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

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.

Molecular Mechanisms of Chitosan Interactions with Fungi and Plants

“…Chitin deacetylases (CDAs) generate chitosan from chitin. Combinations of chitin oligosaccharide deacetylases modify the pattern of polymer deacetylation [5,6,7]. Some fungal plant pathogens generate chitosan [8] during early infection to avoid host defenses [9].…”

Marine chitin upcycling with immobilized chitinolytic enzymes: current state and prospects

“…Table 2 contains a selection of 31 articles from a literature search for work with immobilized chitinolytic enzymes, with the entries split into groups based on the identity of the enzyme used in the studies. 206–236 For the individual reports we include information on the choice of scaffold material, the type of enzyme–scaffold conjugation and the application target, together with some metrics on the performance resulting from the different immobilization procedures, quoting the characteristics reported in the source publications. Note that direct comparison of the metric features in the chart should be made with caution, since protocol standards for trials with immobilized and free enzyme do not yet exist in the field, so extrapolation and interpretations to produce, for instance, a quality ranking may be questionable.…”

Chitin deacetylase: from molecular structure to practical applications

“…So far, random evolution has been used for the modification of CDAs to expand the substrate specificity of CDAs. Pascual and Planas [67] designed a high-throughput screening (HTS) assay for screening the random mutant libraries for catalytic domain of VcCDA, which was based on a fluorescence monitoring assay of the deacetylase activity. A double mutant was selected with a ninefold higher k cat values for the substrates DP4 and DP5, and a retained activity for substrate DP2, which showed an expanded substrate specificity of VcCDA.…”