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Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers.

ABSTRACT: Background:Partially acetylated chito-oligosaccharides (paCOS) have a variety of potential applications in different fields, but to harness their benefits, pure paCOS or well-defined paCOS mixtures are essential. For example, if one could produce fully acetylated (A4) and fully deacetylated (D4) tetramers in abundance, all possible variants of tetrameric paCOS could be generated reliably from them. A promising approach for generating defined paCOS is by enzymatic depolymerization of chitosan polymers using chitosanases, since these enzymes' subsite specificities directly influence the composition of the paCOS produced; however, enzymatic production of e.g. D4 is challenging because the substrate is generally hydrolyzed further by most chitosanases. To overcome this, chitosanases could potentially be engineered so that upon hydrolyzing chitosan, they are unable to hydrolyze certain substrates, leaving well-defined oligomers intact in the hydrolysate. Results:For this purpose, we performed rational protein engineering on the extensively studied GH 8 chitosanase CSN from Bacillus sp. MN. By specifically targeting residues with a predicted function in substrate binding, we created new muteins incapable of efficiently hydrolyzing the fully deacetylated tetramer D4, and we were able to demonstrate efficient large-scale production of D4 with an altered version of CSN. Furthermore, we were able to uncover differences in the substrate positioning and subsite specificities of the muteins, which result in altered paCOS mixtures produced from partially acetylated chitosan polymers, with possibly altered bioactivities. Conclusion:The value of protein engineering as a tool for the more efficient production of pure oligomers and potentially bioactive paCOS mixtures was demonstrated by the results and the suitability of specific muteins for the large-scale production of strictly defined, pure paCOS in a batch process was shown using the example of D4.

SUBMITTER: Gercke D

PROVIDER: S-EPMC6419424 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Rational protein design of <i>Bacillus</i> sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers.

Gercke David D Regel Eva K EK Singh Ratna R Moerschbacher Bruno M BM

Journal of biological engineering 20190312

<h4>Background</h4>Partially acetylated chito-oligosaccharides (paCOS) have a variety of potential applications in different fields, but to harness their benefits, pure paCOS or well-defined paCOS mixtures are essential. For example, if one could produce fully acetylated (A<sub>4</sub>) and fully deacetylated (D<sub>4</sub>) tetramers in abundance, all possible variants of tetrameric paCOS could be generated reliably from them. A promising approach for generating defined paCOS is by enzymatic de ...[more]

PMID: 30918529

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Project description:For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

Dataset Information

Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers.

Publications

Rational protein design of <i>Bacillus</i> sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers.

Similar Datasets

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