Iranian Research Organization for Science and Technology (IROST) Innovative Food Technologies 2783-350X 12 3 2025 04 21 Co-immobilization of alpha-amylase, glucoamylase, and pullulanase by cross-linked enzyme aggregates approach for glucose syrup production from starch Co-immobilization of alpha-amylase, glucoamylase, and pullulanase by cross-linked enzyme aggregates approach for glucose syrup production from starch 287 303 1561 10.22104/ift.2025.7624.2216 FA Homa Torabizadeh Department of Chemical Technologies, Food Science and Technology Group, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran Journal Article 2025 05 21 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. 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. https://jift.irost.ir/article_1561_6c0655d96ac6a888cbb3e22c75ba0d82.pdf