Efficient production of N-acetylglucosamine with chitinolytic enzymes from Myceliophthorathermophila C1

Chitin is a natural biopolymer abundantly occurring in nature and is a main source for production of its monomer - N-acetylglucosamine (GlcNAc). GlcNAc is widely used in medical treatment of joints disorders e.g. osteoarthritis, in skin care and dermatology as moisture agent, in food as a food additive. In this thesis we present the development of an enzymatic process for production of GlcNAc from chitin with use of chitinolytic enzymes Chitinase Chi1 and N-acetylglucosaminidase (NAGase) MthNAG from Myceliophthora thermophila C1.

We homologously overproduced Chitinase Chi1 and MthNAG in a low protease/(hemi-) cellulase free M. thermophila C1-expression host. Overproduced enzymes were purified and their mode of action, catalytic properties and thermostability were characterised. Both enzymes showed remarkable thermostability that has never been observed for this group of fungal enzymes. Chitinase Chi1 and MthNAG synergistically hydrolysed chitin i.e. the products released from polymeric chitin by Chitinase Chi1 were hydrolysed by MthNAG to a final product, GlcNAc. The synergistic action of Chitinase Chi1 and MthNAG and their thermostability was fundamental for the development of an enzymatic production process of GlcNAc.

Furthermore, we investigated the production of GlcNAc from chitin impregnated with H2SO4 and subsequently ball milled (ball-milled-H2SO4-chitin). According to the XRD measurements, the crystallinity of chitin has been drastically decreased upon ball-milling pre-treatment and the accessibility of chitin was considerably improved for the enzymatic action of Chitinase Chi1 and MthNAG. Subsequently, we optimized the production of GlcNAc from ball-milled-H2SO4-chitin based on a catalytic cascade catalysed by Chitinase Chi1 and MthNAG. Using central composite design (CCD) and response surface methodology (RSM), we found conditions that allow production of GlcNAc at high yield. After 6 h incubation at 50 °C, the yield of GlcNAc of 73.2 % from 1.6 % (w/v) ball-milled chitin treated with 25 μg Chitinase Chi1 and 20 μg MthNAG was obtained.

We also applied Chitinase Chi1 as a tool for a fingerprint analysis of (modified)chitosan. Since Chitinase Chi1 showed a wide substrate specificity and was active on chitosans with the degree of deacetylation (DDA) up to 94 %, the enzyme was used for hydrolysis of modified chitosans with different degree of oxidation (OD). The oligosaccharides released by Chitinase Chi1 were identified with MALDI-TOF-MS. Depending on the degree of oxidation (OD), different oligosaccharides were identified. The results were used for the elucidation of the structure of the oxidised chitosans and for understanding the behaviour (structure-function relation) of this polymers.

Finally, we gave an overview on chitin and chitosan as a biomass for monomers in the biobased economy. We showed the valorisation routes for chitin from different types of chitin-containing biomass and we gave an overview on current and potential applications of GlcNAc. Furthermore, we discussed the main drawbacks of the enzyme-based methods for the production of GlcNAc from chitin and we identified the possible solutions. Finally, we present a setup for the process based on hydrolysis of ball-milled-H2SO4 chitin with Chitinase Chi1 and MthNAG and give suggestions for the consideration of the important aspects of the enzymatic production of GlcNAc from chitin.