Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N′-diacetylchitobiose

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401^T, with an ability to degrade crystalline chitin into (GlcNAc)₂ (N,N’-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °C and in the pH range of 5.0–11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)₂, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)₂.     

Preparation of Chitosan Nanocompositeswith a Macroporous Structure by Unidirectional Freezing and Subsequent Freeze-Drying

Chitosan is the N-deacetylated derivative of chitin, a naturally abundant mucopolysaccharide that consists of 2-acetamido-2-deoxy-β-d-glucose through a β (1→4) linkage and is found in nature as the supporting material of crustaceans, insects, etc. Chitosan has been strongly recommended as a suitable functional material because of its excellent biocompatibility, biodegradability, non-toxicity, and adsorption properties. Boosting all these excellent properties to obtain unprecedented performances requires the core competences of materials chemists to design and develop novel processing strategies that ultimately allow tailoring the structure and/or the composition of the resulting chitosan-based materials. For instance, the preparation of macroporous materials is challenging in catalysis, biocatalysis and biomedicine, because the resulting materials will offer a desirable combination of high internal reactive surface area and straightforward molecular transport through broad “highways” leading to such a surface. Moreover, chitosan-based composites made of two or more distinct components will produce structural or functional properties not present in materials composed of one single component. Our group has been working lately on cryogenic processes based on the unidirectional freezing of water slurries and/or hydrogels, the subsequent freeze-drying of which produce macroporous materials with a well-patterned structure. We have applied this process to different gels and colloidal suspensions of inorganic, organic, and hybrid materials. In this review, we will describe the application of the process to chitosan solutions and gels typically containing a second component (e.g., metal and ceramic nanoparticles, or carbon nanotubes) for the formation of chitosan nanocomposites with a macroporous structure. We will also discuss the role played by this tailored composition and structure in the ultimate performance of these materials.     

Physicochemical and Antimicrobial Properties of Thermosensitive Chitosan Hydrogel Loaded with Fosfomycin

Thermosensitive chitosan hydrogels—renewable, biocompatible materials—have many applications as injectable biomaterials for localized drug delivery in the treatment of a variety of diseases. To combat infections such as Staphylococcus aureus osteomyelitis, localized antibiotic delivery would allow for higher doses at the site of infection without the risks associated with traditional antibiotic regimens. Fosfomycin, a small antibiotic in its own class, was loaded into a chitosan hydrogel system with varied beta-glycerol phosphate (β-GP) and fosfomycin (FOS) concentrations. The purpose of this study was to elucidate the interactions between FOS and chitosan hydrogel. The Kirby Bauer assay revealed an unexpected concentration-dependent inhibition of S. aureus, with reduced efficacy at the high FOS concentration but only at the low β-GP concentration. No effect of FOS concentration was observed for the planktonic assay. Rheological testing revealed that increasing β-GP concentration increased the storage modulus while decreasing gelation temperature. NMR showed that FOS was removed from the liquid portion of the hydrogel by reaction over 12 h. SEM and FTIR confirmed gels degraded and released organophosphates over 5 days. This work provides insight into the physicochemical interactions between fosfomycin and chitosan hydrogel systems and informs selection of biomaterial components for improving infection treatment.     

Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. K_m and V_max values for NAB were 30.22 mM and 54.84 U/mg, respectively, and k_cat/K_m was 2.65 s⁻¹ mM⁻¹. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.     

Two Novel Tyrosinase Inhibitory Sesquiterpenes Induced by CuCl2 from a Marine-Derived Fungus Pestalotiopsis sp. Z233

Two new sesquiterpenes, 1β,5α,6α,14-tetraacetoxy-9α-benzoyloxy-7βH-eudesman-2β,11-diol (1) and 4α,5α-diacetoxy-9α-benzoyloxy-7βH-eudesman-1β,2β,11,14-tetraol (2), were produced as stress metabolites in the cultured mycelia of Pestalotiopsis sp. Z233 isolated from the algae Sargassum horneri in response to abiotic stress elicitation by CuCl₂. Their structures were established by spectroscopic means. New compounds 1 and 2 showed tyrosinase inhibitory activities with IC₅₀ value of 14.8 µM and 22.3 µM.     

