*2.3. Biochemical Characterization of ScCDA2*

The investigation of substrate specificity could provide important information for the potential applications of deacetylase. Using a coupled enzyme assay measure the amount of acetate released has been reported to be successfully applied to quantitatively determine the deacetylation activity of a recombinant chitin deacetylase [14]. When determining activity and substrate specific, interestedly, *Sc*CDA2 was observed that it is able to remove about 8% and 20% of the acetyl groups from crystalline chitin, alpha-chitin and beta-chitin (Figure 4). In addition, A*<sup>n</sup>* (A = GlcNAc; *n* = 1, 2, 3, 4, 5 or 6) as substrates also have been measured (Figure S3). To promote the application of *Sc*CDA2 in industry, more detailed physical and chemical properties characterization of CDA is essential. The optimal PH and metal ions of *Sc*CDA2 are pH = 8.0 and 50 ◦C when A4 was used as a substrate (Figure S4). When Co2+ is present, *Sc*CDA2 exhibits the maximum activity on A4. Despite the existence of a conserved zinc-binding triad in the *Sc*CDA2, biochemical data (Figure S4C) and structure-based on sequence alignments (Figure 1) indicate that *Sc*CDA2 as a metal-dependent metalloenzyme with a Co2+ dependence greater than Zn2+. The peptidoglycan deacetylase from *Streptococcus pneumoniae* also shows that the peptidoglycan deacetylase is more metal-dependent on Co2+ than Zn2+. Besides, the reported structures of two distinct acetylxylan esterases of CE4 from *Streptomyces lividans* and *Clostridium thermocellum*, in native and complex forms, show that the enzymes are sugar-specific and metal ion-dependent and possess a single metal (Zn2+) center however with a chemical preference for Co2+ [35].

**Figure 4.** *Sc*CDA2 substrate specificity on chitin. *Sc*CDA2 activity on colloidal chitin, alpha-chitin and beta-chitin. 0.5 mg/mL substrates were incubated with 0. 75 μM *Sc*CDA2 at 37 ◦C for 30 min. The data represent the mean SD values of the results from three independent experiments.

Most of the reported CDAs show only minimal activity or no activity on chitin *in vitro*. For example, CDA from *Cyclobacterium marinum* has been reported to be able to convert acetylglucosamine to glucosamine only with the cooperation of chitinase [17]. However, *Sc*CDA2 can release up to 20.33% of acetyl groups from colloid chitin, as well as 9.16% and 7.29% of acetyl groups from insoluble alpha-chitin and Betabeta-chitin (Figure 4). Previous reported CDAs have no activity or low activity on insoluble chitin, which may be due to poor accessibility of chitin substrates [41]. However, the charge distribution on the surface of *Sc*CDA2 indicates that *Sc*CDA2 has an excessive negative charge in the region that interacts with the longer substrate, which may lead to enhanced substrate accessibility of *Sc*CDA2 to chitin (Figure S2).

#### *2.4. Isolation and Identification of Partially Acetylated Chitooligosaccharides*

Due to its special biological activity, partially acetylated chitosan oligosaccharides have attracted wide interest, and these potential activities are significantly correlated with the degree of polymerization and degree of acetylation of chitooligosaccharides [14,42]. However, the method of preparing and isolating high-purity chitooligosaccharides is time consuming and labor intensive, which severely limits the large-scale production of partially acetylated chitooligosaccharides [43]. Much research into the separation of chitosan oligosaccharides has so far limited to the separation and identification of chitosan oligosaccharides of different degrees of polymerization [44–46]. As far as we know, the method for isolation and identification of partially acetylated chitosan oligosaccharides with a degree of polymerization of four has not been reported.

We have separated and identified the partially acetylated chitosan oligosaccharides with a degree of polymerization of 4. Chitin oligomers were deacetylated with recombinant *Sc*CDA2 to form partially acetylated chitosan oligosaccharides. Three different partially acetylated chitosan oligosaccharides (A1D3, A2D2, A1D3) were obtained. These partially acetylated chitosan oligosaccharides were separated and detected by HPLC-ESI-MS (Figure 5).

**Figure 5.** HPLC-ESI-MS analysis of chitin tetramer (A4) treated with *Sc*CDA2. (**A**) The target peak of the UHPLC-ESI chromatogram began to appear after 14 min, and the deacetylation peak was mainly concentrated between 20 and 26 min. (**B**) The *m*/*z* ratio in the MS spectrum corresponds to the mass of the substrate (A4; *m*/*z* 853.24), its mono-deacetylated products A3D1 (*m*/*z* 811.25), A2D2 (*m*/*z* 768.62) and A1D3 (*m*/*z* 727.13).

#### *2.5. Partially Acetylated Chitooligosaccharides Production Processes*

Exploring the partially acetylated chitooligosaccharides production process (simultaneously or in some order) is important to aid in understanding the action mode of CDA deacetylation. Therefore, the effects of enzyme concentration on the production process of partially acetylated COS have also been determined. As is shown in Figure 6, partially acetylated chitosan oligosaccharides (A1D3, A2D2, A1D3) are gradually produced according to the degree of deacetylation. With the amount of enzymes in the system increases, the types of enzyme reaction products gradually increase. From almost no product generation, to the production of the A3D1 and A2D2, the final substrate is completely consumed at the same time producing A1D3.

**Figure 6.** Partially acetylated chitooligosaccharides production processes. To explore the production processes of partially acetylated chitooligosaccharides 0.25 μM, 0.5 μM, 0.75 μM and 1 μm enzymes were incubated with A4 in 20 mM Tris-Cl buffer (pH 8.0) for 30 min. Then determined by MALDI-TOF-MS.
