Immobilization Of Β-galactosidase Research Articles

Lactase (β-galactosidase) immobilization by complex formation: Impact of biopolymers on enzyme activity

In this study, a novel way to immobilize the enzyme β-galactosidase (Kluyveromyces lactis) through complexation with polysaccharides (sodium alginate (ALG) or Ɩ-carrageenan (CA)) was described. The kinetic parameters of the formation of the complexes and the immobilization efficiency of the enzyme as a function of pH and ratio of biopolymers were studied. The temperature profile, thermal stability, and kinetic parameters of the immobilized and free lactase were also analyzed. At pH 4.0, the results for the zeta potential associated with complex yield showed that the highest complex yield (97.14% ± 2.14) was reached for LAC/ALG at a 5:1 ratio, whereas the highest complex yield (95.26% ± 2.44) was reached an 8:1 ratio for LAC/CA. The process of complexation decreased the activity retained by the enzyme after dissociation. However, this decrease was less substantial for the LAC/ALG (83.2 ± 3.7%) complex than for LAC/CA (69.4 ± 7.6%) complex. The Michaelis–Menten constant (Km) increased from 2.91 mM (native enzyme) to 3.43 mM (LAC/ALG complex) and 4.15 (LAC/CA complex), while the apparent turnover number (Kcat) and the specificity constant (Kcat/Km) of both systems were not significantly different. The immobilization of the enzyme by complexation did not increase its thermal stability. However, the activity of the complexed lactase remained higher than that of native enzyme when exposed to low pH (pH 4.0) after 200 and 250 min for the LAC/ALG and LAC/CA complexes respectively. Our findings suggest that complexation can be a useful method for the immobilization of β-galactosidase.

Use of chitosan heterofunctionality for enzyme immobilization: β-galactosidase immobilization for galacto-oligosaccharide synthesis

Chitosan partially functionalized with aldehyde groups was used for enzyme immobilization, favoring first the enzyme adsorption through its amino groups and then the covalent bonding of the adsorbed catalyst through the aldehyde groups of the support. Using this strategy, immobilized A. oryzae β-galactosidase had a better performance than when only the aldehyde groups were used. The performance was further improved by modifying the support aldehyde group density to 200 μmoles⋅g−1. The biocatalyst under optimized immobilization conditions had 2951 IU⋅g−1 and half-life of 46.3 min at 60 °C, while its agarose counterpart had 2294 IU⋅g−1 and half-life of 59.5 min. Both biocatalysts were applied in galacto-oligosaccharide synthesis. After 10 sequential batches, the cumulative productivity (gGOS⋅h−1ˑgprotein−1) obtained with the chitosan and the agarose biocatalysts were 4.7 and 4.0 times the value when soluble enzyme was used respectively. This methodology had not been reported previously with chitosan, showing the high versatility of this low cost carrier and its high potential for enzyme immobilization.

Hyper-activation of ß-galactosidase from Aspergillus oryzae via immobilization onto amino-silane and chitosan magnetic maghemite nanoparticles

Abstract In the present work, the preparation of magnetic maghemite nanoparticles (MNPs), functionalized with amino-organosilanes and chitosan was investigated for a successful immobilization of enzyme β-galactosidase from Aspergillus oryzae. MNPs were synthesized by co-precipitation of Fe2+ and Fe3+ ions in the presence of ammonium. Immobilization techniques on both carriers were optimized by differing concentration of cross-linking reagent glutaraldehyde (GA) and pentaethylenehexamine (PEHA), where their influence on immobilization efficiency and residual activity of β-galactosidase was studied. Compared to the free enzyme, hyper-activation of enzyme was achieved in both immobilization methods. When functionalizing with amino-organosilanes, residual activity of 154%, 138% and 125% was achieved, using PEHA (20% (v/v)), GA (2.5% (v/v)) and combination of PEHA and GA (30% and 2% (v/v)), respectively. When functionalizing with chitosan, 129% of residual activity was achieved, with combination of PEHA and GA (30% and 0.5% (v/v)), respectively. Such immobilized β-galactosidase opens up a wide range of possibilities for implementation in various cleaner technologies, mostly benefiting in diary industries.

