Abstract
Chitin deacetylase (CDA; EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.
Highlights
In the first PCR, C. japonicus cDNA was used as a template and Chitin deacetylase (CDA) F-1 and CDA R-1 were used as primers
The sequence determined for the cDNA encoding the chitin deacetylase CjCDA was registered with the DNA Data Bank of Japan (DDBJ) database
Group V contains only a signal peptide and catalytic domain, and these CDAs are specific to the midgut [24]
Summary
Chitosan is produced industrially via deacetylation of chitin obtained from the shells of crustaceans, such as shrimp and crab, by heat-treating chitin in a concentrated alkaline solution [9]. This alkali treatment does not lead to complete deacetylation but yields chitosan containing about 30% of GlcNAc at random [3]. CDA was first discovered in the fungus Mucor rouxii [15] [16], and was subsequently found in various fungi and characterized This enzyme was shown to play important roles in various physiological processes, including cell wall formation, spore formation, and fruiting body growth [17] [18] [19]. For the first time, to amplify full-length CDA genes from crabs, which has not been often reported, and to determine the structure and phylogenetic relationships among CDAs
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