Abstract
Chitosans, partially de-N-acetylated derivatives of chitin, are multifunctional biopolymers. In nature, biological activities of partially acetylated chitosan polymers are mediated in part by their oligomeric breakdown products, which are generated in situ by the action of chitosanolytic enzymes. Understanding chitosanolytic enzymes, therefore, can lead to the production of chitosan oligomers with fully defined structures that may confer specific bioactivities. To address whether defined oligomer products can be produced via chitosanolytic enzymes, we here characterized a GH8 family chitosanase from Bacillus spec. MN, determining its mode of action and product profiles. We found that the enzyme has higher activity towards polymers with lower degree of acetylation. Oligomeric products were dominated by GlcN3, GlcN3GlcNAc1, and GlcN4GlcNAc1. The product distribution from oligomers were GlcN3 > GlcN2. Modeling and simulations show that the binding site comprises subsites ranging from (−3) to (+3), and a putative (+4) subsite, with defined preferences for GlcN or GlcNAc at each subsite. Flexible loops at the binding site facilitate enzyme-substrate interactions and form a cleft at the active site which can open and close. The detailed insight gained here will help to engineer enzyme variants to produce tailored chitosan oligomers with defined structures that can then be used to probe their specific biological activities.
Highlights
Chitosans, a family of partially de-N-acetylated derivatives of chitin, are among the most versatile and most promising functional biopolymers, with interesting physicochemical properties and multiple bioactivities such as plant disease protectants[1,2], antimicrobial agents[3], as sustained release nanoparticle carriers for drug, gene, or vaccine delivery[4]
Time course studies using a fully deacetylated chitosan polymer as a substrate showed that larger oligomers appeared gradually at first, smaller oligomers appeared, revealing that the enzyme acts in a non-processive endo-mode (Fig. 1B)
Well-defined chitosan oligomers are required to investigate the biological activity of chitosans
Summary
A family of partially de-N-acetylated derivatives of chitin, are among the most versatile and most promising functional biopolymers, with interesting physicochemical properties and multiple bioactivities such as plant disease protectants[1,2], antimicrobial agents[3], as sustained release nanoparticle carriers for drug, gene, or vaccine delivery[4]. No direct relationship has been established between the above-mentioned chitosanase classes I-IV and the GH families This broad sequence diversity in the chitosanase family, which greatly exceeds that of chitinases, raises the prospect of studying their subsite specificities in the hope of finding more specific enzymes that might be useful for producing chitosan oligomers with more defined PA. To further this goal, in this study we focused on a bacterial chitosanase from Bacillus spec. Merging data obtained from in silico and in vitro experiments provided detailed insights into CSN-MN’s subsite specificities and mode of action; these insights will guide future enzyme engineering[20], to produce well-defined chitosan oligomers that can be used to understand structure-function relationships of partially acetylated chitosan oligomers
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