*2.1. Bioinformatic Analysis and Expression of ScCDA2*

CDA belongs to the carbohydrate esterase family 4 (CE4) according to the classification of the CAZY database (www.cazy.org) [32]. The presence of divalent metal ions, such as Zn2+, Ca2+ and Co2+, have been proved to increase the catalytic activity and stability of the CDAs [30]. The *Colletotrichum lindemuthianum*'s CDA crystal structure indicates that there is a zinc-binding triad (His-His-Asp) around Zn2+ [33].

The sequence of *Sc*CDA2 aligned with deacetylase sequences from marine *Arthrobacter* (*Ar*CE4A, 34%) [34], *Streptomyces lividans* (*Sl*CE4, 33%) [35] and *Streptococcus pneumoniae* (*Sp*PgdA, 29%) [36]

(Figure 1) [37]. The structure-based sequence alignments of *Ar*CE4A, *Sl*CE4 and *Sp*PgdA showed different levels of sequence identities according to their source from different genera and enabled identification the key residues that may contribute to catalysis function, including active site residues (Asp102, His250) and zinc-binding residues (Asp103, His149, His153) (Figure 1). Asp103, His149 and His153 form a zinc-binding triplet (His-His-Asp) around Zn2+, which is similar to chitin deacetylase from *Colletotrichum lindemuthianum* [33], although the CDA sequence from *C. lindemuthianum* only has a 30% similarity to *Sc*CDA2. The full-length open reading frame encoding the *N*-acetylglucosamine deacetylase sequence from *Saccharomyces cerevisiae* was successfully cloned and transformed into *Pichia pastoris* X-33 for highly efficient secretion expression (Figure 2). The molecular weight of *Sc*CDA2, which was digested by *N*-glycosidase F (PNGase F), decreased by about 10 kDa. PNGase F is an amidase working by cleaving between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides. This results in a deaminated protein or peptide and a free glycan [38,39]. Therefore, there are N-glycosylation post-translational modifications in *Sc*CDA2. Glycosylation is one of the most common post-translational modifications of proteins in fungi. It plays an important role in protein activity, thermal stability, proteolytic resistance, folding and secretion [40]. Mass spectrometry showed that *Sc*CDA2 have *N*-glycosylation post-translational modification at positions Asn 181, Asn 199 and Asn 203 (Figure S5).

**Figure 1.** Structure-based on sequence alignments between four chitin deacetylases (CDAs). The sequence of chitin deacetylase from *Saccharomyces cerevisiae* (*Sc*CDA2) was aligned with *Ar*CE4A sequences from a marine *Arthrobacter* species (PDB ID: 5LFZ), the *Sl*CE4 sequence from *Streptomyces lividans* (PDB ID: 2CC0) and the *Sp*PgdA sequence from *Streptococcus pneumoniae* (PDB ID: 2C1G). The conserved motifs are highlighted by a red background and the catalytic amino acids are marked with a yellow triangle. Amino acids capable of forming coordinate bonds with Zn2+ are marked with blue triangles. The symbol above the sequence represents the secondary structure, helices represent α-helices, and the arrow represents the beta fold.

**Figure 2.** Analysis of molecular weight of *Sc*CDA2 protein by 12% SDS-PAGE. (**A**) *Sc*CDA2 crude enzyme; (**B**) purified *Sc*CDA210 μM, 5 μM, 2.5 μM and 1.0 μM, marked as lanes 1, 2, 3 and 4, respectively; (**C**) PNGase F digestion confirmed that the enzyme is glycoprotein. Lane 1, *Sc*CDA2 before being digested by PNGase F; lane 2, PNGase F; lane 3, *Sc*CDA2 has been digested by PNGase F.
