Process for producing the potential food ingredient DFA III from inulin: screening, genetic engineering, fermentation and immobilisation of inulase II.

Difructose anhydride (DFA III) is a new potential sweet food additive. A screening was undertaken to isolate bacterial strains for conversion of inulin to DFA. Of special interest were thermotolerant enzymes. Some 400 strains were investigated, among four of them produce DFA and strain Buo141 expresses an extracellular enzyme which is stable at elevated temperatures. Based on metabolic data and 16S-rRNA-sequencing, the strain was identified as a new Arthrobacter species. For increased enzyme production, the inulase gene was cloned into E. coli XL1-blue, inulase II was expressed and its activity detected. After identifying the cleavage site, the sequence coding for a signal-peptide was eliminated from the plasmid and a beneficial amino acid exchange introduced by error-prone PCR. The recombinant E. coli was fermented to 10.5 g/l and after disruption an activity of 1.76 MioU/l was observed. The enzyme was flocculated from supernatant and entrapped in calcium alginate hydrogels. To enable production of uniform and small beads JetCutter technology was used with a production rate of 5600 beads/(snozzle). The influence of bead diameter on activity was investigated. An activity of 196 U/g was measured for 600-microm beads.