Cloning and Recombinant Expression of Chitosanase Gene from Bacillus Sp. S-1

Abstract

In this work, we report the characterization of a chitosanase-producing bacterium isolated from soil. This strain was grouped under the genus Bacillus by virtue of its morphological, physiological properties and 16S rDNA gene sequence and named it Bacillus sp. S-1. According to the information of chitosanase full-length sequences deposited in NCBI, a pair of degenerated primes was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction (PCR) using Bacillus sp. S-1 genome DNA as the template. A genome walking library was constructed followed as the protocol provided by CLONTECH Company. The flanking sequences of the 5’ and 3’ terminal was obtained by genome walking method and two-step PCR technique. After overlapped and confirmed, the full-length sequence of chitosanase from Bacillus sp. S-1 was achieved and it contained 1362 bp coding 453 amino acids (accession number is EU924147). The predicted amino acid sequence was 96% similar to that of Bacillus cereus ATCC 14579 (accession number is NC_004722). The fusion protein containing BSCHITO was produced in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified rBSCHITO degraded the chitosan (the degree of deacetylation of 99%) to produce mixture of chitooligosaccharides. The BSCHITO is thus an endo-chitosanase.