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1.
Molecules ; 27(4)2022 Feb 12.
Article in English | MEDLINE | ID: mdl-35209024

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Lactobacillus plantarum/enzymology , Xylitol , beta-Galactosidase/chemistry , Catalysis , Xylitol/analogs & derivatives , Xylitol/chemistry
2.
Food Chem ; 355: 129587, 2021 Sep 01.
Article in English | MEDLINE | ID: mdl-33857721

ABSTRACT

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.


Subject(s)
Immunoglobulin E/immunology , Laccase/metabolism , Muramidase/immunology , Ovalbumin/immunology , Phenols/chemistry , Antigen-Antibody Reactions , Biocatalysis , Coumaric Acids/chemistry , Cross-Linking Reagents/chemistry , Humans , Molecular Weight , Muramidase/chemistry , Ovalbumin/chemistry , Oxidation-Reduction
3.
Methods Mol Biol ; 2100: 93-107, 2020.
Article in English | MEDLINE | ID: mdl-31939117

ABSTRACT

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.


Subject(s)
Chemical Engineering , Chemical Phenomena , Enzymes, Immobilized/chemistry , Glyoxylates/chemistry , Sepharose/chemistry , Biotechnology , Enzyme Activation , Enzyme Stability , Hydrogen-Ion Concentration , Oxidation-Reduction
4.
Methods Mol Biol ; 2100: 109-117, 2020.
Article in English | MEDLINE | ID: mdl-31939118

ABSTRACT

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.


Subject(s)
Enzymes, Immobilized/chemistry , Epoxy Compounds/chemistry , Adsorption , Enzyme Activation , Enzyme Stability , Epoxy Resins , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Protein Binding , Proteins/chemistry , Thermodynamics
5.
Methods Mol Biol ; 2100: 129-141, 2020.
Article in English | MEDLINE | ID: mdl-31939120

ABSTRACT

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.


Subject(s)
Enzymes, Immobilized/chemistry , Ions/chemistry , Polymers/chemistry , Adsorption , Cation Exchange Resins , Chemical Phenomena , Enzyme Activation , Enzyme Stability , Hydrogen-Ion Concentration , Osmolar Concentration , Polyethyleneimine/chemistry , Solvents , Temperature
6.
Methods Mol Biol ; 2100: 175-187, 2020.
Article in English | MEDLINE | ID: mdl-31939123

ABSTRACT

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.


Subject(s)
Aldehydes/chemistry , Cross-Linking Reagents/chemistry , Dextrans/chemistry , Enzymes, Immobilized/chemistry , Acetobacter/enzymology , Enzyme Activation , Enzyme Stability , Molecular Structure , Protein Conformation , Protein Multimerization , Proteins/chemistry , Thermodynamics
7.
Int J Biol Macromol ; 116: 1222-1231, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29775711

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Burkholderia , Gene Expression , Triglycerides/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Burkholderia/enzymology , Burkholderia/genetics , Catalysis , Escherichia coli/genetics , Escherichia coli/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
8.
Molecules ; 22(7)2017 Jun 29.
Article in English | MEDLINE | ID: mdl-28788435

ABSTRACT

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.


Subject(s)
Enzymes, Immobilized/chemistry , Glutaral/chemistry , beta-Galactosidase/chemistry , Candida/chemistry , Candida/enzymology , Enzyme Activation , Enzyme Stability , Enzymes, Immobilized/isolation & purification , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Fungal Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , beta-Galactosidase/isolation & purification
9.
Langmuir ; 30(49): 15022-30, 2014 Dec 16.
Article in English | MEDLINE | ID: mdl-25420004

ABSTRACT

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.


Subject(s)
Antibodies, Immobilized/chemistry , Magnetics , Microscopy, Atomic Force , Biosensing Techniques , Models, Biological , Models, Molecular , Particle Size
10.
Methods Mol Biol ; 1051: 43-57, 2013.
Article in English | MEDLINE | ID: mdl-23934797

ABSTRACT

The immobilization of proteins on epoxy activated supports is discussed in this chapter. Immobilization on epoxy supports is carried out as a two-step mechanism: in the first step the adsorption of the protein is promoted and in the second step the intramolecular covalent linkage among epoxy groups and nucleophiles of the protein is produced. Based on this mechanism of the need of a first adsorption of the protein on the support, different epoxy supports are described. The different supports are able to immobilize proteins through different orientations being obtained catalysts with different properties of activity, stability, and selectivity.


