Immobilized enzyme

About: Immobilized enzyme is a research topic. Over the lifetime, 15282 publications have been published within this topic receiving 401860 citations.

Immobilization of naringinase in PVA-alginate matrix using an innovative technique.

Abstract: A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K Mapp) and the maximum reaction velocity (V maxapp) were evaluated for both free (K Mapp = 0.233 mM; V maxapp = 0.13 mM min−1) and immobilized naringinase (K Mapp = 0.349 mM; V maxapp = 0.08 mM min−1). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.

Urease from the soil bacterium Bacillus pasteurii: immobilization on Ca-polygalacturonate.

Abstract: Urease purified from the soil bacterium Bacillus pasteurii was adsorbed and immobilized on a preformed network of Ca-polygalacturonate, a substrate which has a similar composition and morphology to the mucigel present at the root-soil interface. The adsorption proceeded with an essentially quantitative yield, and the immobilized enzyme showed no decrease of specific activity with respect to the free enzyme. The dependence of urease adsorption on NaCl concentration suggested that the enzyme is bound to the carrier gel through electrostatic interactions. The immobilized enzyme showed increased stability, with respect to the free enzyme, with increasing time or temperature, and in the presence of proteolytic enzymes. The pH activity profile revealed that the adsorbed enzyme showed no change in the optimum pH (8.0), but it was more active than the free form in the pH range 5–8. The Michaelis-Menten kinetic pararneters V max and K m were measured for the free ( V max = 1960 ± 250 units ml −1 ; K m = 235 ± 20 mM) and immobilized ( V max = 1740 ± 185 units ml −1 ; K m = 315 ± 25 mM) urease. The substantial similarity of V max in the two cases suggests that there were no conformational changes involving the active site upon enzyme immobilization, while substrate partitioning effects between the bulk solution and the micro-environment surrounding the immobilized enzyme must be operating so as to partly increase its K m . These results suggest that bacterial urease present in plant root mucigel plays a large role in the mobilization of urea N. Its activity is in fact significantly mantained and protected by immobilization on hydrophilic gels such as those produced by root exudates.

Online Microreactors/Capillary Electrophoresis/Mass Spectrometry for the Analysis of Proteins and Peptides

Abstract: We report the use of trypsin and carboxypeptidase Y-modified capillary microreactors (50 μm i.d. and 20 nL volume per centimeter length) in combination with mass spectrometry for peptide molecular mass mapping of various peptides and proteins. Advantages of immobilized enzyme capillary microreactors include picoliter to nanoliter volume requirements, longer enzyme lifetimes, higher stability, and the ability to reuse enzymes conveniently. Additionally, extremely efficient sample handling modes are used, and the reaction products are easily separated from the enzyme reagents. Plasma desorption and matrix-assisted laser desorption/ionization were used for off-line analyses of digestion products. The use of MassMap for the identification of proteins is also discussed. Finally, a trypsin microreactor was integrated on-line with capillary electrophoresis/ion spray mass spectrometry for fast peptide mapping. Digestion of the oxidized insulin B-chain in an on-line trypsin microreactor and electromigration of aliquots from the capillary microreactor into the CE separation capillary allowed the entire peptide mapping procedure to be completed in <1 h. The on-line technique is especially well-suited for the characterization of minute quantities of proteins, because it transfers picoliter to nanoliter volumes of digestion products with minimum sample handling which could lead to losses or contamination.

αs1-Casein Film Prepared Using Transglutaminase

Abstract: Transglutaminase is a Ca2 +-dependent enzyme that covalently polymerizes proteins through the formation of e-(γ-glutamyl)lysyl cross-links. We have reported that highly concentrated protein solutions were firmly gelatinized by transglutaminase. The technique of gelation of an αs1-casein solution with transglutaminase was applied to the preparation of an αs1-casein film. The αs1-casein film obtained showed a high tensile strength (105 g/cm2) and strain (72%). It was insoluble in water, 10% 2-mercaptoethanol, 6.6 m urea, 10% SDS and 6 m guanidine hydrochloride. Even if it was diluted with water and then heated at 100°C for 10 min, it remained insoluble. However, it was gradually hydrolyzed by chymotrypsin. These results suggest the usefulness of the αs1-casein film as a supporting material of immobilized enzymes, a medical polymer and an edible film.