A novel and simple method for the purification of extracellular levansucrase from Zymomonas mobilis.

A new and simple method for the purification of extracellular levansucrase from Zymomonas mobilis from highly viscous fermentation broth was developed. After incubation of the fermentation broth with a fructose-polymer cleaving enzyme preparation (Fructozyme, Novozymes, DK) for 48 h, levansucrase precipitated as aggregates and was redissolved in a 3 M urea solution. By ongoing size-exclusion chromatography on Sephacryl S-300 the final levansucrase preparation was purified 100-fold and exhibited a specific activity of 25-35 U/mg(protein). The levansucrase was stable in 3 M urea solution for at least four months without inactivation. To maximize the enzyme yield the dynamic changes of extracellular levansucrase activity during fermentation were investigated. The highest levansucrase activity was observed during the logarithmic phase of growth (15-19 h of fermentation).

Affinity membranes as a tool for life science applications.

As a matrix for affinity membrane technology we chose a flat-sheet microfiltration membrane based on polypropylene. Using photopolymerization to graft epoxy groups onto the pore surface, we worked with glycidylmethacrylate as a monomer. We developed optimized, efficient, and mild UV irradiation conditions for the two-step photografting process practically preserving the given pore structure of the base membrane. A grafting degree of up to 1.2 mg/cm(2) per surface area of the membrane was obtained. The poly-propylene membrane surface became significantly more hydrophilic. Introduction of epoxy groups allowed a stable covalent immobilization of the protein streptavidin serving as receptor for affinity ligand binding. A relatively high streptavidin immobilization capacity of about 65 micro g/cm(2) per surface area of the membrane was obtained. Apparently, only about two of the binding sites of the immobilized streptavidin were available for biotin recognition. We also found that the oriented immobilization of biotinylated alkaline phosphatase onto the surface via a streptavidin bridge increased the specific enzymatic activity about sixfold compared with random immobilization of this enzyme.

Polymerization of glucans by enzymatically active membranes.

Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release. By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created. Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane. Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size. In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase. Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate. The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated. The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes.