Cloning of chitin deacetylase gene from Phycomyces blakesleeanus and its industrial application

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Chitosan is a biopolymer with unique properties favorable for a broad variety of industrial, biomedical, and agricultural application. The chitosan is produced from chitin which is purified from crab shell waste of can factories. Chemical procedure of chitin to chitosan leaves a lot of alkaline waste at present. If chitin is enzymatically converted to chitosan by chitin deacetylase, no waste is expected to be produced. To develop an enzymatic process for chitosan production that reduces the waste, we have planned to isolate a clone of chitin deacetylase gene from Phycomyces blakesleeanus in which cell wall is composed of mainly chitin and chitosan. The 218bp-DNA fragment amplified from genomic DNA of P. blakesleeanus using PCR primers to two highly conserved sequences among deacetylase for polysaccharide with acetoamido groups within chitin deacetylase gene of Mucor rouxii was the probe for isolation the clone from the cDNA library which we made in the □ZAP vector. A positive cDNA clone which is composed of 1, 658bp was isolated. The deduced protein encoded by this clone is composed of 451 amino acids. The protein sequence comparison revealed that the protein is a membrane protein significantly similar to chitin deacetylase of M. rouxii. These results show that the chitin deacetylase gene has been successfully cloned from P. blakesleeanus. The cloned gene and the putative protein encoded by the gene were named PbCD and PbCDA respectively. The nucleotide sequence of PbCD was registered to GenBank (accession #AB046690). After ligation of the clone to the expression vector pET32 Ek/LIC, chitin deacetylase activity was not detected in the purified His-tagged protein fraction of the crude extract from a E. coli transformant though the expression was identified. Requirements of the protein encoded by the clone for the expression of chitin deacetylase activity will be our next project to be elucidated.