Screening Assay for Directed Evolution of Chitin Deacetylases: Application to Vibrio cholerae Deacetylase Mutant Libraries for Engineered Specificity

Sergi Pascual, Antoni Planas

Research output: Indexed journal article Articlepeer-review

8 Citations (Scopus)

Abstract

Not only the degree of acetylation but also the pattern of acetylation of chitosans and chitooligosaccharides (COS) appear to be critical for their biological activities. Protein engineering may expand the toolbox of chitin deacetylases (CDAs) with defined specificities for the enzymatic production of partially deacetylated COS for biotech and biomedical applications. A high-throughput screening (HTS) assay for screening directed evolution libraries is reported. It is based on a fluorescence monitoring assay of the deacetylase activity on COS substrates after capturing the expressed enzyme variants fused to a chitin binding module with chitin-coated magnetic beads. The assay is applied to the screening of random libraries of a Vibrio cholera CDA for increased activity on longer COS substrates.

Original languageEnglish
Pages (from-to)10654-10658
Number of pages5
JournalAnalytical Chemistry
Volume90
Issue number18
DOIs
Publication statusPublished - 18 Sept 2018

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