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Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

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American Journal of Molecular Biology 2012年第4期2卷 341-350页

作者： Eduardo Mere Del Aguila laidson paes gomes Cristina Tristao Andrade Joab Trajano Silva Vania Margaret Flosi Paschoalin Universidade Federal do Rio de Janeiro Instituto de QuímicaRio de JaneiroBrazil Universidade Federal do Rio de Janeiro Instituto de Macromoléculas Professora Eloisa ManoRio de JaneiroRJBrazil

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.***.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

关键词： Chitin Deacetylase Chitosan Chitin Pichia Pastoris Deacetylation Degree

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Green Synthesis and Physical and Chemical Characterization of Chitosans with a High Degree of Deacetylation, Produced by a Binary Enzyme System

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Journal of Life Sciences 2014年第3期8卷 276-282页

作者： laidson paes gomes Cristina Tristao Andrade Joab Trajano Silva Eduardo Mere Del Aguila Vania Margaret Flosi Paschoalin Institute of Chemistry University Federal of Rio de Janeiro Rio de Janeiro 21941-909 R J Brazil Institute of Macromolecules Professora Eloisa Mano University Federal of Rio de Janeiro Rio de Janeiro 21945-598 R J Brazil

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Pramipexole, a dopamine D3/D2 receptor-preferring agonist, attenuates reserpine-induced fibromyalgia-like model in mice

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Neural Regeneration Research 2022年第2期17卷 450-458页

作者： Carlos Pereira Martins Rodrigo Sebben paes Gabriela Mantovani Baldasso Eduarda gomes Ferrarini Rahisa Scussel Rubya Pereira Zaccaron Ricardo Andrez Machado-de-Avila Paulo Cesar Lock Silveira Rafael Cypriano Dutra Laboratory of Autoimmunity and Immunopharmacology Department of Health SciencesCampus AraranguaUniversidade Federal de Santa CatarinaAraranguáSCBrazil Post-Graduate Program of Neuroscience Center of Biological SciencesUniversidade Federal de Santa CatarinaFlorianópolisSCBrazil Laboratory of Experimental Physiopathology Program of Postgraduate in Science of HealthUniversidade do Extremo Sul CatarinenseCriciumaSCBrazil

Fibromyalgia(FM) is a complex pathology described as persistent hyperalgesia including somatic and mood dysfunctions, depression and anxiety. Although the etiology of FM is still unknown, a significant decrease in biogenic amines is a common characteristic in its pathogenesis. Here, our main objective was to investigate the role of dopamine D3/D2 receptor during the reserpine-induced pain in mice. Our results showed that pramipexole(PPX) – a dopaminergic D3/D2 receptor agonist – inhibited mechanical allodynia and thermal sensitivity induced by reserpine. Relevantly, PPX treatment decreased immobility time and increased the number of grooming in the forced swimming test and splash test, respectively. Animals that received PPX remained longer in the open arms than the reserpine group using elevated plusmaze apparatus. The repeated PPX administration, given daily for 4 days, significantly blocked the mechanical and thermal allodynia during FM model, similarly to pregabalin, although it failed to affect the reserpine-induced thermal nociception. Reserpine administration induced significant downregulation of dopamine concentration in the central nervous system, and repeated treatment with PPX restored dopamine levels in the frontal cortex and spinal cord tissues. Moreover, PPX treatment inhibited oxidants production such as DCFH(2′,7′-dichlorodihydrofluorescein) and nitrite, also decreased oxidative damage(carbonyl), and upregulated the activity of superoxide dismutase in the spinal cord. Together, our findings demonstrated the ability of dopamine D3/D2 receptor-preferring agonist in reducing pain and mood dysfunction allied to FM in mice. All experimental protocols were approved by the Universidade Federal de Santa Catarina(UFSC) Ethics Committee(approval No. 2572210218) on May 10, 2018.

关键词： dopamine dopaminergic system fibromyalgia hyperalgesia pain pramipexole reserpine

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Anatomy and Histochemistry of Araticum(Annona crassiflora Mart.)on Three Annonaceae Rootstocks

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American Journal of Plant Sciences 2014年第15期5卷 2440-2447页

作者： Luis Felipe paes de Almeida Sueli Maria gomes Rebla Goncalves Vasconcelos Osvaldo Kiyoshi Yamanishi Cristina Miranda de Alencar Federal University of Roraima(UFRR) Boa VistaRoraimaBrazil University of Brasilia Federal DistrictBrazil

The cultivation knowledge about the Cerrado native fruits is incipient. These plants are found in wild conditions and its fruits are obtained through extraction, as the case of araticum (Annona crassiflora Mart.), which is a species with the great economic potential. This plant propagation by grafting, among other methods, has proved problematic. Possible incompatibility causes were investigated using histochemical and anatomical studies. Transverse and longitudinal stems sections were analyzed in the araticum grafting area on rootstocks of araticum-de-terra-fria (Annona emarginata (Schltdl.) H. Rainer “Terra-fria”), biribá (Annona mucosa (Jacq.) Baill), and soursop (Annona muricata L.). Araticum graft survival rate was low with these rootstocks, which seems to be associated with anatomical and histochemical factors. The periderm and pith are more developed in araticum than in the other plants, which affects the alignment and juxtaposition of the cambium and vascular bundles in the graft area, hindering a successful graft. The histochemical reactions for phenolic compounds detection were very strong in cortical parenchyma, pith and xylem fibers of araticum (A. crassiflora). The presence of phenolic compounds is increased depending on the cut for grafting and the formation of these compounds is evidenced as an important limiting factor for successful grafting. It is recommended practices for reducing these compounds and a larger diameter of the graft, as a mean to overcome these anatomical difficulties.

关键词： Phenolic Compounds Incompatibility Annona sp.

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