Synthesis of photo-responsive chitosan-cinnamate for efficient entrapment of β-galactosidase enzyme

A modified photo-crosslinkable chitosan cinnamate (CS-CN) bio-polymer was synthesized via functionalization of the main active –NH2 and –OH groups of chitosan using cinnamoyl chloride. The chemical structure of the manufactured bio-polymer was elucidated using instrumental techniques including FTIR and NMR spectra. The obtained photo-active CS-CN was cross-linked upon irradiation with the UV light and the performed photo-crosslinking was monitored using UV–vis light spectra. Moreover, XRD and SEM were respectively utilized to investigate both crystalline and morphological structures of the cross-linked bio-polymer. The obtained CS-CN bio-polymer was successfully employed as a polymeric support for β-galactosidase immobilization via entrapment technique. The entrapped β-galactosidase maintains around 80% of its initial activity and displayed a considerable stability against temperature and pH changes in comparison with the native soluble enzyme form. In addition, the kinetic studies showed an expected elevation of Km along with lowering of vo values after immobilization reflecting a lower enzyme-substrate affinity. However, the reusability experiments indicated that the entrapped enzyme maintains approximately 88% of its activity after 8 reuse cycles, which reveal the value of the performed immobilization technique especially in economic terms.

Production of galacto-oligosaccharides from whey permeate using β-galactosidase immobilized on functionalized glass beads

The conversion of whey permeates to galacto-oligosaccharides (GOS) was studied by the enzymatic action of β-galactosidase from Aspergillus oryzae in a continuous flow packed-bed reactor. A novel method of enzyme immobilization involving covalent immobilization of β-galactosidase on 3-aminopropyl triethoxysilane(3-APTES)-modified glass beads was developed by the cross-linking method. The pH and temperature dependence of the enzymatic efficiency of the glass bead-immobilized enzyme was compared with that of the free enzyme. Increased pH and thermal stabilities were observed for the immobilized enzyme versus the free enzyme. The reusability of the enzyme immobilized packed-bed reactor was studied and only about 4.6% of GOS yield was lost after eight reuses. Repeated cycle reactions were also carried out to improve the formation of GOS. The results showed that the GOS formation increased and a maximum GOS yield of 39.3% with 56.4% lactose conversion was obtained after the 2nd cycle of passing.

Immobilization of β-galactosidase in Chitosan-Alginate composite scaffolds and optimization of lactose hydrolysis

The immobilization of lactase (s-galactosidase) enzyme was achieved by entrapping it in chitosan-alginate composite scaffolds, which were formed by ionotropic pre-gelation followed by a pH increase to synthesize polyelectrolyte complex scaffolds, and subsequent cross-linking and activation by glutaraldehyde. The operational stability upon storage of the enzyme entrapped was checked by performing batch hydrolysis of lactose concentrations ranging from 1mg/ml to 5mg/ml and the resulting glucose produced in every reaction was determined using the glucose oxidase-peroxidase assay. This study also investigates the stability of the free and immobilized enzymes at altered sets of pH and temperature at a fixed concentration of enzyme for the reaction medium. Morphology and structure characterization of lyophilized composite scaffolds were examined using scanning electron microscope and Fourier transform infra-red spectra, respectively.

UTILITY OF FUNCTIONALIZED AGAROSE NANOPARTICLES IN HYDROLYZING LACTOSE IN BATCH REACTORS FOR DAIRY INDUSTRIES

The present study investigates the synthesis of agarose nanoparticles (ANPs) and its surface modification by galactose for the immobilization of β-galactosidase. Galactose modified ANPs retained 91% enzyme activity upon immobilization. Optimum pH (4.5) and temperature (50 oC) remains unchanged after immobilization. However, immobilized enzyme retained greater catalytic activity against lower and higher, pH and temperature ranges. Immobilized β-galactosidase retained 89% biocatalytic activity even at 4% galactose concentration as compared to enzyme in solution, and exhibited 81% activity even after seventh repeated uses. Immobilized enzyme hydrolyzed greater amount of lactose at higher temperatures as compared to β-galactosidase in solution, thereby suggesting its potential application in obtaining lactose-free dairy products at large scale.

Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes

This work is the first study of the immobilization of Aspergillus oryzae β-galactosidase (Gal) on powdered collagen (Col) that had formed a chelate with aluminum (Col-Al-Gal). Other collagen treatments, including those with acetic acid, glutaraldehyde, and a combination of aluminum and glutaraldehyde (Col-Al-Glu-Gal), were also tested. High-yield (superior to 80%) and high-efficiency (superior to 99%) immobilization was obtained for the derivatives Col-Al-Gal and Col-Al-Glu-Gal, even at high protein loads (500–1,000 mg g−1 of support). The storage stability of Gal immobilized on Col-Al and Col-Al-Glu resulted in Gal retaining approximately 60% of its initial activity after 90 days at 4 °C. The half-life values of derivatives Col-Al-Gal and Col-Al-Glu-Gal were higher than those of soluble enzyme at 65, 68, 70, and 73 °C. The derivatives Col-Al-Gal and Col-Al-Glu-Gal retained high enzyme activity in batch hydrolysis of lactose in permeate and lactose solutions for 50 and 60 cycles, respectively. Our results suggest that powdered collagen treated with aluminum, a low-cost support, is a promising support for the immobilization of β-galactosidase.

Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology

β-d-Galactosidase is an important enzyme in the dairy industry, and the enzyme from the yeast Kluyveromyces lactis is most widely used. Here, we report immobilization of the enzyme on a silica/chitosan composite support, devised to have 10% and 20% chitosan (SiQT10 and SiQT20, respectively). Morphological and textural characterizations showed that chitosan is dispersed in micrometric regions in silica. For comparison, a silica organofunctionalized with 3-aminopropyltrimethoxysilane (SiO2aptms) was prepared. Performance of the biocatalysts was tested for lactose hydrolysis, and the enzyme immobilized in SiQT10 and SiQT20 composites showed higher efficiency (62% and 47%, respectively) compared with the enzyme in SiO2aptms. Operational stability in this system was evaluated for the first time. After 200 h of continuous use in a fixed-bed reactor, SiQT10 remained with approximately 90% activity. Thus, in addition to demonstrating compatibility for food processing, these results align the enzyme stabilization properties of chitosan with the mechanical resistance of silica.

Use of silicon dioxide nanoparticles for β-galactosidase immobilization and modulated ethanol production by co-immobilized K. marxianus and S. cerevisiae in deproteinized cheese whey

β-galactosidase from dairy yeast was immobilized on silicon dioxide based nanoparticles for the hydrolysis of whey. The biocatalyst was checked for the optimal thermal and pH stability. Consequently, the immobilized β-galactosidase was recycled 15 times for the hydrolysis of lactose (37 °C and pH 7.0) without loss of a significant amount of catalytic activity. Almost 91% of lactose hydrolysis was obtained from using concentrated cheese whey. Considering the available approaches for efficient bioconversion of hydrolyzed whey into ethanol, co-immobilization strategy was used using Saccharomyces cerevisiae and Kluyveromyces marxianus for fermentation. Same vessel hydrolysis and fermentation process was employed using silicon dioxide nanoparticles based enzyme immobilization along with co-immobilized yeast cell and provided a maximum ethanol titer of 63.9 g/L on concentrated cheese whey (150 g/L). Hence, the nano-biocatalytic system along with immobilized S.cerevisiae could improve the viability of ethanol production from cheese whey.

