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.

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BackgroundIn Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes.ResultsChitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and β-N-acetylglucosaminidase NagA2.ConclusionsC. glutamicum possesses β-N-acetylglucosaminidase. In the wild type, β-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active β-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme activities such as processive chitinase or endochitinase activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0795-3) contains supplementary material, which is available to authorized users.