Immobilization of β-galactosidase from Kluyveromyces lactis onto a polysiloxane–polyvinyl alcohol magnetic (mPOS–PVA) composite for lactose hydrolysis (original) (raw)
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Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers
Applied Biochemistry and Biotechnology, 2009
Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.
Environmental engineering and management journal
galactosidase from Aspergillus oryzae was strongly immobilized on magnetic particles functionalized with amino groups. By simple incubation without any activating agents, electrostatic interactions between amino groups and enzymes allowed obtaining a strong linkage. The immobilization efficiency was studied with the quantification of amino groups of the particles and of immobilized β-galactosidase. Kinetic parameters, especially the maximal velocity Vmax and the affinity Km, were determined with two substrates, o-NPG and lactose, and compared with free enzyme values in order to evaluate the influence of our immobilization methodology on the kinetic behavior of the enzyme. Therefore, magnetic capacity of the functionalized particles allows recovering and reusing the support. Results show efficient immobilization of β-galactosidase (58 µg/mg of support), able to hydrolyze substrates during multiple cycles of use. Thus, magnetic particles functionalized with amino groups represent an a...
Food Chemistry, 2009
The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae b-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40°C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.
Food Chemistry, 2009
The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae b-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40°C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.
Applied Biochemistry and Biotechnology, 2010
β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40°C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.
Journal of Molecular Catalysis B: Enzymatic, 2011
Magnetite particles (<100 m) obtained by coprecipitation of Fe 2+ and Fe 3+ and coated with polyaniline (MAG-PANI) were used to immobilize Aspergillus oryzae -galactosidase via glutaraldehyde. The amount of -galactosidase immobilized onto MAG-PANI was ca. 2.04 mg/g support. This magnetic enzymatic derivative was capable to act on lactose and to produce tri and tetragalactosides (transgalactosylation) and the catalytic properties were similar to the soluble enzyme. For lactose concentrations up to 100 g/L, no differences were observed in the enzyme specific activity between free and immobilized forms (100% of specific activity retention), but for higher lactose concentrations the initial specific reaction rate of the immobilized form was affected by increasing lactose concentrations. The Activation Energy values for both free and immobilized forms were similar, around 16 ± 1.4 KJ/mol. The tri and tetra-galactosides production by both soluble and immobilized enzyme was not affected by temperature in the range of 30-60 • C. As the initial lactose concentration increased from 5% to 50%, the maximum GOS content in the product increased from 11.2% (at 35% conversion) to 26.1% (at 56% conversion) for the free enzyme and from 10.8% (at 33% conversion) to 26.0% (at 52% conversion) for the immobilized enzyme. The MAG-PANI was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, elemental analyzer, scanning electronic microscope, differential scanning calorimeter, thermogravimetric analyzer, vibrating sample magnetometer and thermomagnetization. These analysis showed rhombohedra particles presenting good magnetic response, evidences for the PANI coating and protein immobilization and magnetite as the predominant component. This magnetic -galactosidase derivative presents the following advantages: simple synthesis using low cost reagents, catalytic properties similar to the soluble enzyme and easy removal from the reaction mixture by a magnetic field and reuse.
2009
Magnetized polysiloxane coated with polyaniline (mPOS-PANI) was used as a support for b-galactosidase immobilization via glutaraldehyde. The galactooligosaccharides (GOS) production by this derivative was investigated under different initial lactose concentrations (5-50%) and temperatures (30-60°C). The initial lactose concentration in the reaction media affected the total amounts of produced GOS and their time course production was described as a ''bell-shaped" curve as a result of the balance between transgalactosylation and hydrolysis. No significative difference was observed for the free and immobilized enzymes. The reaction rates for lactose hydrolysis and GOS formation increased with increasing temperature from 30°C to 60°C, but GOS production at all lactose conversion levels was almost unchanged with changing temperature. The mPOS-PANI matrix was also characterized by scanning electronic microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), thermomagnetization, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).
2008
-Galactosidase from Aspergillus oryzae is an enzyme with a wide industrial application, mostly in the hydrolysis of lactose and, more recently, in the synthesis of oligosaccharides. Several advantages are associated with the application of immobilized enzymes on magnetic supports. In this work, -galactosidase was covalently immobilised onto a Polysiloxane-Polyvinyl Alcohol Magnetic Composite (mPOS-PVA), Magnetic Polysiloxane with Polyaniline (mPOS-PANI), Magnetized Dacron (DACRON) and Magnetite with Polyaniline (MAG-PANI) using glutaraldehyde as activating agent being the synthesis of GOS evaluated and compared at different temperatures (30, 40, 50 and 60 o C) and various initial lactose concentration (50, 100, 200, 300, 400 and 500 g/L). The kinetic parameters obtained by fitting the experimental data were compared in order to determine the effect of the immobilization process with different supports on the synthesis of oligosaccharides. These results clearly demonstrate that all supports may be used for -galactosidase immobilization as, besides improving the enzyme hydrolytic and GOS synthesis properties, its separation from the obtained reaction products is easy to accomplish.
Lactose hydrolysis by immobilized β-galactosidase: the effect of the supports and the kinetics
Catalysis Today, 2003
The kinetic behaviour of -galactosidase from Kluyveromices marxianus (Saccharomyces) lactis, immobilized on different oxides supports, such as alumina, silica, and silicated alumina has been studied. We observed a strong dependence of the immobilized enzyme activities on the chemical nature and physical structure of the supports. In particular, when the particle sizes of the supports are increased, the enzymatic activity strongly decreases.