A Chitosanase mutant from Streptomyces sp. N174 prefers to produce functional chitopentasaccharide

The Chitosanase (Genbank No. AAA19865.1, SsCsn46) from Streptomyces sp. N174 was mutated via the deletion of 198A, 199A, 200H and 201D. Mutant chitosanase (m-SsCsn46) and wild chitosanase (SsCsn46) were secretory expressed at high level and purity in Pichia pastoris GS115. The activity of m-SsCsn46 was 30,000 U/mg and reduced by almost 40% of the maximum activity of wild type SsCsn46 (50,000 U/mg). Both enzymes had a high maximum activity at pH 6.0 and 50 °C, and maintained 40% of maximum activity at temperature 40-60 °C at pH 6.0 and or pH 5-7 at 50 °C after 2-hour treatment. The hydrolysis of chitosan by SsCsn46 yielded predominantly chitooligosaccharides with degree of polymerization (DP) 3-5 while m-SsCsn46 preferred to produce chitopentasaccharide from chitosan. Chitopentasaccharide improved gut microbiota significantly (by reducing the proportion of harmful pathogen population and increasing the proportion of the probiotic population) in mice when compared with the chitooligosaccharides with DPs 3-5. Thus, the mutant chitosanase from Streptomyces sp. N174 prefers to produce functional chitopentasaccharide from chitosan and m-SsCsn46 provides a potential tool for producing high-purity functional chitooligosaccharides.