β-galactosidase covalent immobilization over large-pore mesoporous silica supports for the production of high galacto-oligosaccharides (GOS)

Large-pore mesoporous silica supports with different structure and pore sizes distribution have been tested for glyoxyl multipoint covalent immobilization of Pectinex Ultra SP-L, a commercial preparation including the enzyme β-galactosidase from Aspergillus aculeatus. The prepared biocatalysts have been assayed in the production of galacto-oligosaccharides (GOS) from lactose as prebiotic functional food, aiming to optimize the yield to the GOS having the highest presumed prebiotic effect, tri- and tetra- GOS (high-GOS). Immobilization of β-galactosidase over glyoxyl-modified silica supports, with pores, large enough to accommodate the enzyme within their structure, led to an enhancement of the enzyme activity in terms of high-GOS production relative to the free enzyme under the same reaction conditions. Remarkably, such an improvement was achieved without previous purification of β-galactosidase from the commercial source. An enhancement of the transgalactosylation activity over the hydrolytic activity, due to the relatively hydrophobic nature of organically modified silica surface of the supports, led to an increase in the selectivity to high-GOS. The best biocatalyst in the series was that based in hexagonal ultra-large-pore SBA-15 (ULP-SBA-15), which presents the most adequate balance between confinement effect within its pore framework and transgalactosylation activity leading to a remarkable yield to high-GOS of 20.2% vs. 11.2% for the enzyme in free conditions. Furthermore, reusability of these silica-based biocatalysts in three consecutive 24 h reaction-cycles has been successfully performed at 50 °C, temperature that minimizes the thermal deactivation of the enzyme.

Immobilization of β-galactosidase on surface modified cobalt/multiwalled carbon nanotube nanocomposite improves enzyme stability and resistance to inhibitor

The present study aimed to work out a high yield procedure for immobilization of Aspergillus oryzae β-galactosidase on polyaniline cobalt multiwalled carbon nanotubes nanocomposite (PANI/Co/MWCNTNC) by physical adsorption and covalent attachment via glutaraldehyde. The binding was confirmed by scanning and transmission electron microscopy along with Fourier transform-infrared spectroscopy. The immobilization yields obtained for adsorbed and cross-linked enzymes were 93% and 97%, respectively. The covalently immobilized enzyme was remarkably more stable at extremes of pH and temperature compared to native and adsorbed enzymes. The Ki values were found to increase ten-folds for covalently immobilized β-galactosidase indicating that this immobilized enzyme was markedly more resistant to galactose inhibition. The covalently attached enzyme retained 92% activity after its 10th successive reuse compared to the adsorbed β-galactosidase which exhibited 74% of its initial activity. Furthermore, no significant change was noticed in the band intensity of pBR322 plasmid when exposed to free and bound nanocomposite. The covalently bound enzyme exhibited superiority in terms of stability and reusability when compared to the adsorbed and native β-galactosidase. The results, presented here demonstrate an efficient method of immobilization of β-galactosidase on PANI/Co/MWCNTNC in order to construct a convenient and novel biosensor for the detection of lactose concentration.

Functionalized agarose as an effective and novel matrix for immobilizing Cicer arietinum β-galactosidase and its application in lactose hydrolysis

Abstract The present study demonstrates the immobilization of β-galactosidase from Cicer arietinum on a simple and inexpensive matrix, glutaraldehyde functionalized agarose (GFA), to suggest its potential application in hydrolyzing whey lactose in biotechnology industries. The designed matrix provided large surface area for the immobilization of β-galactosidase, apart from exhibiting greater biocatalytic activity in terms of selectivity, loading and stability. GFA retained 83% enzyme activity as a result of immobilization. Soluble and GFA bound Cicer arietinum β-galactosidase showed the same pH and temperature-optima at pH 5.0 and at 50 °C, respectively. However, immobilized enzyme exhibited a greater fraction of activity at both acidic and basic pH, and at higher temperature ranges. GFA bound enzyme lost only 20 % enzyme in the presence of 3% galactose, and retained 70 % activity even after its sixth repeated use. Immobilized enzyme showed pronounced lactose hydrolysis from whey in batch processes at 55 °C as compared to enzyme in solution.

