*3.4. Identification of ScCDA2 Products by MALDI-TOF-MS*

To determine the effect of different enzyme concentrations on the enzyme reaction products, four concentration gradients (0.25 μM, 0.5 μM, 0.75 μM, 1 μM) were prepared under 20 mM pH = 8.0 Tris-HCl. MS spectra were obtained using an Ultraflex™ ToF/ToF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) to analyze the degree of acetylation, as previously described [49].

#### *3.5. Preparation of Partially Acetylated COS*

To analysis the deacetylation pattern of *Sc*CDA2, 20 mM Tris-HCl buffer (pH8.0), including 1 mM CoCl2, 0.5 mg/mL substrate and 0.75 μM purified soluble protein (*Sc*CDA2) was incubated at 37 ◦C for 30 min. Then, 50 μL of the sample was injected into an X-Amide (4.6 mm × 250 mm) column for separation. The column was eluted with 0.3% formic acid and 50 mM ammonium formate buffer at a flow rate of 2 mL/min. The separated sample was analyzed by electrospray ionization mass spectrometry (ESI-MS).

#### *3.6. Acetylation Pattern Analysis of COS*

Reductive amine derivatisation of partially acetylated COS was performed as previously described [50]. In brief, 0.5 mg of the partially acetylated product was dissolved in 10 μL of 0.1 mol/L solution of 2-aminoacridone (AMAC) in acetic acid/DMSO (*v*/*v*, 3/17) and stirred manually for 30 s; then 10 μL of 1 M sodium cyanoborohydride solution was added and stirred for a further 30 s. The mixture was heated at 90 ◦C for 30 min, cooled to −20 ◦C and then completely freeze-dried. The dried sample was dissolved in 200 μL of methanol/water (*v*/*v*, 50/50) solution and sufficiently centrifuged at 12,000× *g*, 4 ◦C for 10 min. Then the supernatant was immediately analyzed by mass spectrometry or stored at −20 ◦C for one month. The method of mass spectrometry to detect the results of reductive amine derivatization was the same as the method of mass spectrometry detection of the enzyme reaction product mentioned previously [51]. MS<sup>2</sup> spectra were used to analyze the acetylation pattern of COS [52].

#### *3.7. Homology Modelling and Molecular Docking*

YASARA software (version 14.12.2) was used to build the homology model of *Sc*CDA2 with three crystal structures (PDB ID: 5LFZ, 2CC0 and 2C1G) as templates, the similarity between *Sc*CDA2 and templates is 34%, 33%, 29%, respectively [53], which are highly homologous to *Sc*CDA2, based on BLAST results using. The 3D structural model was visualized using VMD software (version 1.9.3, University of Illinois; Urbana–Champaign, IL, USA) [54]. The model was further evaluated for protein geometry by SAVES (http://services.mbi.ucla.edu/SAVES/), PROCHECK, ERRAT and VERIFY3D [55]. The chitin molecule structure was acquired from the zinc database (http://zinc.docking.org/). Molecular docking was performed using LeDock software (http://www.lephar.com/) with default parameters [56]. The dimensions of the binding box were set as 10 Å around the active site. The docking center was set at the Zn2+. The number of binding poses of the ligand was 100. Finally, the docking pose that fulfilled the catalytic criteria was chosen as the initial conformation for analysis.

#### **4. Conclusions**

In this study, we firstly report the detailed deacetylation patterns of chitin deacetylase from *Saccharomyces cerevisiae* (*Sc*CDA2). Fully defined chitooligosaccharides (DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA) have been produced by *Sc*CDA2 through multiple attack catalytic mechanisms. In addition, a fast, convenient and online monitoring method has been developed that can be used to separate and detect partially acetylated chitosan oligosaccharides. Enzymatic production of fully defined chitooligosaccharides and on-line monitoring and separation chitooligosaccharides, which solves the time-consuming and labor-intensive problem of isolating high

purity chitooligosaccharides. This bio-enzymatic application could avoid the use of irritating chemicals and allows the production of functional chitosan and COS from crustacean waste chitin.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/2/ 74/s1, **Figure S1:** The model was further evaluated for protein geometry by SAVES (A comprehensive measurement website for the quality of a protein structure). 97.3% Residues in additional allowed regions and 85.57% of the residues have averaged 3D-1D score ≥ 0.2, and the quality factor is 91.2214. **Figure S2:** Compare deacetylase charge distribution. These pictures show the surface charge distributions of chitin deacetylase from *Saccharomyces cerevisiae* (*Sc*CDA2) and chitin deacetylase from *Aspergillus Nidulans* (*An*CDA, PDB ID: 2Y8U) calculated using ABPS (The Adaptive Poisson-Boltzmann Solver to generate electrostatic surface displayed) in VMD. Red represents a negative charge and blue represents a positive charge. **Figure S3:** *Sc*CDA2 substrate specificity on chitin oligomers. 0.5 mg/mL chitin oligomers as substrates were incubated with 0. 75 μM *Sc*CDA2 at 37 ◦C for 30 min. The data represents the mean SD values of the results from three independent experiments. **Figure S4:** Effects of pH, temperature and metal ion on enzyme activity. (**A**) The optimum pH was determined by incubating the 0.75 μM *Sc*CDA2 with A4 chitin oligomer (0.5 mg/mL) for 60 min at pH 3–11 in universal buffer. (**B**) To obtain the optimal temperature, the enzyme (075 μmol) was incubated for 60 min at different temperatures in 50 mM Tris-HCl buffer (pH 8.0) containing chitin oligomer mixture (0.5 mg/mL) as a substrate. (**C**) Relative activity with different metal cations. Proteins were incubated with 1 mm metallized cations, and activity was determined in 50 mM Tris-HCl buffer (pH 8.0) using 0.5 mg/mL A4 as a substrate. **Figure S5:** Spectra of N-glycosylation of *Sc*CDA2. Mass spectrometry showed that *Sc*CDA2 have N-glycosylation post-translational modification at Asn 181, Asn 199 and Asn 203.

**Author Contributions:** General concept and design of studies: H.Y. Experimental concept and data analysis: X.-Y.Z., Y.Z., Experimental conduct and manuscript writing: X.-Y.Z. Manuscript review: H.Y., H.-D.Z., W.-X.W. and H.-H.C. Page: 12. Manuscript finalization: H.Y.

**Funding:** This research was supported by National Natural Science Foundation of China: (31670803, 31770847), National Key Research and Development Project of China: (2017YFD0200902), and H.Y was supported by Dalian city Innovative Support Program for High-Level Talents (2015R010), CAS Youth Innovation Promotion Association (2015144).

**Acknowledgments:** The authors would like to thank L.H.W. for the MALDI-TOF-MS analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.
