MS(n)) and evaporative light scattering detection (ELSD). By MS(n), the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution.

Cross-reactive carbohydrate determinants strongly affect the results of the basophil activation test in hymenoptera-venom allergy.

BACKGROUND: In hymenoptera-venom allergy, sera of up to 60% of patients show in vitro reactivity to honeybee venom (HBV) and yellow jacket venom (YJV). This phenomenon is mainly caused by specific IgE (sIgE) against cross-reactive carbohydrate determinants (CCD). Whether or not these antibodies can induce clinical symptoms is a longstanding debate. OBJECTIVE: The aim of this study was to investigate the biological activity of CCD-sIgE and the suitability of the basophil activation test (BAT) in hymenoptera venom-allergic patients having CCD-sIgE. METHODS: The biological activity of CCD-sIgE was analysed by application of native and CCD-depleted YJV and HBV in BAT with the blood of 62 hymenoptera venom-allergic patients and 16 non-allergic controls. According to results of intracutaneous skin tests (IC) with YJV and HBV and the existence of CCD-sIgE, patients were classified into six subgroups. RESULTS: In patients with mono-positive IC and CCD-sIgE, and thus double-positive sIgE, BAT with native venoms was also double positive in up to 67% of the patients. In contrast, BAT with CCD-depleted venoms was positive only with the IC-positive venom. However, activation of basophils with the IC-negative venom was significantly lower compared with the IC-positive one. In IC mono-positive patients without CCD-sIgE, BAT was mono-positive with the IC-positive venom in the native and in the CCD-depleted form. CCD-positive patients with double-positive IC were a heterogeneous group, with the majority of CCD-positive patients also being double positive with the native forms of both venoms but mono-positive with the CCD-depleted ones. CONCLUSIONS: In vitro BAT clearly demonstrates biological activity of CCD-sIgE. However, because most of the patients showed a mono-positive IC and activation of basophils with the IC-negative venom was significantly lower compared with the IC-positive one, the present data suggest that CCD-sIgE is clinically irrelevant in these patients.

Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust.

When highly resistant wheat (Triticum aestivum L.) varieties are infected by an avirulent race of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Erics. and E. Henn.), penetrated host cells undergo rapid necrotization. This hypersensitive cell death is correlated with cellular lignification which efficiently restricts further fungal growth. Three competitive inhibitors of phenylalanine ammonia-lyase, the first enzyme of the general phenylpropanoid pathway and, thus, of lignin biosynthesis, namely alpha-aminooxyacetate, alpha-aminooxy-beta-phenylpropionic acid, and (1-amino-2-phenylethyl)phosphonic acid, and two highly specific irreversible suicide inhibitors of the lignification-specific enzyme cinnamyl-alcohol dehydrogenase, namely N(O-aminophenyl)sulfinamoyl-tertiobutyl acetate and N(O-hydroxyphenyl)sulfinamoyl-tertiobutyl acetate, were applied to genetically resistant wheat plants prior to inoculation with stem rust. Treatment with any of these inhibitors decreased the frequency of lignified necrotic host cells and concomitantly led to increased fungal growth. The cinnamyl-alcohol dehydrogenase inhibitors were generally more effective than the phenylalanine ammonia-lyase inhibitors, occasionally allowing some sporulation to occur on the resistant wheat leaves. These results clearly point to a causal relationship between the formation of lignin precursors and the resistance of wheat to stem rust.