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

Abstract

1Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase were prepared for 2 the first time with pectin as cross-linking agent. Pectin as biocompatible, biodegradable, non-3 toxic, renewable and macromolecular cross-linker was used instead of traditional micro-4 molecular glutaraldehyde cross-linker. Cross-linker was prepared by controlled sodium 5 metaperiodate oxidation of native pectin. The effects of precipitant type, amount of 6 precipitant and cross-linking on activity recovery of glucoamylase in pectin-CLEAs were 7 studied. After aggregation of glucoamylase with ammonium sulphate, when formed 8 aggregates were cross-linked by pectin, 83% activity recovery was achieved in pectin-9 CLEAs, whereas when cross-linked by traditional cross-linker glutaraldehyde, 64% activity 10 recovery was achieved in glutaraldehyde-CLEAs. After formation of pectin-CLEAs and 11 glutaraldehyde-CLEAs, optimum temperature for glucoamylase activity was shifted from 50 12 to 55°C. The free enzyme and pectin-CLEAs displayed optimal pH 5, whereas optimal pH of 13 glutaraldehyde-CLEAs was shifted to pH 6. Compared with free enzyme and glutaraldehyde-14 CLEAs, lower inactivation rate constant of glucoamylase in pectin-CLEAs within the 15 temperature range of 50-70°C was observed. Moreover, activation energy required for 16 denaturation of glucoamylase in pectin-CLEAs was higher than glutarldehyde-CLEAs and 17 free enzyme. Kinetic studies show that the K m and V max of glucoamylase remained 18 unchanged after pectin-CLEAs formation, whereas K m was increased and V max was decreased 19 after glutaraldehyde-CLEAs formation. Finally upon 10 consecutive uses, pectin-CLEAs 20 retained 55% initial activity and glutaraldehyde-CLEAs retained only 29% initial activity. 21These results suggest that this pectin-CLEA is potentially usable in industrial applications. 22

Self-assembled organic⿿inorganic hybrid glucoamylase nanoflowers with enhanced activity and stability

“…The changes in fractions of secondary structure of free glucoamylase and hybrid glucoamylase nanoflowers were estimated by using FT-IR method according to the previous literature [23]. The FT-IR spectra of free and immobilized glucoamylase were recorded using Shimadzu IRAffinity-1 FT-IR spectrophotometer from 4000 to 400 cm -1 with sample dispersed in the KBr pellets.…”

“…The second derivative FT-IR spectra for free glucoamylase and hybrid nanoflowers were obtained ( Figure S1-2 †).The relative contents of α-helix structure (1650-1658 cm -1 ) (blue), β-sheet structure (1610-1640 cm -1 ) (red), β-turn structure (1660-1700 cm -1 ) (yellow) and random coil structure (1640-1650 cm -1 ) (green) based on the modelled multi-component peak areas calculated according to the previous literature by the software (Figure 6a-b) [23][24].…”

Non-covalent conjugation of cutinase from Fusarium sp. ICT SAC1 with pectin for enhanced stability: Process minutiae, kinetics, thermodynamics and structural study

“…Moreover, there is a need to select the best promising polysaccharide for conjugation with the desired enzyme, as the enzyme interacts in different ways with different polysaccharides [16]. All the polysaccharides used considerably increased the thermal stability of cutinase ( Fig.1) A c c e p t e d M a n u s c r i p t 12 stabilize the glucoamylase [19]. Li et al [15] also observed different thermostability for β-Dglucuronidase with similar molecular structural polysaccharides with different charges.…”

“…Further, conjugation of glucose oxidase and horseradish peroxidase with dextran [13], β-glucuronidase to polysaccharides [15], alcohol dehydrogenase with dextran and gum Arabic [14] enhanced thermal and pH stability. Pectin has been previously reported not only Page 5 of 52 A c c e p t e d M a n u s c r i p t 5 to have a prominent role in aggregation of glucoamylase and folding but also increase its thermal stability and reusability [19].…”

Role of Glutaraldehyde in Imparting Stability to Immobilized β-Galactosidase Systems

“…The process of CLEAs formation involves the treatment of enzymes immobilized on supports that are non-reactive to glutaraldehyde in order to prevent enzyme leakage followed by the crosslinking of enzymes adsorbed on aminated supports. This method was used to prepare CLEAs of trehalose synthase via co-aggregation with polyethyleneimine 11 and pectin CLEAs of glucoamylase 12 . Thus, glutaraldehyde remains the most widely used compound for designing biocatalysts to be utilized in a large number of biomedical and biotechnological applications.…”