Co-immobilization of alpha-amylase, glucoamylase, and pullulanase by cross-linked enzyme aggregates approach for glucose syrup production from starch

In this research, cross-linked enzyme aggregates of thermostable α-amylase (from Bacillus licheniformis), glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus subtilis) that was enriched by calcium and sodium ions and BSA as a proteic feeder were prepared. Initially, acetone, acetonitril, isopropanol, saturated ammonium sulfate, ethanol, and tert-butanol were used for enzyme aggregation. Among them, tert-butanol has the highest enzyme activity compared to compared to other precipitators. The optimum conditions of the immobilization process for these three enzymes and CLEAs formation were: glutaraldehyde concentration: 5mM, enzyme mixture ratios (α-amylase: glucoamylase: pullulanase) was 3: 1: 1, Enzyme/ BSA ratio 1:2, and crosslinking time 2.5 h, at 2-3° C. The optimum temperature and pH for free α-amylase were 95° C, and pH, 5.5, for glucoamylase and pullulanse were 60-62° C, pH 5.5 respectively. CLEAs that was formed by tert-butanol and 1: 2 enzyme/ BSA ratio has the optimum temperature 60-62° C, and optimum pH of 5.5. Kinetic parameters assessment of combi-CLEAs compared to free mixed enzymes revealed that, Km is decreased, Vmax enhanced and catalytic efficiency is increased. Moreover, resulted multi-CLEAs has a reusability about 74% after 10 cycles. Besides, the thermal stability and enzyme half-life of the immobilized enzymes was enhanced about 3 folds than free ones. This raising of activity was owing to the addition of 3: 1 ratio of Ca2+ / Na+ ions to the enzyme mixture during CLEAs formation. This ions addition improves thermostability, functional stability and enzyme half-lives. Accordingly, combi-CLEAs of three amylases introduces as a biocatalyst with ease of operation, enhanced efficacy, eco-friendly and cost effective for a new integrated process design in glucose syrup production from starch.