Secretory expression of κ-carrageenase MtKC16A in Bacillus subtilis for the preparation of κ-carratriose

Carrageenases can degrade carrageenan and produce even-numbered neocarrageenan-oligosaccharides (NCOSs). However, the gel properties of κ-carrageenan limit the maximum concentration of substrate that can be treated by κ-carrageenase. In this study, we first constructed an expression system in Bacillus subtilis and achieved secretion of Nκ4-producing κ-carrageenase MtKC16A, with an activity of 0.016 U/mL. Our further exploration showed that MtKC16A can also cleave the trisaccharide unit from the non-reducing end of κ-carraheptaose, κ-carranonaose, κ-carraundecaose, and κ-carrauntetradecaose. This allowed us to successfully prepare κ-carratriose by combining acid hydrolysis and enzymatic hydrolysis. We were able to obtain 0.47 ± 0.01 g of κ-carratriose from 1 g of κ-carrageenan, with a high yield of 47.0 % (w/w). Furthermore, our study demonstrated that carrageenanoligosaccharides (COSs) prepared by acid hydrolysis combined with enzymatic hydrolysis have excellent in vitro antioxidant activity, significantly higher than polysaccharides. This study presents new methods for the efficient preparation of odd-numbered COSs using κ-carrageenases.