Sensitivity of Neurospora crassa to a Marine-Derived Aspergillus tubingensis Anhydride Exhibiting Antifungal Activity That Is Mediated by the MAS1 Protein

The fungus Aspergillus tubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth (MIC = 330 and 207 μM, respectively) and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.     

Spray Drying of Chitosan Acid Salts: Process Development,Scaling Up and Physicochemical Material Characterization

We investigated a spray drying process for preparing water-soluble salts of high molecular weight chitosan (CH) intended for pharmaceutical excipient applications. CH was derived from chitin of marine lobster origin (Panulirus argus). The effects of organic acid (acetic or lactic acid) and the ratio (difference) of inlet/outlet air temperature (140/90 °C or 160/100 °C) on spray drying were studied. The yield of spray-dried CH salt powders ranged from 50% to 99% in laboratory and industrial-scale processes. The spray-dried dry powder of CH salts consisted of spherical agglomerated particles with an average diameter of 36.2 ± 7.0 µm (CH acetate) and 108.6 ± 11.5 µm (CH lactate). After dispersing the spray-dried CH salt powder samples in purified water, the mean particle sizes obtained for the CH acetate salts were 31.4 nm (batch A001), 33.0 nm (A002) and 44.2 nm (A003), and for the CH lactate salts 100.8 nm (batch L001), 103.2 nm (L002) and 121.8 nm (L003). The optimum process conditions for spray drying were found: an inlet air temperature of 160 ± 5 °C, an outlet temperature of 100 ± 5 °C and an atomizer disk rotational speed of 18,200 min⁻¹. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) results confirmed the amorphous state of the CH salts. The ¹H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectra of CH acetate and lactate salts verified that the spray drying process does not affect the polymer backbone. In conclusion, both laboratory and industrial-scale spray drying methods for preparing water-soluble acid salts of CH are reproducible, and the physicochemical properties of the corresponding CH acid salts are uniform.     

Interaction of Thalassia testudinum Metabolites with Cytochrome P450 Enzymes and Its Effects on Benzo(a)pyrene-Induced Mutagenicity

The aim of the present work was to evaluate the effects of Thalassia testudinum hydroethanolic extract, its polyphenolic fraction and thalassiolin B on the activity of phase I metabolizing enzymes as well as their antimutagenic effects. Spectrofluorometric techniques were used to evaluate the effect of tested products on rat and human CYP1A and CYP2B activity. The antimutagenic effect of tested products was evaluated in benzo[a]pyrene (BP)-induced mutagenicity assay by an Ames test. Finally, the antimutagenic effect of Thalassia testudinum (100 mg/kg) was assessed in BP-induced mutagenesis in mice. The tested products significantly (p < 0.05) inhibit rat CYP1A1 activity, acting as mixed-type inhibitors of rat CYP1A1 (Ki = 54.16 ± 9.09 μg/mL, 5.96 ± 1.55 μg/mL and 3.05 ± 0.89 μg/mL, respectively). Inhibition of human CYP1A1 was also observed (Ki = 197.1 ± 63.40 μg/mL and 203.10 ± 17.29 μg/mL for the polyphenolic fraction and for thalassiolin B, respectively). In addition, the evaluated products significantly inhibit (p < 0.05) BP-induced mutagenicity in vitro. Furthermore, oral doses of Thalassia testudinum (100 mg/kg) significantly reduced (p < 0.05) the BP-induced micronuclei and oxidative damage, together with an increase of reduced glutathione, in mice. In summary, Thalassia testudinum metabolites exhibit antigenotoxic activity mediated, at least, by the inhibition of CYP1A1-mediated BP biotransformation, arresting the oxidative and mutagenic damage. Thus, the metabolites of T. testudinum may represent a potential source of chemopreventive compounds for the adjuvant therapy of cancer.     

