Team:TU Darmstadt/Project/Glykolsaureproduktion Scere

Glykolsäureproduktion in S. cere

Abstract

The chitinase is an enzyme whose ability to break down glycosidic bonds in chitin brings more variability into the molecules. Since not just the grade and pattern of deacetylation, but also the amount of connected chitin monomers influences the entire molecule´s behavior [1], there is a great limitation of the properties and the bioactivity of the products. Its possible implementation in the project shows the future prospects of how chitins and chitosans with all kind of properties can be produced in E. coli.

Introduction

Chitin is a molecule found in fungal cell walls ^[1]. Many plants possess enzymes, so-called chitinases, which are able to break down chitin and thus help along with its digestion. These enzymes play a role in defense mechanisms of plants in case of fungal infections [2]. Even in human tissues chitinases appear, where they defend us against parasites [3]. Chitinases break down the glycosidic bonds between chitin monomer units and are classified as hydrolases. In our project, we decided to focus mainly on chitosan pentamers, synthesized and deacetylated by NodC and NodB respectively. However, chitosan's bioactivity and properties are also defined by its polymer length. For this reason, we want to give a first look at how our project could be extended. The enzyme we use is the ChiA1 from Bacillus circulans [4]. In the strain WL-12, the gene chiA encodes, together with chiB, chiC and chiD, an entire chitinase-system which primarily degrades chitin. This system is made up of at least six different chitinases [5], in which ChiA1 is believed to be the key enzyme [6]. The chiA gene is 1983 base pairs long and translates into an enzyme with a molecular weight of approximately 69.5 kDa [7]. Since ChiA1 has been successfully expressed together with our chitin synthase NodC [8], we originally decided on this specific enzyme. Its enzymatic activity has also previously been tested on chitin pentamers. ChiA1 breaks down these chitin pentamers in two dimers and one N-acetylglucosamine (GlcNAc) unit [8]. Its implementation would then give us a bigger variety of chitosan molecules. The N-terminal domain in the ChiA1 is responsible for its catalytic activity. The C-terminal domain plays an important role in the hydrolysis of chitin, and is the reason ChiA1 has such a high affinity to the substrate [6] [8] [9].

Subheadline

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Mechanism

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Methods

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