Francisco Batista-viera | Universidad de la República (Uruguay) (original) (raw)

Papers by Francisco Batista-viera

Research paper thumbnail of Affinity chromatography

Methods of biochemical analysis, 2011

Research paper thumbnail of Covalent Chromatography

Methods of Biochemical Analysis, 2011

Research paper thumbnail of Isolation of lactoferrin by immobilized metal ion affinity chromatography (IMAC)

Biochemical Education, 1994

Research paper thumbnail of Bone-Bound Glucoamylase as a Biocatalyst in Bench-Scale Production of Glucose Syrups from Liquefied Cassava Starch

Food and Bioprocess Technology, 2008

This research assesses the bench-scale application of a non-conventional support, bone particles,... more This research assesses the bench-scale application of a non-conventional support, bone particles, for glucoamylase (GA) immobilization and its subsequent use in cassava starch hydrolysis. Upon determining the appropriate conditions to immobilize GA onto chicken bone particles, such as pH, ionic strength, particle size, and enzyme load, bench-scale immobilization of commercial GA without further purification was performed. Under the selected conditions, 270 GA units per gram of support were adsorbed. Optimal temperature and thermal stability of immobilized GA were only slightly different from those of the free enzyme, while optimal pH became more acidic by about one unit. The feasibility of the use of this immobilized biocatalyst for high glucose syrup production from liquefied cassava starch, at bench scale in batch process using a stirred-tank reactor, was demonstrated. Repeated use of the GA-bone derivative showed that similar conversions to those achieved with soluble enzyme (dextrose equivalent=98) were reached until the third batch and over 90% until the 25th batch.

Research paper thumbnail of Chromatographic methods for amylases

Journal of Chromatography B: Biomedical Sciences and Applications, 1996

This review surveys recent developments in chromatographic methods for the separation of amylases... more This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of t~and t-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

Research paper thumbnail of Selective adsorption of immunoglobulins and glucosylated proteins on phenylboronate-agarose

Journal of Chromatography A, 1992

Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-... more Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, pH 8.5, selectively adsorbs immunoglobulins and complement factors C3 and C4 from human serum. The selectivity of binding is strongly influenced by the presence of magnesium chloride in the sample buffer. Adsorbed immunoglobulins are quantitatively eluted by sorbitol, but only partially by ethylene glycol or methylcellosolve. Aniline-agarose of a similar degree of substitution shows only weak adsorption of serum proteins under similar experimental conditions, thus indicating the important contribution of the boronate moiety to this interaction. Immunoglobulin adsorption seems not to be due to the cis-diol complexation used extensively for the chromatographic determination of non-enzymatically glucosylated proteins. Hydrophobic and pi-pi interactions with the aromatic structure of the ligand seem also to contribute to protein binding. The behaviour of aminophenylboronate-liganded agarose is, in some respects, rather similar to that of the so-called "thiophilic adsorbents".

Research paper thumbnail of Characterization of Galactosyl Derivatives Obtained by Transgalactosylation of Lactose and Different Polyols Using Immobilized β-Galactosidase from Aspergillus oryzae

Journal of Agricultural and Food Chemistry, 2009

The synthesis of novel galactosides is interesting because of their important role in several bio... more The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.

Research paper thumbnail of Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

Applied Biochemistry and Biotechnology, 1998

A new approach for the control and interruption of enzymatic reactions via selective enzyme immob... more A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: oL-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided with de novo thiol-groups by a two-step reaction involving N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases.

Research paper thumbnail of Studies on aroma generation in lulo ( Solanum quitoense): enzymatic hydrolysis of glycosides from leaves

Food Chemistry, 2003

Five C13-norisoprenoid glycosides, isolated from lulo (Solanum quitoense) leaves, were subjected ... more Five C13-norisoprenoid glycosides, isolated from lulo (Solanum quitoense) leaves, were subjected to enzymatic hydrolysis with a commercial glucosidase (emulsin) and also with an enzymatic preparation having glycosidase activity, isolated from lulo fruit pulp. The volatile compounds generated after reaction were characterized by capillary GC and capillary GC–MS. Lulo fruit glycosidases were extracted by ammonium sulfate precipitation at pH 6.5 and

Research paper thumbnail of Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-lactogl... more Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 µmol of SH groups/g of dried gel). Both reductants allowed an increase of three-(for K. lactis β-galactosidase) and fourfold (for A. oryzae β-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis β-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae β-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in β-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.

