Enzymatic production and fine-tuning of chitin-derived products

Abstract

Chitin is the second moss widespread polysaccharide in the world, and a cheap but valuable resource that can be used in a large range of applications. The derivatives of chitin, chitosan and chitooligosaccharides (CHOS), are of special interest as these compounds can be used for production of high value products in biochemical, medical and industrial sectors. Valorization of chitin today brings about substantial environmental impacts and few options for precise product design of high. Enzymatic valorization of chitin derivatives has the potential to decrease environmental impact while producing higher quality products. In particular, the enzymatic production of longer chain CHOS is of great interest, because there are few enzymes known today that generate these products. This study assessed the possibilities for fine-tuning the production of chitin derived products using a range of chitin-active enzymes.

Five new chitin deacetylases were selected for their deacetylating characteristics based on sequence motifs and identities. These were cloned into the pIB4 expression vector but not expressed and characterized due to a lack of time. Activity assays were performed to analyze the effects of combining chitin-active lytic polysaccharide monooxygenases (LPMOs) with expansins and (previously expressed) chitin deacetylases in one-pot reaction systems. It was found that the expansins GtEXPN, GtCPN and ZtEXLX gave a significant increase in activity for the LPMOs BcLPMO10A, BlLPMO10A and CBP21 when acting on colloidal -chitin, but not for CjLPMO10A and SgLPMO10F. All LPMOs produced longer chain CHOS in the presence of expansin, which shows that expansins may be used to produce new products. Enzymatic reactions with the LPMO CBP21 acting on fungal chitin and -chitin were performed, and the results showed that the LPMO likely is able to fully solubilize the chitin. This may prove to be a useful way to improve subsequent valorization steps, by allowing other enzymes easier access to the substrate. The deacetylases Tk-Dac and VcCDA successfully generated mono-deacetylated products when acting on colloidal -chitin in combination with an LPMO. Combined reactions like this one-pot system could be an important way to increase the efficiency of enzymatic valorization of CHOS.

This study is a step on the way to efficient enzymatic valorization of chitin-derived products because it shows that streamlining the reactions with a one-pot systems may work well for deacetylating CHOS, and that expansins effectively can increase the amount of CHOS generated by LPMOs, while also increasing the chain length.