ABSTRACT
Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.
Subject(s)
Chitin/analogs & derivatives , Oligosaccharides/biosynthesis , Oligosaccharides/chemical synthesis , Acetylglucosamine/chemistry , Aspergillus niger/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbohydrate Sequence , Chitin/biosynthesis , Chitin/chemical synthesis , Chitinases/genetics , Chitinases/metabolism , Crystallography, X-Ray , Glucosamine/chemistry , Hexosaminidases/metabolism , Humans , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Oligosaccharides/chemistry , Serratia/enzymology , Serratia/genetics , Serratia marcescens/enzymology , Serratia marcescens/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Chitin, an insoluble linear polymer of ß-1,4-N-acetyl-d-glucosamine (GlcNAc; A), can be converted to chitosan, a soluble heteropolymer of GlcNAc and d-glucosamine (GlcN; D) residues, by partial deacetylation. In nature, deacetylation of chitin is catalyzed by enzymes called chitin deacetylases (CDA) and it has been proposed that CDAs could be used to produce chitosan. In this work, we show that CDAs can remove up to approximately 10% of N-acetyl groups from two different (α and ß) chitin nanofibers, but cannot produce chitosan.
