Chitosan oligosaccharides (oligomers of (GlcNAc)(x)(GlcN)(y)) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p-nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa-N-acetyl-glucosamine substrate (GlcNAc)(6). Native AxeA and two variants displaying>50% deacetylation of the oligohexamer substrate after reaction at 50°C for 24h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc)(5)(GlcN)(1) to (GlcNAc)(1)(GlcN)(5) and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN)(6). The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.
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