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.

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.