Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A)

A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 degrees C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 degrees C. The K-m and V-max values of this enzyme were 1.32 mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by beta-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by alpha-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)(2). This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.