Abstract
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.
Highlights
Chitin is an unbranched polysaccharide composed of βlinked 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) units found in the shells of crustaceans, cuticles of insects, cell walls of fungi, and in a number of other lower plants and animals [1]
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
A chitin deacetylase protein from S. cerevisiae was cloned and expressed in P. pastoris, and the recombinant enzyme was purified to homogeneity
Summary
Chitin is an unbranched polysaccharide composed of βlinked 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) units found in the shells of crustaceans, cuticles of insects, cell walls of fungi, and in a number of other lower plants and animals [1]. 10 gigatons of chitin is synthesized and degraded each year. Chitin has many useful biological properties such as biocompatibility, biodegradability, hemostatic activity, and wound-healing property, but its use has been limited by its low solubility in water and most common organic solvents [3]. Chitosan, a partially deacetylated form of chitin composed by N-acetyl-D-glucosamine and N-D-glucosamine units that are randomly or block-distributed throughout the biopolymer chain, is readily soluble in dilute acids, rendering it more accessible for utilization and chemical reactions. Chitosan has been used in several biomedical applications, in orthopedic tissue engineering [4], wound dressing and artificial skin, personal-care products [5], and for drug delivery [6]
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