Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases.

Stefanie Nicole Hamer,Stefan Cord-Landwehr,Hendrik Waegeman,Stephan Kolkenbrock,Bruno Maria Moerschbacher,Antoni Planas,Xevi Biarnés

doi:10.1038/srep08716

Stefanie Nicole Hamer, Stefan Cord-Landwehr + Show 5 more

Open Access

https://doi.org/10.1038/srep08716

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Abstract

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

Highlights

Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases
Chitosan oligomers with specific pattern of acetylation (PA) can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available
We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp

Summary

Results and Discussion

The hydrolysis products were characterized by ultra high performance liquid chromatography - evaporative light scattering detection - electrospray ionization - mass spectrometry (UHPLCELSD-ESI-MS), revealing that NodB was not active towards GlcNAc1, but converted GlcNAc2–6 completely into mono-deacetylated chitosan oligomers (Table 1). To our knowledge, this is the first report of obtaining enzymatically active, recombinant NodB without the need of refolding it from insoluble inclusion bodies[20]. The PA of the doubly deacetylated chitosan pentamer was determined using enzymatic/mass spectrometric sequencing which revealed that the first two units from the non-reducing end were deacetylated in the combined as well as in both sequential reactions of NodB and COD (Figure 4).

A2 A3 A4 A5 A6

A3 A4 A5 A6

Methods