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.
Highlights
Chitin, which consists of β-1,4-linked N-acetyl-D-glucosamine residues, is the main component of crustacean shells, such as shrimp, crab and shellfish [1,2]
chitin deacetylases (CDAs) belongs to the carbohydrate esterase family 4 (CE4) according to the classification of the CAZY database [32]
The sequence of ScCDA2 aligned with deacetylase sequences from marine Arthrobacter (ArCE4A, 34%) [34], Streptomyces lividans (SlCE4, 33%) [35] and Streptococcus pneumoniae (SpPgdA, 29%) [36]
Summary
Chitin, which consists of β-1,4-linked N-acetyl-D-glucosamine residues, is the main component of crustacean shells, such as shrimp, crab and shellfish [1,2]. Chitosan and COS produced by chemical methods usually exhibit a randomized pattern of acetylation, making them difficult to control and predict their biological activity [17]. Chitin deacetylase (CDA, E.C. 3.5.1.41) is able to hydrolyse the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, chitin oligosaccharides, chitosan and chitosan oligosaccharides under mild conditions by a specific mode of action. Two genes encoding chitin deacetylases (CDA1 and CDA2 ) have been identified in Saccharomyces cerevisiae in previous reports. The chitin deacetylase (CDA2 ) from Saccharomyces cerevisiae (ScCDA2 ) with a specific mode of action has been characterized and a fast, convenient and online monitoring method has been developed that can be used to separate and detect partially acetylated chitosan oligosaccharides. ScCDA2 is able to remove about 8% and 20% of the acetyl groups from crystalline chitin and colloidal chitin
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