Research paper thumbnail of Affinity chromatography of soybean β-amylase on phenylboronate agarose

Journal of High Resolution Chromatography, 1992

Research paper thumbnail of Bone-Bound Glucoamylase as a Biocatalyst in Bench-Scale Production of Glucose Syrups from Liquefied Cassava Starch

Food and Bioprocess Technology, 2008

This research assesses the bench-scale application of a non-conventional support, bone particles,... more This research assesses the bench-scale application of a non-conventional support, bone particles, for glucoamylase (GA) immobilization and its subsequent use in cassava starch hydrolysis. Upon determining the appropriate conditions to immobilize GA onto chicken bone particles, such as pH, ionic strength, particle size, and enzyme load, bench-scale immobilization of commercial GA without further purification was performed. Under the selected conditions, 270 GA units per gram of support were adsorbed. Optimal temperature and thermal stability of immobilized GA were only slightly different from those of the free enzyme, while optimal pH became more acidic by about one unit. The feasibility of the use of this immobilized biocatalyst for high glucose syrup production from liquefied cassava starch, at bench scale in batch process using a stirred-tank reactor, was demonstrated. Repeated use of the GA-bone derivative showed that similar conversions to those achieved with soluble enzyme (dextrose equivalent=98) were reached until the third batch and over 90% until the 25th batch.

Research paper thumbnail of Improved Glucoamylase Immobilization onto Calcined Chicken Bone Particles

Food and Bioprocess Technology, 2009

Calcination was considered for the first time as an alternative to recover inorganic bone compone... more Calcination was considered for the first time as an alternative to recover inorganic bone component from spent glucoamylase-bone derivatives. The subsequent adsorption of glucoamylase (GA) onto calcined bone particles was assessed. Adsorption capacity of the calcined matrix was found to be from 1.1-to 1.4-fold superior to that of non-calcined supports, and it was dependent on the applied load. Moreover, the expressed activity of GA derivatives on calcined matrix was, at least, 2-fold higher than that of biocatalysts onto non-calcined support. The optimization of the loading allowed the preparation of derivatives with 139 GA units per gram of support, which preserve 52% of the immobilized activity. Additionally, calcination of spent GA biocatalysts on calcined bone particles was performed, and adsorption of glucoamylase onto the bone particles calcined a second time was also found to be efficient. In addition to the improved catalytic properties, the half-life at 55°C of the GA biocatalysts on calcined bone was increased 1.7fold in comparison with that of soluble GA and GA adsorbed onto non-calcined bone particles. Furthermore, the same cassava starch conversion can be achieved batchwise in a stirred-tank reactor using less insoluble biocatalyst, 37% of the GA-bone derivative, which represents an important saving for industrial applications.

Research paper thumbnail of Immobilization of Enzymes: A Literature Survey

Methods in Molecular Biology, 2013

The term immobilized enzymes refers to &a... more The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

Research paper thumbnail of Chromatographic methods for amylases

Journal of Chromatography B: Biomedical Sciences and Applications, 1996

This review surveys recent developments in chromatographic methods for the separation of amylases... more This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of t~and t-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

Research paper thumbnail of Thiolation and reversible immobilization of sweet potato δ-amylase on thiolsulfonate-agarose

Journal of Molecular Catalysis, 1993

Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide... more Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide exchange reactions are unique because, simultaneously, they show a stable covalent bond and the possibility of eluting the protein by reduction when the enzymatic activity decays. The adsorbent can thus be reloaded. In this paper we report the use of the recently developed thiolreactive adsorbent thiolsulfonate-agarose, for the immobilization of sweet potato8-amylase. Since native p-amylase thiol groups were not reactive towards the adsorbent, the enzyme was provided with 'de novo' thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3-(2-pyridy1dithio)propionat.e (SPDP). When the SPDP/B-amylase molar ratio was changed between 3 and 100, up to sixteen exposed thiol groups per mol of enzyme were introduced. This was achieved without affecting the amylolytic activity. The immobilization yield for the intermediate thiolation level was 98%. However, only 19% of the applied enzyme activity was found in the gel suspension. Comparative studies were made on thiolsulfonate-agarose and on a commercial thiol-activated adsorbent (2-pyridyldisulfide-agarose) . The immobilization of the thiolated enzyme through reversible disulfide bonds on both adsorbenta showed similar results. A close analysis reveals that immobilization of proteins on thiolsulfonate-agarose is a very promising technique.

