**1. Introduction**

Chitin, which consists of β-1,4-linked N-acetyl-D-glucosamine residues, is the main component of crustacean shells, such as shrimp, crab and shellfish [1,2]. Chitin, a renewable raw material whose annual production is about 1011 tons, is the second most abundant natural biopolymer after cellulose [3,4]. As a new type of functional material, chitin has attracted wide attention in various fields [5]. However, it is insoluble in water and most organic solvents, this property severely restricts its development and application [3]. On the other hand, chitosan, the deacetylation product of chitin, is soluble in dilute acid solution and has been widely used in agriculture, biomedicine, environmental science and other fields, as a plant inducer, biodegradable hydrogel and sewage treatment agent in antitumor drugs and in other green products [2,6–10]. Chitooligosaccharide (COS), the hydrolytic

product of chitosan, has broader biological activities, such as immunological, antitumor, antioxidant and antibacterial activity [11–13]. Due to its water-soluble ability and broad biological activity, COS has attracted more attention than chitosan. The biological activity of COS are believed to be strongly dependent on the degree of polymerization (DP), the degree of acetylation (DA) and the pattern of acetylation (PA) [14]. Vander et al. reported that COS with different degrees of deacetylation is involved in the induction of phenylalanine ammonia lyase and peroxidase activities, both of which must be activated for lignin biosynthesis [15]. It has previously been observed that the specific recognition of the *N*-acetyl moiety allows *At*CERKl to distinguish chitin and chitosan, which then activate plant immune receptors and elicit a plant immune response [16].

In order to investigate the specific biological activity of COS in a particular acetylation pattern, COS with a completely known structure is required. However, chitosan and COS produced by chemical methods usually exhibit a randomized pattern of acetylation, making them difficult to control and predict their biological activity [17]. Moreover, chitosan produced by chemical methods requires high energy consumption and causes environmental pollution [1]. In contrast, chitin deacetylase (CDA, E.C. 3.5.1.41) is able to hydrolyse the *N*-acetamido groups of *N*-acetyl-D-glucosamine residues in chitin, chitin oligosaccharides, chitosan and chitosan oligosaccharides under mild conditions by a specific mode of action. Previous studies have identified CDAs from bacteria, fungi and insects, such as *Bacillus thuringiensis* [18], *Bacillus amyloliquefaciens* [19], *Colletotrichum gloeosporioides* [20] *Mucor rouxii* [21] *Aspergillus nidulans* [22] *Saccharomyces cerevisiae* [23] *Bombyx mori* [24], *Drosophila melanogaster* [25], *Encephalitozoon cuniculi* [26], *Mamestra configurata* [27]. Although some CDAs have been reported, the deacetylation patterns of deacetylases are poorly understood. CDA from different sources can modify their substrates in different ways: Some being specific for a single position [28], others show showing multiple-attack [29,30]. In addition, COS with specific deacetylation patterns can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the available CDA.

Two genes encoding chitin deacetylases (CDA1 and CDA2) have been identified in *Saccharomyces cerevisiae* in previous reports. And these genes have been proved to be involved in the formation of the ascospores wall of *Saccharomyces cerevisiae* [31]. However, it is very interesting that the deletion of each gene will result in activity decrease of CDA, and the functions of the two genes cannot be replaced by each other [31]. Therefore, the deacetylation mechanisms of these two different chitin deacetylases may be different. However, detailed deacetylation mechanisms of chitin deacetylase from *Saccharomyces cerevisiae* have not been reported so far.

In this study, the chitin deacetylase (CDA2) from *Saccharomyces cerevisiae* (*Sc*CDA2) with a specific mode of action has been characterized and a fast, convenient and online monitoring method has been developed that can be used to separate and detect partially acetylated chitosan oligosaccharides. Mass spectrometry sequencing showed that *Sc*CDA2 can hydrolyze N-acetamido groups rather than the reducing ends of chitin oligosaccharides, producing fully defined chitosan oligosaccharides by a multiple attack mode of action. Furthermore, *Sc*CDA2 is able to remove about 8% and 20% of the acetyl groups from crystalline chitin and colloidal chitin.
