Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

Preparation of lipase-coated, stabilized, hydrophobic magnetic particles for reversible conjugation of biomacromolecules.

This Communication presents the development of a novel strategy for the easy conjugation of biomolecules to hydrophobic magnetic microparticles via reversible coating with previously functionalized lipase molecules. First, the ability of lipase to be strongly adsorbed onto hydrophobic surfaces was exploited for the stabilization of microparticles in aqueous medium by the creation of a hydrophilic surface. Second, the surface amino acids of lipase can be tailored to suit biomolecule conjugation. This approach has been demonstrated by amino-epoxy activation of lipase, enabling the conjugation of different biomolecules to the magnetic particle's surface. For example, it was possible to immobilize 70% of Escherichia coli proteins on the recovered particles. Furthermore, this strategy could be extended to other lipase chemical modification protocols, enabling fine control of biomolecule coupling. These conjugation techniques constitute a modular methodology that also permits the recycling of the magnetic carrier following use.

Combining phenolic grafting and laccase-catalyzed cross-linking: Effects on structures, technofunctional properties and human immunoglobulin E binding capacity of egg white proteins.

The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was positively correlated with protein cross-linking extent, and determined the molecular weight profile and structures of the cross-linked product. While laccase-catalyzed reactions (with or without free FA mediator) did not lead to evident cross-linking of the native proteins, oligomeric (up to 16.4%), polymeric (up to 30.6%) FA-LZMs and oligomeric FA-OVA (5.1-31.1%) were obtained upon the enzymatic treatments. The cross-linking on the grafted FA sites occurred mainly through the formation of 8-5'-noncyclic-dehydro-diferulic linkages. The effects of investigated cross-linking approach on the emulsifying, foaming properties and the immunoglobulin E (IgE) binding capacity of LZM and OVA were also evaluated in relation to the structural properties of cross-linked proteins.

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.

Multi-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Stabilization of Industrial Enzymes.

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes.

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support's possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an "intermolecular" covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme-support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

Very Strong but Reversible Immobilization of Enzymes on Supports Coated with Ionic Polymers.

In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins are shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.

Stabilization of Multimeric Enzymes via Immobilization and Further Cross-Linking with Aldehyde-Dextran.

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternary structure of multimeric enzymes.

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

Covalent immobilization of antibodies on finally inert support surfaces through their surface regions having the highest densities in carboxyl groups.

The correct immobilization of antibodies is one of the most critical steps in the preparation of immunosensors and immunochromatography matrices. In addition, the final support has to be chemical and physically inert to avoid the unspecific adsorption of proteins that can reduce the sensitivity of the biosensor or the purification achieved by the chromatography. The solution to both problems is one of the major challenges in the field. Here, we have presented two different novel and simple alternatives to have the unmodified antibody anionically exchanged to a support, further covalently immobilized with more than 90% of the antibodies bonded to the support by the four subunits, retaining a high functionality and giving a final "inert" surface. The first solution was the use of supports having a low superficial density of amino groups activated with glutaraldehyde. Here, the inertness was achieved by the use of a very low density of amino groups, unable to adsorb proteins at 100 mM sodium phosphate, while immobilization proceeds mainly via a first adsorption of the antibody and a further reaction with the glutaraldehyde groups. The second solution implies the design of a novel support (amino-epoxy). This support again produces a first ionic exchange of the antibody on the support and a further reaction with the epoxy groups, but because the epoxy groups can be finally blocked with aspartic groups (annulling the charge), the initial density of amino-epoxy groups can be as high as possible. Both systems permitted the correct and oriented immobilization of IgG. The immobilized antibody showed high-functionality (65-75%) and a final inert support surface. This immobilized antibody (antiperoxidase) was able to capture fully specifically HRP contaminating a protein crude extract from E. coli.

Oriented covalent immobilization of antibodies on physically inert and hydrophilic support surfaces through their glycosidic chains.

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed two solutions based in tailor-made support surfaces to immobilize antihorseradish peroxidase (HRP). The first solution was the use of supports having a very low amount of amino groups. These amino groups need to be very reactive with the aldehyde groups generated in the protein sugar chains to be efficient. Using supports having 7 micromol EDA/g (e.g., ethylenediamine modified glyoxyl-agarose), the antibody may be immobilized, keeping over 90% of the anti-HRP functionality. Second, by mixing amino groups and carboxylic groups, a neutral surface of the support has been generated. Again, this support has been unable to adsorb proteins while oxidized anti-HRP could be immobilized, giving functional anti-HRP antibodies. Both preparations retained 100% functionality after 2 months of storage at 4 degrees C. This way, the tailoring of the support surfaces has permitted solving some limitations of the immobilization of sugar-chain oxidized antibodies on primary amino supports.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg-1) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n­hexane with an activity of 4 U g-1, maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis.

Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions.

Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.

Partial purification and immobilization/stabilization on highly activated glyoxyl-agarose supports of different proteases from flavourzyme.

The fractioning of some components and their immobilization of Flavourzyme, a commercial protease/aminopeptidase preparation, has been investigated to improve its specificity and stability. Adsorption of Flavourzyme on two ionic exchangers yielded two fractions with endoprotease activity and one fraction containing aminopeptidase activity. The use of an amine agarose gel has made it possible to purify a 43 kDa protein with only endoprotease activity. Immobilization of this endoprotease and the original Flavourzyme preparation onto glyoxyl-agarose provided derivatives that were more thermostable than their soluble counterparts. Tests using immobilized Flavourzyme and immobilized purified endoprotease for the hydrolysis of chickpea proteins showed that both preparations can be used for the production of protein hydrolysates and compare very favorably with the original crude Flavourzyme in terms of reducing the production of free amino acids. This was especially so in the case of immobilized endoprotease, which produced only 0.2% free amino acids. Keeping free amino acids content low is very important in protein hydrolysates for nutritional use to avoid excessive osmotic pressure.

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA.

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.

Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose.

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.

Immobilization and stabilization of a cyclodextrin glycosyltransferase by covalent attachment on highly activated glyoxyl-agarose supports.

Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40 degrees C. Other immobilized preparations (obtained via other immobilization protocols) exhibit less than 10% of activity after incubation under similar conditions. Optimized glyoxyl-agarose immobilized preparation expressed a high percentage of catalytic activity (70%). Immobilization using any technique prevents enzyme inactivation by air bubbles during strong stirring of the enzyme. Stabilization of the enzyme immobilized on glyoxyl-agarose is higher when using the highest activation degree (75 micromol of glyoxyl per milliliter of support) as well as when performing long enzyme-support incubation times (4 h) at room temperature. Multipoint covalent immobilization seems to be responsible for this very high stabilization associated to the immobilization process on highly activated glyoxyl-agarose. The stabilization of the enzyme against the inactivation by ethanol seems to be interesting to improve cyclodextrin production: ethanol strongly inhibits the enzymatic degradation of cyclodextrin while hardly affecting the cyclodextrin production rate of the immobilized-stabilized preparation.

Simple purification of immunoglobulins from whey proteins concentrate.

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.