Research paper thumbnail of beta-Galactosidase from Kluyveromyces lactis immobilized on to thiolsulfinate/thiolsulfonate supports for lactose hydrolysis in milk and dairy by-products

Biotechnology Techniques, 1998

After reversible immobilization of neutral beta-galactosidase from Kluyveromyces lactis on to thi... more After reversible immobilization of neutral beta-galactosidase from Kluyveromyces lactis on to thiolsulfinate/thiolsulfonate supports, more than 80 % of the activity was retained. Blocking the remaining reactive groups with glutathione increased the thermal stability of the derivatives almost two-fold. These derivatives achieved a high degree of conversion (85-90 %) of lactose (50g / l) in saline solution, whey, whey permeates, and

Research paper thumbnail of A novel extracellular β-glucosidase from Issatchenkia terricola: Isolation, immobilization and application for aroma enhancement of white Muscat wine

Process Biochemistry, 2011

Since most Saccharomyces strains show no ␤-glucosidase activity, the importance of non-Saccharomy... more Since most Saccharomyces strains show no ␤-glucosidase activity, the importance of non-Saccharomyces ␤-glucosidases in the development of wine aroma has been highlighted. However, only a few enzymes from yeasts isolated from grape-must and enological ecosystems are active in the stringent conditions of wine and the wine-making process (low pH, high concentrations of ethanol or glucose). A purified extract of extracellular ␤-glucosidase from Issatchenkia terricola, proved to be very active in the presence of glucose (100 g/L), ethanol (18%) and metabisulfite (60 mg/L). It is also active and relatively stable at acidic pH over 3.0. Immobilization onto Eupergit C greatly improved its stability, allowing the aromatization of white Muscat wine over a 16-day experiment. The released aroma compounds of control and treated wine were analyzed by GC-MS. The enzymatic treatment significantly increased the amount of monoterpenes and norisoprenoids, showing the potential of the immobilized enzyme for aroma development in wines.

Research paper thumbnail of Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

Journal of Molecular Catalysis B: Enzymatic, 1998

. The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two differ... more . The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose Ž . via a cyanylating agent CDAP was optimized. Gel-bound protein and gel-bound activity were both measured directly, Ž . allowing the determination of apparent specific activities S.A. . Higher amounts of b-gal were immobilized on the activated Ž y1 . CPC-silica maximum capacity, 23 mg ml of packed support than on the CDAP-activated agarose. For the lower enzyme Ž y1 . loading assayed 12.6 mg ml packed support , 100% of the enzyme was immobilized but only 34% of its activity was Ž y1 expressed. This inactivation during immobilization was confirmed by the S.A. values 22-29 EU mg for the CPC-deriva-y1 . Ž . tives and 80 EU mg for soluble b-gal . The K 3.4 mM for the CDAP-derivative with ONPG as substrate was higher app Ž . than the K value for soluble b-gal 2 mM . When the enzyme loading was increased five-fold, the K increased M app four-fold, to 13 mM. The V values for the CPC-derivatives were remarkably lower than the V for soluble app max b-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 48C, the activity of both derivatives remained stable for at least 2 months. Ž . Both derivatives displayed high percentages of lactose conversion 90% in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved. q 1998 Elsevier Science B.V. All rights reserved.

Research paper thumbnail of Development of a continuous solid phase process for reduction and thiol-dependent immobilization of yeast β-galactosidase

Journal of Molecular Catalysis B: Enzymatic, 2009

... Karen Ovsejevi Corresponding Author Contact Information , a , E-mail The Corresponding Author... more ... Karen Ovsejevi Corresponding Author Contact Information , a , E-mail The Corresponding Author , Karina Cuadra a and FranciscoBatista-Viera a. ... In addition, it could be reused four times without losing its lactose hydrolysis capacity. ...

Research paper thumbnail of Affinity chromatography

Methods of biochemical analysis, 2011

Research paper thumbnail of Covalent Chromatography

Methods of Biochemical Analysis, 2011

Research paper thumbnail of Isolation of lactoferrin by immobilized metal ion affinity chromatography (IMAC)

Biochemical Education, 1994

Research paper thumbnail of Bone-Bound Glucoamylase as a Biocatalyst in Bench-Scale Production of Glucose Syrups from Liquefied Cassava Starch

Food and Bioprocess Technology, 2008

This research assesses the bench-scale application of a non-conventional support, bone particles,... more This research assesses the bench-scale application of a non-conventional support, bone particles, for glucoamylase (GA) immobilization and its subsequent use in cassava starch hydrolysis. Upon determining the appropriate conditions to immobilize GA onto chicken bone particles, such as pH, ionic strength, particle size, and enzyme load, bench-scale immobilization of commercial GA without further purification was performed. Under the selected conditions, 270 GA units per gram of support were adsorbed. Optimal temperature and thermal stability of immobilized GA were only slightly different from those of the free enzyme, while optimal pH became more acidic by about one unit. The feasibility of the use of this immobilized biocatalyst for high glucose syrup production from liquefied cassava starch, at bench scale in batch process using a stirred-tank reactor, was demonstrated. Repeated use of the GA-bone derivative showed that similar conversions to those achieved with soluble enzyme (dextrose equivalent=98) were reached until the third batch and over 90% until the 25th batch.

Research paper thumbnail of Chromatographic methods for amylases

Journal of Chromatography B: Biomedical Sciences and Applications, 1996

This review surveys recent developments in chromatographic methods for the separation of amylases... more This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of t~and t-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

Research paper thumbnail of Selective adsorption of immunoglobulins and glucosylated proteins on phenylboronate-agarose

Journal of Chromatography A, 1992

Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-... more Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, pH 8.5, selectively adsorbs immunoglobulins and complement factors C3 and C4 from human serum. The selectivity of binding is strongly influenced by the presence of magnesium chloride in the sample buffer. Adsorbed immunoglobulins are quantitatively eluted by sorbitol, but only partially by ethylene glycol or methylcellosolve. Aniline-agarose of a similar degree of substitution shows only weak adsorption of serum proteins under similar experimental conditions, thus indicating the important contribution of the boronate moiety to this interaction. Immunoglobulin adsorption seems not to be due to the cis-diol complexation used extensively for the chromatographic determination of non-enzymatically glucosylated proteins. Hydrophobic and pi-pi interactions with the aromatic structure of the ligand seem also to contribute to protein binding. The behaviour of aminophenylboronate-liganded agarose is, in some respects, rather similar to that of the so-called "thiophilic adsorbents".

Research paper thumbnail of Characterization of Galactosyl Derivatives Obtained by Transgalactosylation of Lactose and Different Polyols Using Immobilized β-Galactosidase from Aspergillus oryzae

Journal of Agricultural and Food Chemistry, 2009

The synthesis of novel galactosides is interesting because of their important role in several bio... more The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.

Research paper thumbnail of Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

Applied Biochemistry and Biotechnology, 1998

A new approach for the control and interruption of enzymatic reactions via selective enzyme immob... more A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: oL-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided with de novo thiol-groups by a two-step reaction involving N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases.

Research paper thumbnail of Studies on aroma generation in lulo ( Solanum quitoense): enzymatic hydrolysis of glycosides from leaves

Food Chemistry, 2003

Five C13-norisoprenoid glycosides, isolated from lulo (Solanum quitoense) leaves, were subjected ... more Five C13-norisoprenoid glycosides, isolated from lulo (Solanum quitoense) leaves, were subjected to enzymatic hydrolysis with a commercial glucosidase (emulsin) and also with an enzymatic preparation having glycosidase activity, isolated from lulo fruit pulp. The volatile compounds generated after reaction were characterized by capillary GC and capillary GC–MS. Lulo fruit glycosidases were extracted by ammonium sulfate precipitation at pH 6.5 and

Research paper thumbnail of Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-lactogl... more Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 µmol of SH groups/g of dried gel). Both reductants allowed an increase of three-(for K. lactis β-galactosidase) and fourfold (for A. oryzae β-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis β-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae β-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in β-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.