Subject(s)
Cross-Linking Reagents/chemistry , Enzymes, Immobilized/chemistry , Epoxy Compounds/chemistry , Adsorption , Enzyme Stability , Escherichia coli Proteins/chemistry , Fungal Proteins/chemistry , Hydrogen-Ion Concentration , Ion Exchange , Penicillin Amidase/chemistry , Polymers/chemistry , beta-Galactosidase/chemistry
11.
Methods Mol Biol ; 1051: 59-71, 2013.
Article in English | MEDLINE | ID: mdl-23934798

ABSTRACT

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 has 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.


Subject(s)
Enzymes, Immobilized/chemistry , Escherichia coli Proteins/chemistry , Glyoxylates/chemistry , Penicillin Amidase/chemistry , Sepharose/chemistry , Amines/chemistry , Cross-Linking Reagents/chemistry , Enzyme Stability , Hydrogen-Ion Concentration , Lysine/chemistry , Protein Binding , Schiff Bases/chemistry
12.
Methods Mol Biol ; 1051: 73-88, 2013.
Article in English | MEDLINE | ID: mdl-23934799

ABSTRACT

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contained the maximal concentration of glyoxyl groups to promote maximization of covalent immobilization and groups' capability to adsorb proteins by various mechanisms (e.g., ionic exchange, metal-chelate formation). Immobilization on various supports makes it possible to orientate and rigidify an enzyme in various regions of its surface. The use of different heterofunctional supports allowed for obtaining catalysts with different activity, stability, and selectivity properties.


Subject(s)
Cross-Linking Reagents/chemistry , Enzymes, Immobilized/chemistry , Glyoxylates/chemistry , Sepharose/chemistry , Adsorption , Animals , Bacterial Proteins/chemistry , Carboxylic Ester Hydrolases/chemistry , Chymotrypsin/chemistry , Enzyme Assays , Enzyme Stability , Hydrogen-Ion Concentration , Lipase/chemistry , Stereoisomerism , Substrate Specificity
13.
Biomacromolecules ; 14(3): 602-7, 2013 Mar 11.
Article in English | MEDLINE | ID: mdl-23406524

ABSTRACT

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.


Subject(s)
Coated Materials, Biocompatible/chemistry , Lipase/chemistry , Adsorption , Amino Acids/chemistry , Enzymes, Immobilized/chemistry , Hydrophobic and Hydrophilic Interactions , Magnetics , Nanoparticles/chemistry , Surface Properties
14.
Biotechnol Prog ; 27(3): 717-23, 2011.
Article in English | MEDLINE | ID: mdl-21509953

ABSTRACT

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.


Subject(s)
Chromatography/methods , Enzymes, Immobilized/isolation & purification , Lipase/isolation & purification , Staphylococcus/enzymology , Adsorption , Detergents/chemistry , Hydrophobic and Hydrophilic Interactions , Lipase/chemistry , Lipase/metabolism
15.
Colloids Surf B Biointerfaces ; 84(2): 556-60, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21382701

ABSTRACT

Sucrose laurate is a detergent that is useful for various biochemical applications because it is a green compound and is easily degradable after hydrolysis with a lipase or esterase. One problem observed in the process of sucrose laurate degradation is that most commercial detergent preparations are impure, necessitating the hydrolysis of all of the sucrose esters present in the preparation, all of them with detergent properties. In this article, a highly active catalyst, which is able to perform the hydrolysis of commercial sucrose laurate, is presented. The use of glyoxyl agarose preparations of a previously aminated Thermomyces lanuginosa lipase (TLL) enabled complete hydrolysis, in less than 30 min, of all of the compounds that comprise the mixture. In addition, this derivative is stable in the presence of 20% ethanol, which is necessary to prevent microbial contamination.


