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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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Quality of Semi-Prepared Products from Rainbow Trout Waste(Onchorynchus mykiss)by Using Different Technological Strategics

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Food and Nutrition Sciences 2014年第6期5卷 571-580页

作者： Karoline Ribeiro Palmeira Eliane Teixeira Mársico Laís Doro Mosar Lemos Claudia Emília Teixeira vania margaret flosi paschoalin Maria Lúcia Guerra Monteiro Carlos Adam Conte Júnior Department of Food Technology Fluminense Federal UniversityRio de JaneiroBrazil Department of Biochemistry Chemistry InstituteFederal University of Riode JaneiroRio de JaneiroBrazil

The consumption of freshwater fish and fish products has gradually grown worldwide over the last decades, generating a proportional waste increase. The objective of the present study was to assess the chemical and bacteriological quality of restructured fish product, meatball-type, prepared with rainbow trout (Onchorynchus mykiss) waste added of 1% transglutaminase (MTG), 4% textured soy protein (TSP) and replacing part of the sodium chloride with potassium chloride (75%/25%) as described below: T1—starch addition (control);T2—MTG addition (1%);T3—soy protein addition (4%);T4—soy protein addition (4%) and MTG addition (1%);T5—soy protein addition (4%), MTG addition (1%) and partial replacement of salt (75% NaCl/25% KCl). Total aerobic mesophilic bacteria (TAMB), 2-thiobarbituric acid reactive substances (TBARS), pH determination and quantification of biogenic amines were performed on the day after manufacturing (P0) and after 60 days of storage (P1) at -25℃?± 2℃. The results showed that there was no significant difference (p < 0.05) of microbiological quality, TBARS and pH after storage. T4 presented the lowest total biogenic amine content (256.84 mg/kg) whereas T3 and T5 had the highest value (791.36 and 707.19 mg/kg, respectively) in this parameter. Putrescine was the biogenic amine that presented the highest concentration (504.00 mg/kg) in T3 and cadaverine that presented the smallest concentration (0.36 mg/kg) in T4. The use of technological strategies for developing new products with non-commercial fillets kept the most standards, having changes only in some biogenic amines.

关键词： Oncorhynchus mykiss Soy Protein Microbial Transglutaminase Salt Replacer Biogenic Amine

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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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