Sachin Talekar

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.

Carrier free co-immobilization of glucoamylase and pullulanase as combi-cross linked enzyme aggregates (combi-CLEAs)

Herein we report the successful preparation of carrier free co-immobilized form of glucoamylase and pullulanase using combi-CLEAs method from commercial multi-enzyme preparation OPTIMAX® 7525 HP. Glucoamylase and pullulanase were precipitated using ammonium sulfate, followed by cross-linking for 8 h with 2% (v/v) glutaraldehyde to produce insoluble catalytically active co-immobilized form of enzymes. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that co-immobilized form of enzymes was of spherical structure. For starch hydrolytic activity, shift in optimum pH from 5 to 7 and temperature from 60 to 70 °C were observed after co-immobilization of enzymes. After starch hydrolysis reaction in batch mode, 100, 80 and 30% conversions were obtained with co-immobilized enzymes, mixture of separate CLEAs and free enzymes, respectively. Co-immobilization also enhanced the thermal stability and storage stability. After eight reuses with 30 min reaction time, co-immobilized glucoamylase and pullulanase retained 90 and 85% of its initial activity, respectively.

Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase

Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase were prepared for the first time with pectin as cross-linking agent. Pectin as a biocompatible, biodegradable, non-toxic, renewable and macromolecular cross-linker was used instead of traditional micro-molecular glutaraldehyde cross-linker. The cross-linker was prepared by controlled sodium metaperiodate oxidation of native pectin. The effects of precipitant type, amount of precipitant and cross-linking on activity recovery of glucoamylase in pectin-CLEAs were studied. After aggregation of glucoamylase with ammonium sulphate, when formed aggregates were cross-linked by pectin, 83% activity recovery was achieved in pectin-CLEAs, whereas when cross-linked by traditional cross-linker glutaraldehyde, 64% activity recovery was achieved in glutaraldehyde-CLEAs. After formation of pectin-CLEAs and glutaraldehyde-CLEAs, the optimum temperature for glucoamylase activity was shifted from 50 to 55 °C. The free enzyme and pectin-CLEAs displayed an optimal pH of 5, whereas the optimal pH of glutaraldehyde-CLEAs was shifted to pH 6. Compared with the free enzyme and glutaraldehyde-CLEAs, lower inactivation rate constant of glucoamylase in pectin-CLEAs within the temperature range of 50–70 °C was observed. Moreover, the activation energy required for denaturation of glucoamylase in pectin-CLEAs was higher than glutaraldehyde-CLEAs and free enzyme. Kinetic studies show that the Km and Vmax of glucoamylase remained unchanged after pectin-CLEAs formation, whereas Km was increased and Vmax was decreased after glutaraldehyde-CLEAs formation. Finally upon 10 consecutive uses, pectin-CLEAs retained 55% initial activity and glutaraldehyde-CLEAs retained only 29% initial activity. These results suggest that this pectin-CLEA is potentially usable in industrial applications.

An integrated green biorefinery approach towards simultaneous recovery of pectin and polyphenols coupled with bioethanol production from waste pomegranate peels

An integrated biorefinery, incorporating hydrothermal processing of waste pomegranate peels (WPP), was proposed for the acid and organic solvent-free simultaneous recovery of pectin and phenolics with bioethanol production. The hydrothermal treatment (HT) was optimized using Box-Behnken design and the maximum recovery of pectin (18.8-20.9%) and phenolics (10.6-11.8%) were obtained by hydrothermal treatment at 115 °C for 40 min with a liquid-solid ratio of 10. The WPP pectin was characterized by IR, 1H NMR, and TGA which showed close similarity to commercial pectin. Depending on WPP cultivar type the degree of esterification, galacturonic acid content and molecular weight of pectin were in the range of 68-74%, 71-72%, and 131,137-141,538 Da, respectively. The recovered phenolics contained 57-60% punicalagin. Enzyme digestibility of WPP improved using HT with 177 g glucose produced per kg dry mass which was fermented to obtain 80 g ethanol with 88% of theoretical yield.