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

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) 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.

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

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.