RESUMO
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 â¼80 % 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.
Assuntos
Celulose , Enzimas Imobilizadas , Celulose/química , Enzimas Imobilizadas/química , Catálise , beta-Galactosidase/química , Fenômenos MagnéticosRESUMO
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.
Assuntos
Lactose , Leite , Animais , Lactose/química , Hidrólise , Leite/química , Leite/metabolismo , beta-Galactosidase/química , Fenômenos Magnéticos , Enzimas Imobilizadas/metabolismoRESUMO
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.
Assuntos
Enzimas Imobilizadas , Lactose , Celulose , Estabilidade Enzimática , Enzimas Imobilizadas/química , Hidrólise , Lactose/química , beta-Galactosidase/químicaRESUMO
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.
Assuntos
Celulose , Lactose , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Fenômenos Magnéticos , beta-Galactosidase/metabolismoRESUMO
The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.
Assuntos
Histidina/química , Lactose/química , Níquel/química , beta-Galactosidase/metabolismo , Queijo/análise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Nanopartículas de Magnetita , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Soro do Leite/química , beta-Galactosidase/químicaRESUMO
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.
Assuntos
Reatores Biológicos , Escherichia coli , Celulose , Escherichia coli/genética , Lactose , beta-Galactosidase/genéticaRESUMO
OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. ß-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-ß-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant ß-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant ß-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.
Assuntos
Proteínas Fúngicas , Lactose , Proteínas Recombinantes , Soro do Leite/química , beta-Galactosidase , Reatores Biológicos/microbiologia , Queijo , Meios de Cultura/química , Meios de Cultura/farmacologia , Indústria de Laticínios , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/enzimologia , Kluyveromyces/genética , Lactose/química , Lactose/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Enzymes are proteins specialized in catalyzing biological reactions. However, factors such as cost and operational limitations could limit their applications in the industrial sector. An alternative to these limiting factors is enzyme immobilization, which enables reuse and increases biocatalyst stability. Cellulose can be employed in enzyme immobilization, and is an outstanding alternative due to availability and cost. Additionally, this material might undergo several chemical treatments, thus obtaining cellulose nanocrystals and nanofibers. The use of nanomaterials at an industrial scale requires more refined unit operations to separate them, a setback that can be solved by combining these materials to magnetic nanoparticles. This review shows important aspects for the synthesis and application of nanocellulose and magnetic nanoparticles. It also reports new trends and strategies to associate these materials. Magnetic cellulose is a versatile support for enzyme immobilization, so much so that different immobilization methods might be conducted using this material.
Assuntos
Proteínas de Bactérias/química , Celulose/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Nanopartículas de Magnetita/química , Nanotecnologia/métodos , Biocatálise , Emulsões , Estabilidade Enzimática , Humanos , Micro-Ondas , SonicaçãoRESUMO
Hydrolysis efficiency of ß-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial ß-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnßGal), Aspergillus oryzae (AoßGal), Bacillus circulans (BcßGal), Bifidobacterium bifidum (BbßGal), and Kluyveromyces lactis (KlßGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnßGal), Asn140Ala and Asn199Ala (AoßGal), Arg111Ala and Glu355Ala (BcßGal), Arg122Ala and Phe358Ala (BbßGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlßGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcßGal, BbßGal, and KlßGal. The double mutations in BcßGal and BbßGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlßGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of ß-galactosidases and rendering them promising for industrial applications.
Assuntos
Galactose/química , Lactose/química , beta-Galactosidase/genética , Aspergillus niger/enzimologia , Aspergillus oryzae/enzimologia , Bacillus/enzimologia , Bifidobacterium bifidum/enzimologia , Catálise , Hidrólise , Cinética , Kluyveromyces/enzimologia , Mutação Puntual/genética , beta-Galactosidase/química , beta-Galactosidase/ultraestruturaRESUMO
We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-NC) or alkaline treatment (OH-NC) of microcrystalline cellulose, which was subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) ß-galactosidases. The mean size of magnetic nanocellulose particles was approximately 75 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with a coercivity of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose either in permeate or in cheese whey for 30× and it reached hydrolysis higher than 50%. Furthermore, using a continuous process in a column packed-bed reactor, the derivative OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or whey after 24 h of reaction. Fungal ß-galactosidases immobilized on magnetic nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.