Chitin Deacetylase from Bacillus aryabhattai TCI-16: Heterologous Expression, Characterization, and Deacetylation Performance.

Chitin deacetylase (CDA) removes the acetyl group from the chitin molecule to generate chitosan in a uniform, high-quality deacetylation pattern. Herein, BaCDA was a novel CDA discovered from our previously isolated Bacillus aryabhattai strain TCI-16, which was excavated from mangrove soil. The gene BaCDA was cloned and overexpressed in Escherichia coli BL21 (DE3) to facilitate its subsequent purification. The purified recombinant protein BaCDA was obtained at a concentration of about 1.2 mg/mL after Ni2+ affinity chromatography. The molecular weight of BaCDA was around 28 kDa according to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In addition, BaCDA exhibited a significant deacetylation effect on colloidal chitin, and the deacetylation degree was measured from the initial 25.69 to 69.23% by Fourier transform infrared (FT-IR) spectroscopy. Scanning electron microscopy (SEM) observation showed that the surface of colloidal chitin after enzymatic digestion was rough, the crystal fibers disappeared, and the chitin structure was loose and porous with grooves. The results of electrospray ionization mass spectrometry (ESI-MS) showed that BaCDA had full-deacetylation activity against (GlcNAc)4. Molecular docking revealed that BaCDA had an open active pocket capable of binding to the GlcNAc unit. This study not only provides a novel enzymatic resource for the green and efficient application of chitin but also helps to deepen the understanding of the catalytic mechanism of CDA.

Assessment of the properties of chitin deacetylases showing different enzymatic action patterns.

Chitin deacetylases are a group of enzymes catalysing the conversion of chitin to chitosan. Obtaining chitosan with established deacetylation degree and pattern is important for biomedical and biotechnological applications. Understandings of the structural properties of chitin deacetylases and the specificity of their interactions with chitin may conduct to the control of the pattern of deacetylation of chitosan. Our study is focused on the characterization and comparison of the structural and physicochemical properties of chitin deacetylases from fungi and marine bacteria. Despite the low sequences identity for the investigated chitin deacetylases, there are amino acids belonging to their active sites that are strongly conserved. Moreover, they reveal an increased structural similarity of their catalytic domains, reflecting the common biological function of these enzymes. The studied enzymes present dissimilar local physicochemical properties of their catalytic cavities that could be responsible of their distinct deacetylation patterns. Molecular docking studies reflect that deacetylation efficiency is also distinct for the chitin and partially deacetylated chitin oligomers and that N-acetylglucosamine units and some partially deacetylated chitin oligomers could have inhibitory effect against chitin deacetylases belonging to fungi and marine bacteria.

Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases.

Carbohydrate esterases family 4 (CE4 enzymes) includes chitin and peptidoglycan deacetylases, acetylxylan esterases, and poly-N-acetylglucosamine deacetylases that act on structural polysaccharides, altering their physicochemical properties, and participating in diverse biological functions. Chitin and peptidoglycan deacetylases are not only involved in cell wall morphogenesis and remodeling in fungi and bacteria, but they are also used by pathogenic microorganisms to evade host defense mechanisms. Likewise, biofilm formation in bacteria requires partial deacetylation of extracellular polysaccharides mediated by poly-N-acetylglucosamine deacetylases. Such biological functions make these enzymes attractive targets for drug design against pathogenic fungi and bacteria. On the other side, acetylxylan esterases deacetylate plant cell wall complex xylans to make them accessible to hydrolases, making them attractive biocatalysts for biomass utilization. CE4 family members are metal-dependent hydrolases. They are highly specific for their particular substrates, and show diverse modes of action, exhibiting either processive, multiple attack, or patterned deacetylation mechanisms. However, the determinants of substrate specificity remain poorly understood. Here, we review the current knowledge on the structure, activity, and specificity of CE4 enzymes, focusing on chitin deacetylases and related enzymes active on N-acetylglucosamine-containing oligo and polysaccharides.

Chitin Deacetylases: Structures, Specificities, and Biotech Applications.

Depolymerization and de-N-acetylation of chitin by chitinases and deacetylases generates a series of derivatives including chitosans and chitooligosaccharides (COS), which are involved in molecular recognition events such as modulation of cell signaling and morphogenesis, immune responses, and host-pathogen interactions. Chitosans and COS are also attractive scaffolds for the development of bionanomaterials for drug/gene delivery and tissue engineering applications. Most of the biological activities associated with COS seem to be largely dependent not only on the degree of polymerization but also on the acetylation pattern, which defines the charge density and distribution of GlcNAc and GlcNH2 moieties in chitosans and COS. Chitin de-N-acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. The deacetylation patterns are diverse, some CDAs being specific for single positions, others showing multiple attack, processivity or random actions. This review summarizes the current knowledge on substrate specificity of bacterial and fungal CDAs, focusing on the structural and molecular aspects of their modes of action. Understanding the structural determinants of specificity will not only contribute to unravelling structure-function relationships, but also to use and engineer CDAs as biocatalysts for the production of tailor-made chitosans and COS for a growing number of applications.