Enzymatic Modification of Native Chitin and Conversion to Specialty Chemical Products

Abstract

: Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher M_w due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.

Keywords: biotechnology; chitin; chitin deacetylase; chitinase; chitooligosaccharides; chitosan; chitosanase; enzymatic modification; lytic polysaccharide monooxygenase.

Figure 1

Chemical and biotechnological chitin extraction methods from crustacean shell waste. Since native chitin from marine sources is a composite of calcium carbonate, protein, carotenoids, and small amounts of lipids, it has to be processed for industrial applications. After mincing and cleaning of the crustacean shell waste, it has to be freed of minerals and proteins. This can be achieved chemically (red, left side) by the employment of HCl and NaOH, respectively, while biotechnological approaches (green, right side) utilise lactic acid bacteria fermentation for mineral removal and protease cocktails for the excision of residual protein. To obtain the industrially relevant end products of chitosan and its partially acetylated chitooligosaccharides (paCOS), chitin is depolymerised by acid-hydrolysis or chitinases in a first step. Afterwards, the obtained chitooligosaccharides (COS) are N-deacetylated with 50% NaOH at room temperature or 120 °C, conventionally, or through chitin oligosaccharide deacetylases (COD), enzymatically. If high M_w chitosan is desired, the isolated chitin can be converted directly through incubation with hot alkali or chitin deacetylases. The figure was created with BioRender.

Figure 2

Chemical structures of chitin and chitosan. (a) Chitin is a homo-polymer assembled of N-acetyl-D-glucosamine monomers (GlcNAc), covalently bound through β-(1-4)-glycosidic linkages. In nature, it occurs as a co-polymer comprised of both GlcNAc and non-acetylated D-glucosamine (GlcN) subunits, with a molar GlcNAc fraction > 50%. (b) Chitosan is the deacetylated derivative of chitin, defined by a ratio of GlcN to GlcNAc monomers of > 1,0. It exhibits solubility in aqueous acetic solvents. (c) Chitin has three different allomorphs, which differ in the orientation of the respective polymer chains within the micro-fibril macro structure. The most abundant and resilient α-chitin is formed by antiparallel aligned polysaccharide chains. In β-chitin, the sugar chains are ordered in a parallel manner, therefore exhibiting weaker intramolecular interactions. The γ-allomorph of chitin is characterized by a mixture of both antiparallel and parallel aligned chains, which leads to a polymer with fractions of higher and lower levels of crystallinity. The figure was created with BioRender.

Figure 3

Enzymes with catalytic activity towards chitin and chitosan. The crystal structures of the chitinase A from Serratia marcescens [95], the lytic polysaccharide monooxygenase (LPMO) from Bacillus thuringiensis [96], the chitin deacetylase (CDA) from Colletotrichum lindemuthianum [97] and the chitosanase from Streptomyces sp. N174 [77], derived from the RCSB protein data bank (PDB), are illustrated as exemplary enzymes of their respective catalytic activity. Red coloured parts are α-helices, while β-sheets are indicated by yellow. Chitinases generally hydrolyse the β-(1,4)-glycosidic links between two GlcNAc monomers. Their modes of action, amino sequence, and catalytic sites can vary substantially. LPMOs are copper-dependent enzymes, which cleave chitin by oxidation of C1 or C4. They can recruit either H₂O₂ or O₂ as co-substrate and need an external reducing agent. The NodB-related CDAs hydrolyse the acetamido groups of GlcNAc monomers with a catalytic water molecule. Each enzyme exhibits specificity for target substrate sequences and creates unique deacetylation patterns. Chitosanases generally hydrolyse the β-(1,4)-glycosidic links between two GlcN monomers with a retaining or inverting mechanism. The specificity towards an additional substrate besides GlcN-GlcN is common. The figure was created with BioRender.