Proteases Production and Chitin Preparation from the Liquid Fermentation of Chitinous Fishery By-Products by Paenibacillus elgii

Chitinous fishery by-products have great application in the production of various bioactive compounds. In this study, Paenibacillus elgii TKU051, a protease-producing bacterial strain, was isolated using a medium containing 1% squid pens powder (SPP) as the sole carbon/nitrogen (C/N) source. P. elgii TKU051 was found to produce at least four proteases with molecular weights of 100 kDa, 57 kDa, 43 kDa, and 34 kDa (determined by the gelatin zymography method). A P. elgii TkU051 crude enzyme cocktail was optimally active at pH 6–7 and 60 °C. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and α-glucosidase inhibitory activity of the hydrolysates obtained from the hydrolysis of shrimp shell powder, shrimp head powder, shrimp meat powder, fish head powder and soya bean powder catalyzed by the P. elgii TkU051 crude enzyme cocktail were also evaluated. P. elgii TKU051 exhibited a high deproteinization capacity (over 94%) on different kinds of shrimp waste (shrimp heads and shells; fresh and cooked shrimp waste; shrimp waste dried by oven and lyophilizer), and the Fourier-transform infrared spectroscopy profile of the chitin obtained from the deproteinization process displayed the characteristic of chitin. Finally, the obtained chitin exhibited an effect comparable to commercial chitin in terms of adsorption against Congo Red (90.48% and 90.91%, respectively). Thus, P. elgii TKU051 showed potential in the reclamation of chitinous fishery by-products for proteases production and chitin extraction.     

6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH₄C₁ cells (IC₅₀ 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca²⁺-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC₅₀ ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.     

Carijoside A,a Bioactive Sterol Glycoside from an Octocoral Carijoa sp. (Clavulariidae)

A new bioactive sterol glycoside, 3β-O-(3′,4′-di-O-acetyl-β-d-arabinopyranosyl) -25ξ-cholestane-3β,5α,6β,26-tetrol-26-acetate) (carijoside A, 1), was isolated from an octocoral identified as Carijoa sp. The structure of glycoside 1 was established by spectroscopic methods and by comparison with spectral data for the other known glycosides. Carijoside A (1) displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils and this compound exhibited moderate cytotoxicity toward DLD-1, P388D1, HL-60, and CCRF-CEM tumor cells.     

Lipid Inhibitory Effect of (−)-loliolide Isolated from Sargassum horneri in 3T3-L1 Adipocytes: Inhibitory Mechanism of Adipose-Specific Proteins

Sargassum horneri (S. horneri) is a well-known brown seaweed widely distributed worldwide. Several biological activities of S. horneri have been reported. However, its effects on lipid metabolism and the underlying mechanisms remain elusive. In the present study, we examined the inhibitory effect of the active compound “(−)-loliolide ((6S,7aR)-6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one (HTT))” from S. horneri extract on lipid accumulation in differentiated adipocytes. MTT assays demonstrated that (−)-loliolide is not toxic to 3T3-L1 adipocytes in a range of concentrations. (−)-loliolide significantly reduced intracellular lipid accumulation in the differentiated phase of 3T3-L1 adipocytes as shown by Oil Red O staining. Western blot analysis revealed that (−)-loliolide increased the expression of lipolytic protein phospho-hormone-sensitive lipase (p-HSL) and thermogenic protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1). Additionally, (−)-loliolide decreased expression of adipogenic and lipogenic proteins, including sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), and fatty acid-binding protein 4 (FABP4) in 3T3-L1 adipocytes. These results indicate that (−)-loliolide from S. horneri could suppress lipid accumulation via regulation of antiadipogenic and prolipolytic mechanisms in 3T3-L1 cells. Considering the multifunctional effect of (−)-loliolide, it can be useful as a lipid-lowering agent in the management of patients who suffer from obesity.     

