α-l-Rhamnosidase of Aspergillus terreus immobilized on ferromagnetic supports

Abstract

α-L-rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity-pH and activity-temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40-70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K (m) of α-L-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).

Synthesis of hesperetin 7-O-glucoside from flavonoids extracted from Citrus waste using both free and immobilized α-l-rhamnosidases

“…In a previous study, the same RAT was immobilized covalently onto ferromagnetic hydrazide-Dacron activated via the cross-linking with glutaraldehyde. In that study, the specific activity measured in the same conditions was estimated in 0.53 nkat/μg-protein (Soria et al, 2012), thus indicating that the current method constitutes an improved approach for RAT immobilization with high enzymatic capacity retained.…”

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

“…The amount of protein in the supernatant (S) and washing buffers (WB) was subtracted from the protein contained in the initial enzyme load (IEL), giving the immobilized protein ([P]IEL 2 ([P]S 1 [P]WB)). Concentration of immobilized protein was expressed as mg mL 21 .…”

“…After changing levels of total reactants' volume, the quantity of FE offered to the activated support (AS) was studied (1.0, 1.25, 2.0, 3.0, 4.0, 5.0, and 4.2 g FE g 21 AS ), and for each of these conditions, 6 levels of immobilized enzyme were analyzed (10-60 mg immobilized enzyme g 21 substrate ) to stablish optimum enzymatic activity.…”

“…Figure 3a shows the monosaccharide ions, lactose, and/or transgalactosylated disaccharides (218.9 and 365.1 m/z, respectively), whereas Figure 3b shows the trisaccharides and tetrasaccharide ions (527.2 and 689.2 m/z, respectively). We hypothesize that the ion with m/ z ratio of 689.2 could correspond to a tetrasaccharide (molecular weight 666.58 g mol 21 ), and considering a sodium adduct, the m/z goes up to 689.2. Detection of m/z of a compound not detected by HPLC may be possible, considering that mass detector is more sensitive than the refractive index detector, according to Macrae et al 40 Mozaffar et al 36 immobilized b-galactosidase onto Merckogel and observed the same behavior in regard to the difference of products from the action of free and immobilized 38 enzyme, reporting only trisaccharides for the former and di-, tri-, tetra-, and pentasaccharides for the latter.…”

<b>PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography

“…Many organic supports, natural or synthetics, have been proposed for immobilization of proteins, mainly because of their versatility for participating in a broad number of different reactions, favoring their activation (Soria, Ellenrieder, Oliveira, Cabrera, & Carvalho, 2012). Polyvinyl alcohol (PVA) is an organic support and one of the most polyester studied and has been used for different purposes in biotechnology and biochemistry (Bai et al, 2014).…”