Deacetylation of chitin and chitooligosaccharides (COS) renders chitosans (with variable degrees of acetylation (DA)) and partially deacetylated oligosaccharides (paCOS) with considerable industrial interest as biocompatible, biodegradable, and non-toxic functional materials with a wide variety of biotechnological and biomedical applications. The degree of acetylation (DA) strongly influences the physicochemical and biological properties of chitosans and paCOS, but the role of the pattern of acetylation (PA), which defines the distribution of charged D-glucosamine (GlcNH₂) residues along the N-acetyl-D-glucosamine (GlcNAc) polymeric/ oligomeric chain, on the specific interactions with biological receptors and target structures remains largely unknown.
Chitin de-N-acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. The deacetylation pattern exhibited by CDAs and related carbohydrate esterase (CE4) enzymes active on COS is diverse, some being specific for a single position, others showing multiple attack mechanisms. After solving the first 3D structure of a Vibrio cholera CDA CE4 deacetylase in complex with substrates , we are addressing structural and biochemical studies with the goal of understanding the determinants of substrate specificity of CDAs and related CE4 enzymes active on COS [1-5]. The search of new enzymes and the engineering of substrate specificity, either by structure-guided and directed evolution approaches will extend the toolbox of selective enzymes for the biotechnological production of tailored and sequence-defined COS to evaluate their biological functions and develop novel biotech and biomedical applications.
Here, we report the design and development of a HTS assay in microplates format using a medium scale robotic platform for the screening of directed evolution libraries of CDA and other CE4 enzymes active on COS and its proof-of-concept application to the engineering of substrate specificity of VcCDA. The assay is based on fusion of the target CE4 catalytic domain to be evolved to a chitin binding module (CBM), capture of the expressed proteins from cell-free extracts with chitin-coated magnetic beads, and evaluation of the deacetylase activity of the immobilized enzyme variants on COS substrates by monitoring product formation with a coupled assay leading to a fluorescence readout . Results on the identification and characterization of novel enzyme variants from random libraries with engineered specificity will be reported.
Work supported by EU-FP7 project Contract Number 613931 (NANO3BIO project), and BFU2016-77427-C2-1-R, MINECO, Spain. S. Pascual acknowledges a predoctoral contract from Nano3Bio.
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