Optimizing the preparation conditions and characterization of a stable and recyclable cross-linked enzyme aggregate (CLEA)-protease

Abstract

Background: Cross-linked enzyme aggregate (CLEA) is considered as an effective technique in the production of immobilized biocatalysts for its industrially attractive advantages. Simplicity, stability, low cost, time saving and reusability are proved to be some of CLEA's main advantages. Results:In this study, an active, stable and recyclable CLEA-protease from the viscera of channel catfish Ictalurus punctatus has been prepared. Optimization of the preparation parameters is carried out with the help of Response Surface Methodology. This methodology helped in studying the interaction between the most contributing factors such as cross-linker, precipitant and the additive concentrations. The optimum specific activity for CLEA-protease of 4.512 U/mg protein has shown a high stability against the denaturation forces such as temperature and pH as compared to free protease. It is further found from the study that the highest activity was achieved at the pH of 6.8 and at the temperature of 45 °C. After six cycles, CLEA-protease maintained 28 % of its original activity. Additionally, Michaelis-Menten models were used to determine the kinetic parameters i.e. K m and V max that helped in showing a significant difference after immobilization as compared to free protease. Conclusion:This work found that this novel CLEA-protease can be used as a very active biocatalyst in industrial applications.

Cross‐linked α‐galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose‐type oligosaccharides in soymilk

“…There are various reports concerning the preparation, characterization and application of CLEAs of different enzymes, e.g. lipase, glucose oxidase, and proteases …”

“…The combination of enzyme and glutaraldehyde ties all available amino groups on the surface of the enzyme together; hence, the basic groups on the enzyme surface gave a positive charge to the enzyme protein and consequently shifted the optimum pH to the lower side. That is, an increase in the number of basic groups occurred after immobilization and the enzyme acquired a more polycationic character …”

“…That is, an increase in the number of basic groups occurred after immobilization and the enzyme acquired a more polycationic character. 29,30 For the determination of pH stability, the free enzyme and CLEAs were incubated in 100 mmol L −1 citrate and phosphate buffer at various pH values. As can be seen from Fig.…”

Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose

“…This enhancement in thermal stability of the immobilized XCA colloids was even better than enzyme immobilized in alginate beads without cross-linking. This is probably due to the rigid structure by covalent intra-crosslinking among enzyme aggregates [33][34][35].…”

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

“…The majority of CLEAs described in the literature involve hydrolases, probably because they are the enzymes that have the most industrial applications and are the simplest enzymes to work with. These include CLEAs from lipases [63], proteases [64], esterases [65,66], amidases [67], nitrilases [68], epoxide hydrolases [69], and glycosidases.…”