Abstract
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 29kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45°C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30°C. The Km and Vmax values of this enzyme were 1.32mg/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 β-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-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.
Highlights
Chitosan is a kind of linear polymer which was composed of β-1, 4-linked D-glucosamine (GlcN) units
The protein sequences of SaCsn46A and other chitosanases belonging to four different glycoside hydrolase (GH) families were selected from the GenBank database to constructed a phylogenetic tree (see Fig. 1(a))
Homology model of SaCsn46A was built by the Swiss-Model server with the chitosanase from Streptomyces sp
Summary
Chitosan is a kind of linear polymer which was composed of β-1, 4-linked D-glucosamine (GlcN) units. GlcN has gained its popularity as nutraceutical having application in weight loss and in treatment of osteoarthritis, knee pain and back pain[9] It is a popular compound in medicine, agriculture and rises interest in converting chitosan into COS and GlcN[24]. COS and GlcN can be prepared from chitosan by a variety of methods, such as chemical, physical and enzymatic[10]. Enzymatic conversion to produce COS and GlcN produced has attracted extensive attention as an environmentally friendly, high efficiency and controllable and alternative to chemical and physical production. Many enzymes have been reported to hydrolyze chitosan to produce chitooligosaccharides (COS) such as chitosanase, chitinase[22], protease[20], cellulase, lipase[28]. Chitosanase (EC. 3.2.1.132) is the most attractive because of its high activity and specificity
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