Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties.

A partial cDNA sequence from Anacardium occidentale CCP 76 was obtained, encoding a GH19 chitinase (AoChi) belonging to class VI. AoChi exhibits distinct structural features in relation to previously characterized plant GH19 chitinases from classes I, II, IV and VII. For example, a conserved Glu residue at the catalytic center of typical GH19 chitinases, which acts as the proton donor during catalysis, is replaced by a Lys residue in AoChi. To verify if AoChi is a genuine chitinase or is a chitinase-like protein that has lost its ability to degrade chitin and inhibit the growth of fungal pathogens, the recombinant protein was expressed in Pichia pastoris, purified and biochemically characterized. Purified AoChi (45 kDa apparent molecular mass) was able to degrade colloidal chitin, with optimum activity at pH 6.0 and at temperatures from 30 °C to 50 °C. AoChi activity was completely lost when the protein was heated at 70 °C for 1 h or incubated at pH values of 2.0 or 10.0. Several cation ions (Al3+, Cd2+, Ca2+, Pb2+, Cu2+, Fe3+, Mn2+, Rb+, Zn2+ and Hg2+), chelating (EDTA) and reducing agents (DTT, ß-mercaptoethanol) and the denaturant SDS, drastically reduced AoChi enzymatic activity. AoChi chitinase activity fitted the classical Michaelis-Menten kinetics, although turnover number and catalytic efficiency were much lower in comparison to typical GH19 plant chitinases. Moreover, AoChi inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, causing several alterations in hyphae morphology. Molecular docking of a chito-oligosaccharide in the substrate-binding cleft of AoChi revealed that the Lys residue (theoretical pKa = 6.01) that replaces the catalytic Glu could act as the proton donor during catalysis.

Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Dwarf-cashew resistance to whitefly (Aleurodicus cocois) linked to morphological and histochemical characteristics of leaves.

BACKGROUND: The cashew whitefly (CW), Aleurodicus cocois, is an important pest of cashew in Brazil. The use of resistant plants may be an effective strategy for the control of this pest. In a preliminary assay, we found that dwarf-cashew clones show different levels of resistance to CW. Here, we hypothesized that such resistance is associated with morphological characteristics of cashew leaves and their content of phenolic compounds. RESULTS: We determined (i) the attractiveness and suitability for oviposition of five dwarf-cashew clones towards CW, (ii) the leaf morphology and chemistry of those clones, and (iii) the relationship between leaf characteristics and resistance to CW. In greenhouse multiple-choice assays, PRO143/7 and CCP76 showed, respectively, the lowest and highest counts of both CW adults and eggs. Scanning electron microscopy (SEM) analysis revealed that PRO143/7 and EMBRAPA51 have, respectively, the highest and lowest numbers of leaf glandular trichomes. We found a negative correlation between number of trichomes in the abaxial surface of cashew leaves and CW oviposition. In addition, confocal microscopy analysis and histochemical tests with ferrous sulfate indicated a higher accumulation of phenolic compounds in the resistant clone PRO143/7 relative to the other clones. Dwarf-cashew clones did not significantly differ based on the number of leaf epicuticular striations, and the thickness of both leaf lamina and the epidermal layer. CONCLUSION: The resistance of dwarf-cashew plants to CW is associated with an elevated number of trichomes and accumulation of high levels of phenolics in leaves. Additionally, the contribution of epicuticular striation density and thickness of leaf lamina/epidermal layer are insignificant. © 2019 Society of Chemical Industry.

Zein films with unoxidized or oxidized tannic acid.

BACKGROUND: Corn zein is a predominatly hydrophobic protein, forming films with relatively good water resistance. Tannic acid, especially in its oxidized form, is supposed to cross-link proteins including zein, which may be explored to further enhance the water resistance of zein films. The effects of different contents (0-8 wt%) of unoxidized and oxidized tannic acid (uTA and oTA, respectively) on the properties of zein films at different pH values (4-9) were studied, according to central composite designs. RESULTS: Increasing tannic acid contents and pH values resulted in decreased water solubility and increased tensile strength and modulus of films. The presence of tannic acid provided the films with a yellowish color and increased opacity. Paired t-tests indicated that oTA films presented higher tensile strength, lower water vapor permeability and lower water solubility than uTA films. CONCLUSION: Higher tannic acid contents and pH values resulted in films with better overall physical properties, which might be ascribed to cross-linking, although the films were still not water resistant. The resulting films have potential to be used for food packaging and coating applications. © 2017 Society of Chemical Industry.

The ultrastructure of shelled and unshelled cashew nuts.

Cashew nuts have many attributes, including sensory, nutritional and health appeal, which contribute to their worldwide acceptance. We demonstrate details of the microstructure of shelled and unshelled cashew kernels with regard to pericarp and cotyledon organization. This study also provides evidence of the colonization of these kernels by filamentous fungi. Nuts were examined by scanning electron and confocal scanning laser microscopy. Staining with acridine orange was performed. A tight lignified palisade layer adjacent to the exocarp surface explains the hardness of the shell's pericarp. The mesocarp contains large secretory cavities that confer a spongy property to this tissue. Papillose cells, which are responsible for secreting CNSL (cashew nutshell liquid), were observed to cover the inner wall of these cavities. Lipid components are readily released from the parenchyma and appear as oil droplets. The outer surface of the shelled samples exhibited a dense Aspergillus infestation.