Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable alpha-amylases and pullulanases.

For the production of cell-free thermostable alpha-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full as well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60 degrees C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10(12) cells up to 700 U/10(12) cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450,000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes.