Cloning，Expression，and Characterization of Chitosanase

Abstract:

A chitosanase gene BUT from the Butyrivibrio sp. MC2013 was selected from the NCBI database，which contains 903 bp and encodes a protein with 301 animo acids. Research has suggested that the chitosanase belongs to the GH-46 by sequence alignment in NCBI datebase and phylogenetic analysis. It is a novel chitosanase which shares 59% identities with any other chitosanases. The genes weresynthesized after codon optimization and inserted into pET21a（+），then the recombinant plasmid，pET-21a-BUT，was transformed into E. coli BL21（DE3）. The chitosanase was induced in ZYP-5052 and purified with the affinity chromatography of Ni-NTA. The molecular weight was estimated to be 35 kDa by SDS-PAGE. The maximum activity of purified enzyme was 146.0 U/mg. The enzymatic properties of recombinant chitosanase showed that the optimal temperature and pH of the BUT were 45 ℃ and 8.0，respectively. The stability in alkaline conditions was higher than that in acidic conditions. Mn2+ could significantly enhance the enzymatic activity，while SDS，Fe3+，Cu2+ and Zn2+ could inhibit the activity strongly. The analysis of hydrolysates by TLC demonstrated that chitosan was degraded into（GlcN）2，（GlcN）3，and（GlcN）4.