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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

Chitosan is a biopolymer obtained from chitin, where the N-acetylglucosamine monomer is in its deacetylated form; this polymer is useful for a wide variety of industrial applications. The properties and uses of chitosan depend on its physical and chemical characteristics, which result from the treatments used for its production. In this study, we report the preparation and characterization ofchitosan oligosaccharides by a green synthesis from crystalline shrimp chitin, using a sequential enzyme treatment by chitinase and chitin deacetylase. Chitinases were purified from grapes and used to rupture the crystalline shrimp chitin structure, modifying the crystallinity index from 57.6% to 15.9%. The resultant polymers were deacetylated using a recombinant chitin deacetylase from Saccharomyces cerevisiae, which was cloned and expressed in Pichia pastoris. The chitosans produced showed an estimated DA （degree of acetylation） of approximately 20%, and the molecular weights ranged from -7,600 to -3,700 after treatment in pH 3.0 and pH 6.0 for 10 min and 40 min, respectively. Physical and chemical characterization of the products indicated that enzyme fragmentation of chitin probably makes the acetamide groups more accessible to deacetylation, forming homogeneous polymers that are free of hazardous sub-products, have defined low molecular weights, and are highly deacetylated.

关键词： Chitosan binary enzyme system chitinases recombinant chitin deacetylase low acetylation degree low molecular weight.

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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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L-Arginine Supplementation and Nitric Oxide Production:No Additional Effect When Associated to Exercise

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Food and Nutrition Sciences 2013年第8期4卷 779-784页

作者： Diego dos Santos Baiao Carlos Adam Conte Jr joab trajano silva Vânia Margaret Flosi Paschoalin Thiago Silveira Alvares Department of Biochemistry Chemistry InstituteFederal University of Rio de JaneiroRio de JaneiroBrazil Department of Food Technology Fluminense Federal UniversityNiteróiBrazil Department of Basic Nutrition&Dietetics Federal University of Rio de JaneiroRio de JaneiroBrazil

L-arginine is an amino acid semiessencial considered a precursor of nitric oxide, a gas mainly produced in endothelial cells. Nutritional supplements based on the amino acid L-arginine have been broadly marketed in order to increase vasodilation and the blood supply to muscle in order to optimize metabolic responses induced by exercise. The main objective of this study was to evaluate the effect of L-arginine supplementation on nitric oxide production in response to exercise. Furthermore, the biochemical parameters of muscle fatigue were assessed. Fourteen trained runners were divided in two groups, supplemented with L-arginine (ARG) and placebo (PLA). Blood samples were collected before supplementation (T0), immediately after the first exercise session (T1), immediately after the second exercise session (T2), and after 20 minutes of rest (T3). Plasma cyclic guanosine monophosphate was assessed as a marker of nitric oxide production. The biochemical parameters of muscle fatigue analyzed were plasma lactate and ammonia. There was significant increase in plasma cyclic guanosine monophosphate in both groups in response to exercise: ARG (T0: 3.6 ± 1.4;T1: 17.9 ± 5.8;T2: 15.9 ± 5.3;T3: 7.3 ± 2.5 pmol/mL) and PLA (T0: 4.1 ± 1.1;T1: 18.8 ± 9.9;T2: 16.1 ± 3.5;T3: 9.3 ± 3.7 pmol/mL). A significant reduction in plasma lactate and ammonia were observed in the recovery period after exercise (T3). However, no significant difference was observed between groups in the variables studied. Therefore, L-arginine supplementation was unable to increase the effects of exercise on nitric oxide production and did not improve the metabolic responses to exercise in runners.

关键词： Nutritional Supplements L-Arginine Nitric Oxide Exercise Cyclic Guanosine Monophosphate

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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（美国分子生物学期刊） 2012年第4期

作者： Eduardo Mere Del Aguila Laidson Paes Gomes Cristina Tristão Andrade joab trajano silva Vânia Margaret Flosi Paschoalin

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Potentially dangerous fish of the Paraiba Estuary: Identification and envenomation mechanisms

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Journal of Coastal Life Medicine 2017年第11期5卷 459-462页

作者： Anderson Kelvin Saraiva Macêdo Jicaury Roberta Pereira Da silva Samara Pereira De Oliveira Vidal Haddad Jr Ana Lúcia Vendel Centro de Ciências Biológicas e Sociais Aplicadas Universidade Estadual da ParaíbaCampus VRua Horácio TrajanoCristo Redentor58070-450 Joao PessoaParaíbaBrasil Faculdade de Medicina de Botucatu Universidade Estadual PaulistaCaixa Postal 55719618-000 BotucatuSao PauloBrasil

Objective: To present venomous and poisonous fish species in the Paraiba Estuary, Paraiba State, Brazil, with a description of the envenomation mechanisms and characteristics of the toxins. Methods: Fishes were caught by beach seine in the dry and rainy season (November 2013 and June 2014) and bimonthly between May 2016 and March 2017. Results: Eight potentially dangerous species belonging to the families Batrachoididae, Ariidae and Tetraodontidae were identified. The representatives of the former two families, toadfish and catfish, respectively had specific venom inoculating apparatuses that could penetrate tissues, releasing toxins that caused injuries mainly to fishermen. The family Tetraodontidae had poisonous species, once the pufferfish could accumulate biotoxins (especially tetrodotoxin, which is a neurotoxin produced by bacteria). Despite being toxic for consumption, these fish were often consumed by fishermen and their families. Conclusions: The present study is the first record of venomous and poisonous species in the Paraiba Estuary and contributes to the knowledge of the identification of the species and the mechanisms that cause severe envenomation and even deaths in artisanal fishing communities. Such knowledge is fundamental to future campaigns aimed at the prevention of injuries involving fish in the region and in other estuarine environments, where such fishes are common.

关键词： Catfish Poisonous fish Pufferfish Toadfish Venomous fish

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