Abstract

The aim of our project is the production of a chitosan hydrogel which uses chitin as source material. This N-acetylglucosamine oligosaccharide (chitin) can be produced in E. coli via a Chitin Synthase (CHS). The enzyme used here is CHS NodC from the bacteria Rhizobium Leguminosarum. NodC is an N-acetylglucosaminyl transferase which catalyzes the formation of chitin tetramers and pentamers using activated N-acetylglucosamine monomers [Samain et al., 1997]. Originally, chitin is extracted chemically from crustacean shells, which uses a lot of chemicals and produces chitin oligosaccharides of unspecified length [Kurita, 2006]. To avoid this chemical process, we produce chitin oligosaccharides in E. coli by insertion of the nodC gene with the BioBrick system [Knight et al., 2003]. NodC reliably produces short oligosaccharides of certain lengths which can be directly further processed in vivo.[Samain et al., 1997]

Introduction

Besides cellulose, chitin is the most common natural polysaccharide in nature. Chitin is composed of β(1 -> 4) linked 2-acetamido-2-deoxy-β-D-glucose (N-acetylglucosamine, Figure 1). The polymer is a white, hard nitrogenous polysaccharide and is a component of fungi cell walls and of the exoskeletons of insects and crustaceans, like crabs or shrimps. [Dutta et al., 2004; Kumar, 2000]

The extraction of chitin from crustaceans produces a lot of waste and uses a lot of chemicals. The waste of the seafood-processing industry, mostly the shells of crustaceans, contains 14 – 40 % chitin. This waste is treated with alternate acid and alkali to remove other components from the shells of the crustacean and to extract the chitin. The unnecessary components and the chemicals are waste. [Kurita, 2006] One approach to produce the polymer in an environmentally friendly way, are bacteria like E. coli which can produce chitin via a CHS.
The production of chitin appears to be important as it is a useful substance which finds applications in medicinal, industrial and biotechnological research. Chitin, and its derivate chitosan, is non-toxic, biocompatible and biodegradable. Their bioactivities are for example the promotion of wound healing or hemostatic activity, immune enhancement, eliciting biological responses, and antimicrobial activity. [Kurita, 2006]
Chitin oligomers are also of great biological interest as they elicit biological responses in plants and form the backbone of substituted lipochitoologosaccharides which induce the nodulation in leguminous plants. [Samain et al., 1997]
There are different kinds of CHS from several organisms. One interesting enzyme is NodC originating from the gram-negative bacterium Rhizobium Leguminosarum and is a homologue to the chitin synthase from yeast (Strucutre see Figure 2). [Debelle et al., 1992]

Rhizobium leguminosarum bv viciae, where our enzyme originates from, is found to live in symbiosis with plants of the genera Pisum and Vicia of the family Fabaceae. [Long, 1996] Rhizobium species live in symbiosis with legumes, where the bacteria form nitrogen-fixing nodules in the legume roots. The symbiotic interaction leads to an activation of the bacterial nodulation (nod) genes and the secretion of Nod factors. These nod genes create and modify the Nod factors, to which NodC belongs. The Nod factors have a backbone consisting of β-1,4-N-acetylglucosamine oligosaccharides, most often tetra – or pentasaccharides with an acyl chain at C2 of the non-reducing end instead of an acetyl group. [Barny et al., 1993; Debelle et al., 1993]

The NodC protein has strongly hydrophobic domains which indicate that it is an integral or transmembrane protein (Figure 3). Interestingly it was only found in the inner but not outer membrane of Rhizobium leguminosarum. [Barny et al., 1993] NodC belongs to the class of glycosyltransferases which catalyse the transfer of sugar components from an activated donor molecule to a specific acceptor molecule. [Dorfmueller et al., 2014] .

Mechanism

NodC is involved in the synthesis of chitin oligosaccharides, but only with a polymerization degree up to five. In earlier studies it was shown that researching this CHS in E.coli is possible with good results. [Kamst et al., 1995] NodC uses UDP-N-acetylglucosamine (UDP-GlcNAc) as sugar donor, which is a precursor for the biosynthesis of peptidoglycan and therefore present in growing bacterial cells.
Another advantage is the unique property of the NodC which allows it to produce chitinpentaoses in living E.coli without exogenous acceptor. [Samain et al., 1997] If an acceptor molecule and the substrate are added to the purified enzyme, the reaction can also be done in vitro. The mechanism of elongation proceeds by a successive inverting nucleophilic substitution reaction at C1 of the UDP-GlcNAc – molecule (Figure 4). UDP departs when the O4 atom of the growing sugar chain attacks as a nucleophile. [Dorfmueller et al., 2014] With a low concentration of UDP-GlcNAc NodC produces a mixture of trimers, tetramers and pentamers and with high concentrations of UDP-GlcNAc it produces pentamers solely. It almost exclusively directs the formation of pentasaccharides. [Samain et al., 1997]

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