Abstract

Introduction

Besides cellulose, chitin is the most common 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 [4][5].

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 [2]. 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 [2].
Chitin oligomers are also of great biological interest as they elicit biological responses in plants and form the backbone of substituted lipochitooligosaccharides which induce the nodulation in leguminous plants [1].
There are different kinds of CHS from several organisms. The enzyme of our interest is the NodC which originats from the gram-negative bacterium Rhizobium leguminosarum. It is a homologue to the chitin synthase from yeast (Structure see Figure 2)[6].

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 [7]. 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 [6][8].

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 [8]. NodC belongs to the class of glycosyltransferases which catalyse the transfer of sugar components from an activated donor molecule to a specific acceptor molecule [9].

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 [10]. 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 [1]. 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 [9]. 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 [1].

Methods

We ordered the nodC gene via IDT sequencing and inserted this gene into the pSB1C3 vector via the BioBrick system and verify this via sequencing. One pSB1C3 vector has an AraC promoter system (BBa_K808000) and the other an Anderson Promotor with defined cleavage sites (BBa_K2380025). Both vectors have the RBS BBa_K2380024. Afterwards we transformed the vector in E.coli BL21 for expression studies and started the expression, once by induction with 100 µL arabinose for the AraC promoter system and the other without induction by the constitutive Anderson promoter. To examine the successful expression, an SDS-PAGE was done. The next step was the purification of the protein and the verification of the enzyme function. To purify the enzyme, a side-directed mutagenesis was done at the C-Terminus to add a His-taq and the protein was purified via an ÄKTA in combination with a 1mL HisTrap column by GE Healthcare. The activity the NodC enzyme was evaluated via two different assays.

Thin-layer Chromatography

The first was a thin-layer chromatography (TLC) to show that chitin is produced. TLC utilizes the principle of separation based on the different affinity of several compounds inside a mixture towards the stationary planar phase and the mobile phase. That means in detail the compounds run within the stationary phase driven by capillary effects and under influence of their differing affinity towards the mobile phase. Thereby, small differences in the affinity towards the mobile phase are sufficient for getting a clear fractionation.

UDP-GloTM Glycosyltransferase Assay>/i>

The other assay was the UDP-GloTM Glycosyltransferase Assay from Promega. The Assay was used in order to test the functionality of the NodC enzyme. NodC is an N-acetylglucosamine transferase that uses UDP-GlcNAc as donor molecule. The NodC transfers N acetylglucosamine from the UDP-GlcNAc to single N-acetylglucosamine bricks and UDP is set free. The free UDP is converted to ATP via a UDP Detection Reagent. This ATP generates light in a luciferase reaction which can be measured using a luminometer.