*Article* **Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides**

**Xian-Yu Zhu 1,2, Yong Zhao 1, Huai-Dong Zhang 1,3, Wen-Xia Wang 1, Hai-Hua Cong <sup>2</sup> and Heng Yin 1,\***


Received: 19 December 2018; Accepted: 16 January 2019; Published: 22 January 2019

**Abstract:** Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from *Saccharomyces cerevisiae*, *Sc*CDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. *Sc*CDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that *Sc*CDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. *Sc*CDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore *Sc*CDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

**Keywords:** chitin deacetylase; deacetylation patterns; chitooligosaccharides; separating; detecting
