Immobilization of ß-galactosidase by halloysite-adsorption and entrapment in a cellulose nanocrystals matrix.

BACKGROUND: Immobilization allows easy recovery and reuse of enzymes in industrial processes. In addition, it may enhance enzyme stability, allowing prolonged use. A simple and novel method of immobilizing ß-galactosidase is reported. Effects of immobilization on the enzyme characteristics are explained. ß-Galactosidase is well established in dairy processing and has emerging applications in novel syntheses. METHODS: ß-Galactosidase was immobilized by physical adsorption on halloysite, an aluminosilicate nanomaterial. Optimal conditions for adsorption were identified. The optimally prepared halloysite-adsorbed enzyme was then entrapped in a porous matrix of nanocrystals of sulfated bacterial cellulose, to further enhance stability. RESULTS: Under optimal conditions, 89.5% of the available protein was adsorbed per mg of halloysite. The most active and stable final immobilized biocatalyst had 1 part by mass of the enzyme-supporting halloysite particles mixed with 2 parts of cellulose nanocrystals. Immobilization raised the optimal pH of the catalyst to 7.5 (from 6.0 for the native enzyme) and temperature to 55 °C (40 °C for the native enzyme). During storage at 25 °C, the immobilized enzyme retained 75.8% of initial activity after 60 days compared to 29.2% retained by the free enzyme. CONCLUSION: The immobilization method developed in this work enhanced enzyme stability during catalysis and storage. Up to 12 cycles of repeated use of the catalyst became feasible. GENERAL SIGNIFICANCE: The simple and rapid immobilization strategy of this work is broadly applicable to enzymes used in diverse bioconversions.

Improvement of lipase biochemical properties via a two-step immobilization method: Adsorption onto silicon dioxide nanoparticles and entrapment in a polyvinyl alcohol/alginate hydrogel.

In this study, the factors affecting lipase adsorption onto SiO2 nanoparticles including SiO2 nanoparticles amounts (8, 19 and 30 mg/mL), lipase concentrations (30, 90 and 150 µg/mL), adsorption temperatures (5, 20 and 35 °C) and adsorption times (1, 12.5 and 24 h) were optimized using central composite design. The optimal conditions were determined as a SiO2 nanoparticles amount of 8.5-14 mg/ml, a lipase concentration of 106-116 µg/mL, an adsorption temperature of 20 °C and an adsorption time of 12.5 h, which resulted in a specific activity and immobilization efficiency of 20,000 (U/g protein) and 60 %, respectively. The lipase adsorbed under optimal conditions (SiO2-lipase) was entrapped in a PVA/Alg hydrogel, successfully. FESEM and FTIR confirmed the two-step method of lipase immobilization. The entrapped SiO2-lipase retained 76.5 % of its initial activity after 30 days of storage at 4 °C while adsorbed and free lipase retained only 43.4 % and 13.7 %, respectively. SiO2-lipase activity decreased to 34.43 % after 10 cycles of use, while the entrapped SiO2-lipase retained about 64.59 % of its initial activity. Compared to free lipase, the Km values increased and decreased for SiO2-lipase and entrapped SiO2-lipase, respectively. Vmax value increased for both SiO2-lipase and entrapped SiO2-lipase.

Enhancement of biochemical aspects of lipase adsorbed on halloysite nanotubes and entrapped in a polyvinyl alcohol/alginate hydrogel: strategies to reuse the most stable lipase.

Entrapment of halloysite nanotubes (HNTs) loaded with enzyme, into a polymer matrix (PVA/Alg), is a way to produce an environment surrounding the adsorbed enzyme molecules which improves the enzyme properties such as storage and operational stability. Hence, in this study, we optimised the factors affecting lipase adsorption onto halloysite nanotubes including halloysite amounts (5, 42.5 and 80 mg), lipase concentrations (30, 90 and 150 µg/ml), temperatures (5, 20 and 35 °C) and adsorption times (30, 165 and 300 min). The optimal conditions were determined as an halloysite amount of 50 to 80 mg, a lipase concentration of 30 to 57 µg/ml, an adsorption temperature of 20 °C and an adsorption time of 165 min, which resulted in a specific activity and adsorption efficiency of 15,000 (U/g protein) and 70%, respectively. Then, lipase adsorbed under optimal conditions was entrapped in a PVA/Alg hydrogel. The formation mechanism of immobilized lipase was investigated by FESEM and FTIR. Subsequent entrapment of adsorbed lipase improved the lipase storage and operational stability. Km, Vmax, Kcat and Kcat/Km values showed an increase in the entrapped HNT-lipase performance in comparison with the free and adsorbed lipase.