title: A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation.
abstract: Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.
  
  
  
  We successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycete Puccinia graminis f. sp. tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Shoa
authors: S
authors: Naqvi
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Stefan
authors: S
authors: Cord-Landwehr
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Ratna
authors: R
authors: Singh
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Frank
authors: F
authors: Bernard
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Stephan
authors: S
authors: Kolkenbrock
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany.
authors: Nour Eddine
authors: NE
authors: El Gueddari
authors: Institute for Biology and Biotechnology of Plants (IBBP), WWU Münster University, Münster, Germany moersch@uni-muenster.de.
authors: Bruno M
authors: BM
authors: Moerschbacher
mesh_terms: D000107:Acetylation; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics*; D002686:Chitin / Q000378:metabolism*; D048271:Chitosan / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000235:genetics; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D058105:Polymerization; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D002686:Chitin; D048271:Chitosan; D000581:Amidohydrolases; C019866:chitin deacetylase