Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70–75 °C, with higher stability in pH 3.0–4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb⁺² and citrate and total with Cu⁺², Hg⁺², and Co⁺². Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.

     

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 l-Arabinose Isomerase for d-Tagatose Synthesis

A recombinant l-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with K_m values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

     

Ion exchange using poorly activated supports, an easy way for purification of large proteins

Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.     

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.