CBH-ChitoMech

The project CBH-ChitoMech investigates the mechanisms behind the hydrolysis of chitosan by the Cellobiohydrolase I from Hypocrea jecorina (HjCBH). Chitosan is a polysaccharide commonly found in crustacean shells but also in fungi. The natural product gains more and more attention for medicinal applications due to its antimicrobial activity and biocompatibility. Especially the hydrolysis products of chitosan, chito oligosaccharides (COSs), are of great interest since latest research results dedicate these compounds several properties beneficial to the health. However the production of COSs remains a challenge since chemical methods result in heterogeneous product pools, which can hardly be characterized. The enzymatic hydrolysis of chitosan is the remedy to obtain more homogeneous COSs with defined structural properties. Recent studies on chitosan degrading enzymes concentrate on chitosan specific enzymes, which are to date very expensive and not economically feasible. Previous studies revealed the potential of readily available, commercial cellobiohydrolases from the fungi Trichoderma to produce bioactive COSs, such as HjCBH. Despite the great potential to fulfill this task, further mechanistic studies of HjCBH acting on chitosan are required to exploit the full potential of this enzyme. Mechanistic studies of enzymes require a well-defined set of model compounds to perform kinetic studies and more. Within this project COS model compounds are synthesized using enzymes specifically designed for their synthesis and are provided by the host Prof. Withers. Once model compounds of different structural composition are prepared, detailed kinetic studies are performed, which unveil the preferred structural properties of chitosan by HjCBH. This information will lay the foundation for an optimized COS production by HjCBH since key parameters of the hydrolysis process can be accordingly adjusted. The multidisciplinary approach followed in this project will be transferred to the home institution and is the basis of further studies of this enzymes class on chitosan. Within the return phase enzymes of similar structural properties are produced and tested regarding their chitosan hydrolyzing activity. Previous learned approaches are therefore applied, but also their role of so called carbohydrate binding modules in the hydrolysis process is investigated, a core competence of the lab of Prof. Gübitz (home institution). The presented study will improve enzymatic production procedures following the path of using chitosan unspecific enzymes. In addition an educated guess will be provided why CBH from Trichoderma evolved a chitosan hydrolyzing side activity and can give insight into additional biological properties this enzyme class may have in fungi.

Cellobiohydrolases (CBHs) are essential constituents of the enzymatic cocktail of fungi responsible for the degradation of cellulose. Recent studies unveiled a remarkable side hydrolysis activity of CBH I from Hypocrea jecorina (HjCBH) on chitosan. The hydrolysis products of chitosan, chito oligosaccharides (COSs), show various bioactivities including antimicrobial and plant resistance stimulating properties. To date, the production of COSs still relies on the use of specific enzymes, which are expensive and impede an economically feasible production of these valuable compounds. HjCBH instead is used in the biofuel industry and thus commercially available in bulk, which renders this enzyme a potential candidate for the low-cost production of COSs. The project aimed to increase the understanding of the catalytic mechanism of HjCBH hydrolyzing chitosan, as the basis to implement and improve an enzymatic process for COS production. Chitosan is a heterogenous polysaccharide consisting of two monosaccharides, glucosamine (GlcN) and, to a minor degree, N-acetyl glucosamine (GlcNAc). Previous studies revealed that the percentage and distribution of GlcNAc in chitosan greatly alters the hydrolysis efficiency of HjCBH. Using chitosan model compounds with a known composition regarding the mentioned monosaccharides, we could show that a GlcNAc content of around 50% and an even distribution of GlcNAc in the chitosan polymer is required to harness to full potential of HjCBH hydrolyzing chitosan. Cellulose and chitosan are structurally very similar; however, one hydroxy group of the monosaccharides found in cellulose is replaced by an amine or N-acetyl group in chitosan. This little change is responsible for the remarkably different properties of these two polysaccharides. We tried to get insights into the promiscuity of HjCBH to understand why this enzyme can hydrolyze both compounds and used computational simulation approaches, showing that HjCBH can also accommodate and bind the bulkier N-acetyl group in the active site. We further calculated binding energies from our computational model which revealed that HjCBH prefers GlcNAc units in proximity of the cleavage site. Further insights in the catalytic mechanism of HjCBH hydrolyzing chitosan was obtained in this project which lays the foundation to implement this enzyme in large scale processes to produce COSs. The increased availability of defined COSs will foster research and furthermore facilitate the implementation of these promising compounds in various disciplines, ranging from medicine to crop protection.