Where two are fighting, a third rejoices

Scientists can argue about strange things: One says: “The acetic acid molecules in chitosan are randomly distributed!”, the other says: “No, in blocks!”. And they argue about this, each with their own supporters, in public, for decades. The last time prominently during a large international meeting on chitin and chitosan in Seville, where Prof. Bruno Moerschbacher from the Institute for Biology and Biotechnology of Plants at the University of Münster, then freshly elected President of the European Chitin Society EUCHIS, started his presentation with a statement that all currently commercially available chitosans have a random distribution of acetic acid molecules, while only those produced biotechnologically by his own team have blockwise distributions. And only minutes later, Dr. Mats Andersson, founder of the company Flexichem in Sweden, started his presentation with the statement that all currently commercially available chitosans have a blockwise distribution of acetic acid molecules, while only those produced homogeneously by his company have random distributions. The discussion became heated, but led to no conclusion. And now, a young scientist comes along and proves that both parties are wrong: “On the contrary, they are surprisingly distributed fairly evenly!” And she publishes her findings in the highly prestigious journal “Nature Communications”.

What is it all about? Chitosans are long chain molecules, so-called biopolymers, made up of many sugar molecules, some of which carry an acetic acid molecule, while others do not. Chitosans are rare in nature and - so far, science believes, but wait and see! - only occur in the cell walls of some fungi, the yoke fungi. But similar biopolymers, in which each sugar molecule carries an acetic acid molecule, are very common in nature: they are called chitin. Chitin is found in the cell walls of practically all fungi, but also in the shells of crabs and crayfish, insects and spiders and in many other invertebrates. Chitin can therefore be easily isolated from crab shells, which are produced in huge quantities in the fishing industry. And then you can remove a few of the acetic acid molecules with a chemical treatment, voilà: chitosan!

If you take away more or less acetic acid molecules, you get different chitosans. And these have unusual properties and interesting biological activities. Some chitosans have an antimicrobial effect, others strengthen plants, again others heal wounds. For this, it is important that the long chains, the polymers, are broken down into shorter chains, oligomers, on site, i.e. in the respective target organism - because it is apparently these oligomers that are responsible for the bioactivities. They can consist of different numbers of sugar molecules with different numbers of acetic acid molecules, and the acetic acid molecules can also be distributed differently. For example, there are 16 different oligomers made of exactly four sugar molecules with zero to four acetic acid molecules, 32 different ones made of exactly five and 64 made of exactly six sugar molecules, etc. etc. - an almost infinite variety! And there are probably certain oligomers that are decisive for each bioactivity, while others are inactive or even inhibit the active ones. And which oligomers are formed during the chain scission of the polymers, active or inactive, depends on how the acetic acid molecules are distributed in the polymer and how the respective enzyme that carries out the scission reacts to the presence or absence of these acetic acid molecules. So you have to use exactly the right chitosan polymer with the right distribution of acetic acid molecules so that the target organism's chitosan-cleaving enzymes produce the desired bioactive chitosan oligomers and not the undesirable, inhibitory ones.

And here's the thing: although this chemical method of producing chitosan from chitin is already a century old, it is still unclear how it works and what exactly is produced! This is because the chitin is initially insoluble, but becomes soluble chitosan during the reaction. It is a so-called heterogeneous reaction in which the chitosan chunks are gradually converted to chitosan from the outside in. Depending on how long the reaction is allowed to take place and under which reaction conditions, different mixtures of chitosans with varying degrees of deacetylation, i.e. free of acetic acid, are produced. Some scientists argue that the parts of the chitin chains that are further out are already deacetylated, while other parts of the same chains that are closer to the center of the chunks remain completely acetylated. This would result in blocks of sugar molecules without acetic acid and other blocks with acetic acid in the chains. Other scientists believe that during the process, those chitin polymers that are further out are more deacetylated than those that are further in, which only come into contact with the reaction solution later in the reaction. As a result, the chitosan chains would ultimately differ in their degree of acetylation, i.e. the percentage of sugar units still carrying acetic acid, but the distribution of acetic acid molecules in all chains would be purely random, because the chemical reaction of deacetylation is a stochastic, random process. So what now? Blocks or random?

This is a matter of great debate as long as there are no good analytical methods to determine the distribution of acetic acid molecules. Our colleague Prof. Kjell Vårum from Norway developed a method for this analysis decades ago, but it is difficult to interpret and ultimately failed to settle the debate. Interestingly, both parties invoke Kjell's authority! In recent years, Dr. Stefan Cord-Landwehr has developed new methods for chitosan analysis in Prof. Bruno Moerschbacher's working group in Münster: enzymatic mass spectrometric fingerprinting of chitosans, which allows much more detailed insights. And his doctoral student Margareta Hellmann has now used these methods for a very systematic analysis of a large number of chitosans produced under different reaction conditions. And obtained surprising results. It quickly became clear that the traditionally produced chitosans exhibit neither a random nor a blockwise distribution of acetic acid molecules! But it still took some creativity, numerous discussions with specialists such as Prof. Laurent David, new chitosans produced according to Margarita's wishes by Dr. Stéphane Trombotto and Dr. Dominique Gillet and additional experiments with special enzymes carried out by Sonja Raetz as part of her Master's thesis before it slowly became clear that traditionally produced chitosans - and these are practically all commercially available chitosans - disproportionately often carry an acetic acid on every third sugar molecule. But how can this be? To understand this, Margareta had to do a lot of reading and to develop computer models for the deacetylation reaction before she could come up with a hypothesis. This, we have now jointly published prominently - and we are looking forward to hearing what our colleagues and past disputants have to say about it!

And before you ask: yes, this is important! Because - remember! - the distribution of the acetic acid molecules determines which oligomers are produced in the target organism. If I spray a plant with chitosan to activate its immune system so that it can better defend itself against pathogens and better cope with stress situations such as heat or drought, then I have to use the right chitosan with the right distribution of acetic acid molecules so that the plant enzymes can produce the plant-strengthening chitosan oligomers from it. And if I want to produce a wound dressing from chitosan under which even extensive third-degree burns can heal without scarring, then the choice of the right chitosan is also crucial. This has already worked well for the plants: we - that is our former doctoral students Dr. Sruthi Sreekumar and Dr. Carolin Richter as well as Dr. Anne Vortkamp and Dr. Philipp Lemke with the support of Prof. Bruno Moerschbacher - are currently in the process of using these new and other findings from our more than thirty years of chitosan research to found biotech start-ups. We want to use our basic research for the development of agri-biologics, which are urgently needed for the reliable production of high-quality food in sustainable agriculture. We haven't got that far with the wound dressings yet. But Margareta has also investigated in detail the three human enzymes that can cleave chitosan chains - this will be her next publication. And together with our colleague Dr. Christian Gorzelanny from the University Medical Center Eppendorf in Hamburg, she has been granted a project as part of the DFG Priority Program Codeχ to continue working on this question as a post-doctoral researcher in our group. In the Codeχ program, a dozen research groups in Germany are currently working on deciphering the chitosan code, i.e. understanding the influence of the acetylation pattern of chitosans on their material properties and biological activities - and then exploiting it. Exciting times!

further information
https://www.uni-muenster.de/Biologie.IBBP/agmoerschbacher/index.html
https://www.nature.com/articles/s41467-024-50857-1
https://codechi.de/