Carrier free co-immobilization of alpha amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity.

A tri-enzyme biocatalyst "combi-CLEAs" with starch hydrolytic activity was prepared from commercially available alpha amylase, glucoamylase and pullulanase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for 4.5h with 40 mM glutaraldehyde. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that tri-enzyme biocatalyst was of spherical structure. For one pot starch hydrolytic activity, shift in optimum pH from 6 to 7 and temperature from 65 to 75 °C were observed after co-immobilization of enzymes. After one pot starch hydrolysis reaction in batch mode, 100%, 60% and 40% conversions were obtained with combi-CLEAs, separate CLEAs mixture and free enzyme mixture, respectively. Co-immobilization also enhanced the thermal stability of enzymes. Finally, the catalytic activity of enzymes in combi-CLEAs during one pot starch hydrolysis was well maintained up to five cycles without performance changes.

Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase.

Novel magnetic cross-linked enzyme aggregates of alpha amylase were prepared by chemical cross-linking of enzyme aggregates with amino functionalized magnetite nanoparticles which can be separated from reaction mixture using magnetic field. Of the initially applied alpha amylase activity 100% was recovered in magnetic CLEAs, whereas only 45% was recovered in CLEAs due to the low content of Lys residues in alpha amylase. Scanning electron microscopy analysis showed that CLEAs and magnetic CLEAs were spherical structures. The CLEAs and magnetic CLEAs displayed a shift in optimal pH towards the acidic side, whereas optimal temperature of magnetic CLEAs was improved compared to free enzyme and CLEAs. Although V(max) of enzyme in CLEAs and magnetic CLEAs did not change, substrate affinity of the enzyme increased. The magnetic CLEAs also enhanced the thermal stability and storage stability. Moreover, the magnetic CLEAs retained 100% initial activity even after 6 cycles of reuse.