Magnetic core-shell cellulose system for the oriented immobilization of a recombinant ß-galactosidase with a protein tag.

The objective of this study was to immobilize a recombinant ß-galactosidase (Gal) tagged with a cellulose-binding domain (CBD) onto a magnetic core-shell (CS) cellulose system. After 30 min of reaction, 4 U/capsule were immobilized (CS@Gal), resulting in levels of yield and efficiency exceeding 80 %. The optimal temperature for ß-galactosidase-CBD activity increased from 40 to 50 °C following oriented immobilization. The inhibitory effect of galactose decreased in the enzyme reactions catalyzed by CS@Gal, and Mg2+ increased the immobilized enzyme activity by 40 % in the magnetic CS cellulose system. The relative enzyme activity of the CS@Gal was 20 % higher than that of the soluble enzyme activity after 20 min at 50 °C. The CS support and CS@Gal capsules exhibited an average size of 8 ± 1 mm, with the structure of the shell (alginate-pectin-cellulose) enveloping and isolating the magnetic core. The immobilized ß-galactosidase-CBD within the magnetic CS cellulose system retained ∼80 % of its capacity to hydrolyze lactose from skim milk after 10 reuse cycles. This study unveils a novel and promising support for the oriented immobilization of recombinant ß-galactosidase using a magnetic CS system and a CBD tag. This support facilitates ß-galactosidase reuse and efficient separation, consequently enhancing the catalytic properties of the enzyme.

Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant ß-galactosidase immobilized on magnetic core-shell capsules.

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.

Application of cellulosic materials as supports for single-step purification and immobilization of a recombinant ß-galactosidase via cellulose-binding domain.

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of ß-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/gsupport, expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na+, K+, Mg2+, and Ca2+), magnesium provided the highest increase in the enzymatic activity of ß-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a ß-galactosidase on cellulose via CBD.

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability.

For the first time, this work reported the one-step purification and targeted immobilization process of a ß-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after ß-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between ß-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. ß-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of ß-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes.

This study aimed to produce and characterize a recombinant Kluyveromyces sp. ß-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl ß-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant ß-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.