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.

Automated summary

The novel glycoside hydrolase family 46 chitosanase SaCsn46A from Streptomyces avermitilis has been successfully cloned and functionally expressed. This enzyme shows high stability and activity in moderate temperature and pH conditions, with the ability to hydrolyze a variety of polysaccharides linked by beta-1,4-glycosidic bonds. Moreover, it can be enhanced by divalent metal ions like Mg2+ and Mn2+, making it a promising candidate for producing glucosamine and chitooligosaccharides from chitosan.