Biocatalytic production of chitosan polymers from shrimp shells, using a recombinant enzyme produced by pichia pastoris英文文献资料.docVIP

American Journal of Molecular Biology, 2012, 2, 341-350 AJMB /10.4236/ajmb.2012.24035 Published Online October 2012 (http://www.SciRP.org/journal/ajmb/) Biocatalytic production of chitosan polymers from shrimp shells, using a recombinant enzyme produced by Pichia pastoris Eduardo Mere Del Aguila , Laidson Paes Gomes , Cristina Trist?o Andrade , Joab Trajano Silva , 1 1 2 1 1 Vania Margaret Flosi Paschoalin 1 2 Universidade Federal do Rio de Janeiro, Instituto de Química, Rio de Janeiro, Brazil Universidade Federal do Rio de Janeiro, Instituto de Macromoléculas Professora Eloisa Mano, Rio de Janeiro, Brazil Email: emda@iq.ufrj.br Received 4 August 2012; revised 4 September 2012; accepted 19 September 2012 ABSTRACT crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequen- tial enzyme treatment is performed. Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 1) catalyzes the hydrolysis of the N-ace- tamido 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 Saccha- romyces 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 puri- fied 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 col- orimetric method based on the ?-phthal