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

Abstract

BackgroundPartially 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.ResultsFor 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s13036-019-0152-9) contains supplementary material, which is available to authorized users.

Enzymatic Modifications of Chitin, Chitosan, and Chitooligosaccharides

“…They recommended a chitosanases classification system which is based on specificity and preferences toward subsite (−2) to (+2). Gercke et al (2019) used rational protein engineering methods to produce modified chitosanase from Bacillus sp. The obtained enzyme was specific toward subsite (−3) to (+3) and able to produce DP4 COS by hydrolysing fully deacetylated substrates.…”

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives

“…Altered substrate specificity was also obtained by site directed mutagenesis of residues involved in substrate binding of the Bacillus sp. MN chitosanase (E309R and N319E), leading to muteins able to bind N-acetyl- d -glucosamine [ 98 ] and unable to hydrolyze the fully deacetylated chito-oligosaccharide tetramer [ 99 ]. Moreover, a series of seven mutations (V119D, S262K, N291D, D293T, G319S, D358G, and D368H) induced in alpha-gliadin peptidase by a computational protein design approach that enhanced the number of hydrogen bonds within substrate binding pockets led to an increased specificity for the immunogenic fragments of gluten peptides by 877-fold, with putative application in biosensing [ 100 ].…”

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

“…Alternatively, A4 can be produced by GlcNase digestion of the α-mono-deacetylated pentameric paCOS DAAAA, synthesized itself by an E. coli cell factory expressing NodC and NodB from Rhizobium , as described above. There is also an alternative route to the fully de-acetylated tetramer D4 using a mutant of the chitosanase Bsp CsnMN which has been engineered using site directed mutagenesis, to reduce its ability to hydrolyse the tetramer which consequently is obtained as a product of polyglucosamine digestion, of course also requiring SEC purification [ 70 ].…”

“… Biotechnological preparation of the fourteen possible tetrameric paCOS using CDAs in forward or reverse mode on the fully acetylated or fully de-acetylated tetramers AAAA and DDDD, respectively, as described previously [ 34 ]. Both substrates can be prepared biotechnologically in good yields, AAAA by an E. coli cell factory expressing NodC and NodB to produce DAAAA followed by GlcNase treatment to yield AAAA, DDDD by digestion of polyglucosamine using an engineered chitosanase unable to cleave tetrameric substrates [ 70 ]. Both AAAA and DDDD need to be purified before converting them to paCOS using CDAs.…”