Effective development of a biotechnical process: Screening, genetic engineering, and immobilization for the enzymatic conversion of inulin to DFA IIIon industrial scale

The bioconversion of renewable resources for industrial applications demand s the availability of suitable enzymes and technologies for their usage. Th is paper presents results obtained from the development of a process design ed to convert inulin into a difructose anhydrid (DFA III). A broad screening programme was carried out to isolate bacterial strains wi th the necessary enzymatic activity (inulase H). Of special interest were t hermotolerant enzymes, i. e. enzymes which are stable at leastwise 60 degre es C for prolonged time. Using a mineral salt medium with inulin as the sol e source for carbon and energy some 400 strains were investigated. Four str ains were found to pro duce DFA III and the strain Buo141 expresses an enzy me which is stable for weeks at elevated temperatures. Using metabolic data and 16S-rRNA-sequencing the strain was identified to be a new Arthrobacter species. Inulase II is secreted as an extracellular enzyme. To increase the formation of enzyme the inulase gene was cloned into E. col i XL I -blue, inulase IJ was expressed and its activity was detected. After identifying the cleavage site the DNA sequence coding for the original sig nal peptide was eliminated in the expression vector. An amino acid exchange was induced by error-prone PCR. The recombinant E. coli was fermented to 1 0,5 g/L (dry matter) and cells were disrupted. An activity of 1.76 Mio U/L was observed. The enzyme was flocculated from cell-free extract and entrapped in calcium alginate hydrogels for better retention under continuous operation. To enab le the production of uniform and small bead shaped particles novel JetCutte r technology was used with a production rate of 5600 beads/(s-nozzle). The influence of bead diameter on the activity was investigated and an activity of 196 U/g was measured for 600 rectanglem beads. The presented work covers the complete range of developing a biotechnical p rocess and provides the basics for establishing an industrial process for p roducing DFA from inulin at low cost.