Production of terminally n-deacetylated oligomers of chitosane using recombinant chitooligosacharide deacetylase NodB of bacteria Mesorhizobium loti expressed in Escherichia coli

Chitin and chitosan oligomers affect the growth and development of plants and are able to induce plant resistance to infection with phytopathogens, which determines the interest in the preparation and use of these compounds. The influence of chitosan oligomers on the plant directly depends on the degree of deacetylation, but it is difficultly to obtain compounds with necessary structure using hydrolysis of the polymer or chemical synthesis. Such problems can be solved in the process of biosynthesis of chitooligosaccharides, when enzymes with specific activity are used. The selectivity of the chitooligosaccharide deacetylase (EC 3.5.1.-) of rhizobia to carry out the mono-deacetylation of the chitooligosaccharides at the terminal position of the molecule causes interest in studying the possibility to use this enzyme for the synthesis of such compounds. In current work we have developed approaches for the synthesis of mono-deacetylated chitopentaose (tetra-N-acetylchitopentaose) using Mesorhizobium loti CIAM1026 enzyme chitooligosaccharide deacetylase. Heterologous expression of the nodB gene encoding the M. loti chitooligosaccharide deacetylase in Escherichia coli XL1-Blue MRF' and SHuffle express strains using the modified pOPE101mod- nodB vector with deleted pelB sequence resulted in soluble enzyme preparation. The amount of soluble enzyme was higher in SHuffle express strain, which was specially developed for correct formation of disulfide bonds in synthesized proteins. Studying the properties of the enzyme purified on Ni-NTA agarose showed its ability to deacetylate penta-N-acetylchitopentaose at the terminal position. Mass spectrometric analysis confirmed the use of practically the entire substrate for the preparation of deacetylated tetra-N-acetylchitopentaose. Methods for the separation and purification of deacetylated chitooligosaccharides by ion exchange chromatography followed by desalination have been developed. Synthesis of terminally N-deacetylated chitosan oligomers may be a necessary step in the preparation of their conjugates with biologically active compounds.