Engineering chitin deacetylase AsCDA for improving the catalytic efficiency towards crystalline chitin

Chitin deacetylase (CDA) catalyzing the deacetylation of crystal chitin is a crucial step in the biosynthesis of chitosan, and also a scientific problem to be solved, which restricts the high-value utilization of chitin resources. This study aims to improve the catalytic efficiency of AsCDA from Acinetobacter schindleri MCDA01 by a semi-rational design using alanine scanning mutagenesis and saturation mutagenesis. The quadruple mutant M11 displayed a 2.31 and 1.73-fold improvement in kcat/Km and specific activity over AsCDA, which can remove 68 % of the acetyl groups from α-chitin. Furthermore, structural analysis suggested that additional hydrogen bonds, contributing the flexibility of amino acids and increasing the negative charge in M11 increased the catalytic efficiency. The microstructure changes of α-chitin pretreated by the mutant M11 were observed and evaluated using 13C CP/MAS NMR spectroscopy, FT-IR spectroscopy, XRD and SEM, and the results showed that M11 more efficiently catalyzed the release of acetyl groups from α-chitin. This study would provide a theoretical basis for the molecular modification of CDAs and accelerate the process of industrial production of chitosan by CDAs.