Paper accepted: “Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers”

Today, the manuscript of David Gercke and Eva Regel on engineering ‘our’ chitosanase to generate fully deacetylated chitosan tetramer, GlcN4, when hydrolyzing polyglucosamine was accepted for publication in the Journal of Biological Engineering. Supported by our bioinformatician and co-author Dr. Ratna Singh, our Master candidate David and our doctoral researcher Eva (financially supported by her doctoral fellowship from DBU) had designed a series of single amino acid exchange mutants of the Bacillus chitosanase originally isolated by our former doctoral researcher Malathi Nampally. Ratna as well as our former doctoral researcher Tobias Weikert had characterized structure-function relationships of this enzyme in great detail. The wildtype enzyme, like most chitosanases as well as other polysaccharide hydrolases, yields mostly dimeric and trimeric products, because the tetramer is the smallest substrate that is efficiently hydrolyzed, when accommodated in the substrate binding cleft from subsites (-2) to (+2). The idea was to weaken substrate-enzyme interactions at either subsite (-2) or (+2) so that the tetramer does not bind firmly enough anymore to be cleaved, while larger oligomers and polymers, by interacting at the extremities of the binding cleft with subsites (-3) and/or (+3)/(+4), can still be cleaved. And this worked! Two of the muteins designed cannot hydrolyze the tetramer anymore, and efficiently convert polyglucosamine into GlcN oligomers, including ca. 40% of tetramer. We are now converting this fully deacetylated tetramer into defined partially acetylated chitosan tetramers using chitin deacetylases ‘in reverse’, as recently described by our doctoral researcher Lea Hembach. And hopefully, we can soon report on using these oligomers to begin deciphering the ‘language of chitosans’.