Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides

Abstract

Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from Saccharomyces cerevisiae, ScCDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. ScCDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that ScCDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. ScCDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore ScCDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

Chitin Deacetylase from Bacillus aryabhattai TCI-16: Heterologous Expression, Characterization, and Deacetylation Performance

“…The deacetylation process of BaCDA can be revealed through ESI-MS analysis of enzymatic hydrolysis products . As can be seen from Figure S4, ESI-MS detection of the product after 5 min of BaCDA hydrolysis revealed the presence of (GlcNAc) 3 GlcN (A3D1) and (GlcNAc) 4 (A4), indicating the beginning of the deacetylation reaction (Figure S4a).…”

Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes

“…The Tyr255(ChDaI)/Phe269(ChDaII) (MT3) together with Asp164(ChDaI)/Asp178(ChDaII) (MT1) and His320(ChDaI)/His334(ChDaII) (MT5) residues are directly involved in the amide bond hydrolysis reaction ( Nakamura et al, 2017 ; Tuveng et al, 2017 ). Moreover, highly conserved residues Asp165(ChDaI)/Asp179(ChDaII) (MT1), His214(ChDaI)/His228(ChDaII) (MT2) and His218(ChDaI)/His232(ChDaII) (MT2) form the so-called an metal-binding triad that coordinate the metal atoms; typically one of Zn 2+ , Co 2+ , Ca 2+ ; which are co-factors of chitin deacetylases ( Zhu et al, 2019 ). Moreover, the performed multi-sequence comparison allowed for the identification of six loops in the amino acid sequences of ChDaI and ChDaII proteins.…”

Research of saccharides and related biocomplexes: A review with recent techniques and applications

“…It was not the case in MALDI-MS since matrix removal was advantageous for the separation of glucose-bound lipids and peptides [236]. They could be identified from native and methylated gangliosides with high sensitivity, although usually the saccharides are patterned with RP-HPLC-Orbitrap-MS [46,94,119,243,246]. Tables S4 [106,[244][245][246][247][248][249] and S5 [75,129,250,251] give fragmentation and identification of saccharides with specific m/z fragments (data correlates with their isolation from plants).…”