Subject(s)
Ascomycota/enzymology , Enzymes, Immobilized/chemistry , Lipase/chemistry , Sucrose/analogs & derivatives , Hydrolysis , Sucrose/chemistry , Time Factors
16.
Biomacromolecules ; 11(11): 3112-7, 2010 Nov 08.
Article in English | MEDLINE | ID: mdl-20945834

ABSTRACT

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.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Chymotrypsin/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Glyoxylates/chemistry , Lipase/metabolism , Sepharose/chemistry , Adsorption , Animals , Carboxylic Ester Hydrolases/chemistry , Chymotrypsin/chemistry , Enzyme Stability , Geobacillus/enzymology , Hydrogen-Ion Concentration , Lactobacillus plantarum/enzymology , Lipase/chemistry , Pancreas/enzymology , Surface Properties , Swine
17.
Colloids Surf B Biointerfaces ; 78(1): 140-5, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20307961

ABSTRACT

A new anion exchanger support has been designed for the selective adsorption of small proteins. This has been achieved activating an aminated support with glutaraldehyde and further coating the support surface with bovine serum albumin (BSA). In this support, "wells" are generated by two neighborhoods BSA molecules, on the bottom of those "wells" glutaraldehyde groups are exposed out ready to react with small molecules that have a size small enough to be accommodated between two BSA molecules on the pre-existing support. However, the BSA surface was not inert enough adsorbing many proteins, thereby reducing the selectivity of the system. A further solution was coating the immobilized BSA molecules with dextran, reducing the adsorption of protein on the BSA surface. This new matrix has been evaluated in the selective adsorption of the very small beta-lactoglobulins and alpha-lactalbumin from dairy whey, achieving the selective adsorption of both small proteins while other larger proteins from dairy whey remained in the supernatant. Moreover, a protein crude extract has been offered to the new matrix, and only small proteins could be adsorbed on the support (as probed by gel filtration). Thus this amino-glutaraldehyde-BSA-dextran-Sepharose is a matrix that may be used to selectively ionically adsorb proteins that were smaller than BSA (62 kDa). This strategy may be used for any other kind of adsorbing groups (chelating agents, boronic acid, etc.), or using proteins with different sizes to coat the support, designing tailor-made supports that may permit the fractioning of proteins following their sizes and by adsorption/desorption on different matrices.


Subject(s)
Anion Exchange Resins/metabolism , Milk Proteins/metabolism , Particle Size , Serum Albumin, Bovine/metabolism , Adsorption , Animals , Cattle , Chromatography, Gel , Complex Mixtures , Dextrans/metabolism , Escherichia coli , Glutaral/chemistry , Immobilized Proteins/metabolism , Porosity , Sepharose/chemistry , Whey Proteins
18.
Biomacromolecules ; 10(4): 742-7, 2009 Apr 13.
Article in English | MEDLINE | ID: mdl-19267470

ABSTRACT

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).


Subject(s)
Enzymes, Immobilized/chemistry , Formate Dehydrogenases/chemistry , Glutamate Dehydrogenase/chemistry , Polyethyleneimine/chemistry , Polymers/chemistry , Protein Structure, Quaternary , Cation Exchange Resins/metabolism , Coated Materials, Biocompatible , Enzyme Stability , Enzymes, Immobilized/metabolism , Formate Dehydrogenases/metabolism , Glutamate Dehydrogenase/metabolism , Glutaral/metabolism , Pseudomonas/enzymology , Sodium Chloride/metabolism , Thermus thermophilus/enzymology
19.
Appl Microbiol Biotechnol ; 80(1): 49-58, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18545996

ABSTRACT

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.


Subject(s)
Bacterial Proteins/chemistry , Glutamate Dehydrogenase/chemistry , Thermus thermophilus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Enzyme Stability , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glutamate Dehydrogenase/genetics , Glutamate Dehydrogenase/metabolism , Hydrogen-Ion Concentration , Kinetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Temperature , Thermus thermophilus/chemistry , Thermus thermophilus/genetics
20.
Biomacromolecules ; 9(8): 2230-6, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18558741

ABSTRACT

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.


Subject(s)
Antibodies/chemistry , Adsorption , Animals , Antigen-Antibody Complex/immunology , Chromatography/methods , Escherichia coli/metabolism , Glutaral/chemistry , Horseradish Peroxidase/chemistry , Immunoassay , Immunoglobulin G/chemistry , Phosphates/chemistry , Rabbits , Surface Properties
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