Cloning and Expression of a Novel Chitosanase Gene (choK) from β-Proteobacterium KNU3 by Double Inverse PCR

The DNA sequence of the chitosanase gene (choK) from β-Proteobacterium KNU3 showed an 1,158-bp open reading frame that encodes a protein of 386 amino acids with a novel 74 signal peptide. The degenerated primers based on the partial deduced amino acid sequences from MALDI-TOF MS analyses yielded the 820 bp of the PCR product. Based on this information, double inverse PCR cloning experiments, which use the two specific sets of PCR primers rather than single set primers, identified the unknown 1.2 kb of the choK gene.