Preparation of Nano-Sized Mesoporous Silica and Its Application in Immobilization of β-galactosidase from Aspergillus Oryzae

In alkaline conditions, monodisperse nano-sized mesoporous silica was synthesized using cetyl trimethyl ammonium bromide (CTAB) as template and tetraethoxysilane (TEOS) silica as source in ethanol / water cosolvent conditions. Using method of nitrogen adsorption, specific surface area of the dried monodisperse nano-sized mesoporous silica was about 1591 m2/g and the pore size was about 3.8 nm. The field-emission scanning electron microscope (SEM) micrographs showed that the silica particles obtained were spherical with an approximate diameter of 160 nm and of good dispersion. Transmission electron microscopy (TEM) revealed that the carrier had an excellent cellular structure with disordered multi-channels and smooth surface. The nano-sized mesoporous silica above was employed to immobilize β-galactosidase from aspergillus oryzae for the first time. At the experimental conditions in section 2.4, the enzyme activity and the activity yield were 535.11 U/g dry carrier and 79.63%, respectively. Kinetic data of the immobilized enzyme such as optimum temperature, pH, and thermal and pH stability among other valuable results were also determined.

Activity of enzymes immobilized on plasma treated polyester

The aim of this study was to investigate the effect of 3 different surface plasma treatments on the immobilization of β-galactosidase on a fibrous PET nonwoven membrane. Two methods 1. Entrapment in a thin calcium alginate coating and 2. Direct sorption, were used to immobilize the enzyme. The three different plasma treatments for surface activation of PET nonwovens were: 1. Air atmospheric DBD plasma, 2. Cold remote plasma-CRP with 100% N2 and 3.- CRP with a mixture of N2/O2 gases. Plasma treatment of the PET fiber surface increased the quantity of immobilized enzyme using the entrapment method, and the degree of alginate film cross-linking highly influenced the enzyme activity. Highest enzyme activity was reached for the PET treated with air atmospheric plasma and cross-linked with 0.25g/l of CaCl2. With the direct sorption method, greater amounts of enzyme were immobilized as compared to the entrapment method, but a considerable proportion of enzyme lost their catalytic activity. Only with the CRP N2/O2 plasma treatment, up to 90% of sorbed enzyme maintained their activity. Reusability study showed that for the optimized entrapment method, a progressive decrease in activity was observed after each use cycle. With the optimized sorption method using N2/O2 CRP plasma, no decrease in enzyme activity was detected, and the immobilized enzyme could be used over more than 15 cycles.

Kluyveromyces lactis β-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose

ABSTRACT: One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis β-galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35°C. Almost 89% lactose hydrolysis was attained for concentrated whey (100g/L) and retained 89% catalytic activity after 15cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9g/L was achieved during fermentation at 35°C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

Elucidating the binding efficacy of β-galactosidase on graphene by docking approach and its potential application in galacto-oligosaccharide production.

Herein, we propose the synthesis and characterization of graphene for the immobilization of β-galactosidase for improved galacto-oligosaccharide (GOS) production. The size of synthesized graphene was observed to be 25 nm by TEM analysis while interaction of enzyme with the nanosupport was observed by FTIR spectroscopy. Docking was obtained using molecular docking program Dock v.6.5 while the visual analyses and illustration of protein-ligand complex were investigated by utilizing chimera v.1.6.2 and PyMOL v.1.3 softwares. Immobilized β-galactosidase (IβG) showed improved stability against various physical and chemical denaturants. Km of IβG was increased to 6.41 mM as compared to 2.38 mM of soluble enzyme without bringing significant change in Vmax value. Maximum GOS content also registered an increase in lactose conversion. The maximum GOS production was achieved by immobilized enzyme at specific temperature and time. Hence, the developed nanosupport can be further exploited for developing a biosensor involving β-galactosidase or for immobilization of other industrially/therapeutically important enzymes.

Graphene based magnetic nanocomposites as versatile carriers for high yield immobilization and stabilization of β-galactosidase

The present study demonstrates an efficient method for high yield immobilization ofAspergillus oryzaeβ-galactosidase onto graphene-iron oxide nanocomposites (Gr@Fe3O4NCs) by a simple adsorption mechanism.

Studies of β-Galactosidase Production and Immobilization Using Novel Bacteria Isolated from the Soil Enriched with Whey

Studies of β-Galactosidase Production and Immobilization Using Novel Bacteria Isolated from the Soil Enriched with Whey