The Glycolytic Enzymes Activity in the Midgut of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) adult and their Seasonal Changes

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is an important pest of maize. The diet of the D. virgifera imago is rich in starch and other polysaccharides present in cereals such as maize. Therefore, knowledge about enzymes involved in digestion of such specific food of this pest seems to be important. The paper shows, for the first time, the activities of main glycolytic enzymes in the midgut of D. virgifera imago: endoglycosidases (α-amylase, cellulase, chitinase, licheninase, laminarinase); exoglycosidases (α- and β-glucosidases, α- and β-galactosidases) and disaccharidases (maltase, isomaltase, sucrase, trehalase, lactase, and cellobiase). Activities of α-amylase, α-glucosidase, and maltase were the highest among assayed endoglycosidases, exoglycosidases, and disaccharidases, respectively. This indicates that in the midgut of D. virgifera imago α-amylase, α-glucosidase and maltase are important enzymes in starch hydrolysis and products of its digestion. These results lead to conclusion that inhibition of most active glycolytic enzymes of D. virgifera imago may be another promising method for chemical control of this pest of maize.     

24(S)-Saringosterol Prevents Cognitive Decline in a Mouse Model for Alzheimer’s Disease

We recently found that dietary supplementation with the seaweed Sargassum fusiforme, containing the preferential LXRβ-agonist 24(S)-saringosterol, prevented memory decline and reduced amyloid-β (Aβ) deposition in an Alzheimer’s disease (AD) mouse model without inducing hepatic steatosis. Here, we examined the effects of 24(S)-saringosterol as a food additive on cognition and neuropathology in AD mice. Six-month-old male APPswePS1ΔE9 mice and wildtype C57BL/6J littermates received 24(S)-saringosterol (0.5 mg/25 g body weight/day) (APPswePS1ΔE9 n = 20; C57BL/6J n = 19) or vehicle (APPswePS1ΔE9 n = 17; C57BL/6J n = 19) for 10 weeks. Cognition was assessed using object recognition and object location tasks. Sterols were analyzed by gas chromatography/mass spectrometry, Aβ and inflammatory markers by immunohistochemistry, and gene expression by quantitative real-time PCR. Hepatic lipids were quantified after Oil-Red-O staining. Administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice without affecting the Aβ plaque load. Moreover, 24(S)-saringosterol prevented the increase in the inflammatory marker Iba1 in the cortex of APPswePS1ΔE9 mice (p < 0.001). Furthermore, 24(S)-saringosterol did not affect the expression of lipid metabolism-related LXR-response genes in the hippocampus nor the hepatic neutral lipid content. Thus, administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice independent of effects on Aβ load and without adverse effects on liver fat content. The anti-inflammatory effects of 24(S)-saringosterol may contribute to the prevention of cognitive decline.     

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate

Background:There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods:The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results:Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. {"type":"entrez-protein","attrs":{"text":"WP_071210493.1","term_id":"1098418292"}}WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: TheDNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii. Key Words: Acinetobacter baumannii, Amino acid substitution, Colistin, Mutation     

Ovarian Stimulation by Exogenous Gonadotropin Decreases the Implantation Rate and Expression of Mouse Blastocysts Integrins

Background: Integrins are heterodimeric glycoprotein receptors that regulate the interaction of cells with extracellular matrix and may have a critical role in implantation. The aim of this study was to investigate the effect of ovulation induction on the expression of α4, αv, β1, and β3 integrins in mouse blastocyst at the time of implantation. Methods: The ovarian stimulated and non-stimulated pregnant mice were sacrificed on the morning of 5^th day of pregnancy. The blastocysts were collected, and the expression of αv, α4, β1, and β3 integrins was examined using real-time RT-PCR and immunocytochemical techniques, then their ovarian hormones were analyzed at the same time. The implantation sites in uterine horns of other pregnant mice in both groups were determined under a stereomicroscope on the 7^th day of pregnancy. Results: The results showed that the expression of αv, β1, and β3 integrins in both mRNA and protein levels was significantly lower in the ovarian stimulated group than the control group, and the maximum ratio of expression was belonged to β1 molecule (P>0.05). Conclusion: The implantation rate in superovulated mice was significantly lower than control mice. It was suggested that ovulation induction decreased the expression of αv, β1, and β3 integrins of mouse blastocysts. Key Words: Blastosyst, Integrins, Implantation