Immobilized Enzymes Availability for Glycerol – 1,3 Propanediol Bioconversion

The increasing abundance of glycerol, its renewability, and attractive pricing make it an appealing platform chemical. Glycerol can be utilized in various ways of biological transformation to industrially valuable products. There are some important derivatives among these such as 3-hydroxypropionaldehyde ((3-HPA) commercial name: reuterin), dihydroxyacetone (DHA), and 1,3-propanediol (PDO). PDO has the largest potential need. Several bacteria are able to ferment glycerol to PDO under anaerobic conditions. A new recombinant technology was worked out with the leadership of DuPont and Genecor in the last decade, in which a recombinant Escherichia coli converts glucose directly to PDO. Microbiological production of a molecule has always lower yield than an enzymatic method and usually produces a lot of by-products that can be avoided using enzymatic bioconversion. For years we have been working on a new enzymatic process in order to produce PDO and DHA simultaneously from glycerol. For this enzymatic way three key enzymes are needed: Glycerol-dehydratase (GDHt), 1,3-propanediol-oxydoreductase (PDOR), glycerol-dehydrogenase (GDH). These enzymes must be produced by microbial fermentation. We would like to present the results of developing the fermentation media in economic aspect. Our pleriminary results (not publicated) indicated that the 2xYT medium is perfect for our Clostridium strain. However in economic aspect the concentrations of yeast extract and bactotryptone are too high to scale-up this fermentation procedure. The first purpose of this study was to optimize the fermentation media. Optimized media concentrations are yeast extract, 6 g∙l-1 and bactotryptone, 2 g∙l-1 (instead of 10 and 16 g∙l-1 respectively). The other aim of our work was to develop immobilized enzymes suitable for glycerol to propanediol bioconversion and develop a method to measure the immobilized enzymes activity. We tried a covalent method applying chitosan matrix activated with glutaraldehyde. Chitosan – a natural polyaminosaccharide obtained from chitin – was chosen as support material to bind the three key enzymes from the crude enzyme solution. We could detect that PDOR and GDH enzyme bind to chitosan beads. Immobilized enzymes activity was higher than of the soluble enzymes.