Benevides C. Pessela

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.

Improvement of enzyme properties with a two-step immobilizaton process on novel heterofunctional supports.

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative.

Immobilization of Lactase from Kluyveromyces lactis Greatly Reduces the Inhibition Promoted by Glucose. Full Hydrolysis of Lactose in Milk

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of β‐galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki ≈ 15 000 and >40 000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.

Reversible and Strong Immobilization of Proteins by Ionic Exchange on Supports Coated with Sulfate-Dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate‐dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate‐dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic‐dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (β‐galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermus sp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Studies on the activity and the stability of β-galactosidases from Thermus sp strain T2 and from Kluyveromyces fragilis

Abstract The activity and the stability of the β-galactosidases from Thermus sp strain T2 and Kluyveromyces fragilis have been compared. Both enzymes have been partially purified by gel permeation chromatography, determining their molecular weights too. The influence of several metal cations and some buffers on the activity of the enzymes has been tested. The specificity of the enzymes for galactosyl moieties and β-bonds has been established by testing their activity on several synthetic chromogenic substrates and disaccharides. Also, it has been determined that both enzymes showed a remarkable hydrolytic activity and a weak transgalactosilation activity, even in the presence of high concentrations of lactose. The stability of both enzymes in soft and extreme conditions of pH and temperature and in the presence of aggressive chemicals (organic miscible solvents, oxygen peroxide, surfactants and urea) was studied. The thermophilic enzyme showed a higher resistance to hydrophobic agents and a higher stability at different temperatures, pHs and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, showing that some residues important for its stability were affected by oxidation. Kinetic studies on the ONPG hydrolysis with both enzymes were carried out in a wide range of temperatures and substrate and product concentrations. The data obtained at all the temperatures were fitted by a nonlinear technique to different kinetic models and two of them were selected to describe the reaction catalysed by the enzymes. The enzyme from K. fragilis was strongly inhibited by o-nitrophenol in a acompetitive way but it was weakly and competitively inhibited by galactose. The thermophilic enzyme was competitively inhibited by galactose much strongly than its mesophilic counterpart but the inhibition did not change with the temperature.

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine.

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE‐agarose, Q1A‐Sepabeads, and Sepabeads EC‐EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI‐Sepabeads than in Q1A‐Sepabeads or DEAE‐supports, requiring very high ionic strength to remove glucoamylase from the PEI‐supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).

Stabilization of a multimeric β-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads‐epoxy support with other Sepabeads‐epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable β‐galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads‐epoxy‐chelate, Sepabeads‐epoxy‐amino, or Sepabeads‐epoxy‐boronic to 5% using Sepabeads‐epoxy‐IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized β‐galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long‐term alkaline incubation, the enzyme immobilized on Sepabeads‐epoxy‐boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS‐PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross‐linking with aldehyde‐dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 °C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.

Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

ABSTRACT A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.

New cationic exchanger support for reversible immobilization of proteins

New tailor‐made cationic exchange resins have been prepared by covalently binding aspartic‐dextran polymers (e.g. MW 15 000–20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 °C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic‐dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic‐dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes ( Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. β‐galactosidases, Candida antarctica Blipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an α‐galactosidase and a β‐galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.