Research paper thumbnail of Affinity chromatography of soybean β-amylase on phenylboronate agarose

Journal of High Resolution Chromatography, 1992

Research paper thumbnail of Bone-Bound Glucoamylase as a Biocatalyst in Bench-Scale Production of Glucose Syrups from Liquefied Cassava Starch

Food and Bioprocess Technology, 2008

This research assesses the bench-scale application of a non-conventional support, bone particles,... more This research assesses the bench-scale application of a non-conventional support, bone particles, for glucoamylase (GA) immobilization and its subsequent use in cassava starch hydrolysis. Upon determining the appropriate conditions to immobilize GA onto chicken bone particles, such as pH, ionic strength, particle size, and enzyme load, bench-scale immobilization of commercial GA without further purification was performed. Under the selected conditions, 270 GA units per gram of support were adsorbed. Optimal temperature and thermal stability of immobilized GA were only slightly different from those of the free enzyme, while optimal pH became more acidic by about one unit. The feasibility of the use of this immobilized biocatalyst for high glucose syrup production from liquefied cassava starch, at bench scale in batch process using a stirred-tank reactor, was demonstrated. Repeated use of the GA-bone derivative showed that similar conversions to those achieved with soluble enzyme (dextrose equivalent=98) were reached until the third batch and over 90% until the 25th batch.

Research paper thumbnail of Improved Glucoamylase Immobilization onto Calcined Chicken Bone Particles

Food and Bioprocess Technology, 2009

Calcination was considered for the first time as an alternative to recover inorganic bone compone... more Calcination was considered for the first time as an alternative to recover inorganic bone component from spent glucoamylase-bone derivatives. The subsequent adsorption of glucoamylase (GA) onto calcined bone particles was assessed. Adsorption capacity of the calcined matrix was found to be from 1.1-to 1.4-fold superior to that of non-calcined supports, and it was dependent on the applied load. Moreover, the expressed activity of GA derivatives on calcined matrix was, at least, 2-fold higher than that of biocatalysts onto non-calcined support. The optimization of the loading allowed the preparation of derivatives with 139 GA units per gram of support, which preserve 52% of the immobilized activity. Additionally, calcination of spent GA biocatalysts on calcined bone particles was performed, and adsorption of glucoamylase onto the bone particles calcined a second time was also found to be efficient. In addition to the improved catalytic properties, the half-life at 55°C of the GA biocatalysts on calcined bone was increased 1.7fold in comparison with that of soluble GA and GA adsorbed onto non-calcined bone particles. Furthermore, the same cassava starch conversion can be achieved batchwise in a stirred-tank reactor using less insoluble biocatalyst, 37% of the GA-bone derivative, which represents an important saving for industrial applications.

Research paper thumbnail of Immobilization of Enzymes: A Literature Survey

Methods in Molecular Biology, 2013

The term immobilized enzymes refers to &a... more The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

Research paper thumbnail of Chromatographic methods for amylases

Journal of Chromatography B: Biomedical Sciences and Applications, 1996

This review surveys recent developments in chromatographic methods for the separation of amylases... more This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of t~and t-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

Research paper thumbnail of Thiolation and reversible immobilization of sweet potato δ-amylase on thiolsulfonate-agarose

Journal of Molecular Catalysis, 1993

Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide... more Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide exchange reactions are unique because, simultaneously, they show a stable covalent bond and the possibility of eluting the protein by reduction when the enzymatic activity decays. The adsorbent can thus be reloaded. In this paper we report the use of the recently developed thiolreactive adsorbent thiolsulfonate-agarose, for the immobilization of sweet potato8-amylase. Since native p-amylase thiol groups were not reactive towards the adsorbent, the enzyme was provided with 'de novo' thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3-(2-pyridy1dithio)propionat.e (SPDP). When the SPDP/B-amylase molar ratio was changed between 3 and 100, up to sixteen exposed thiol groups per mol of enzyme were introduced. This was achieved without affecting the amylolytic activity. The immobilization yield for the intermediate thiolation level was 98%. However, only 19% of the applied enzyme activity was found in the gel suspension. Comparative studies were made on thiolsulfonate-agarose and on a commercial thiol-activated adsorbent (2-pyridyldisulfide-agarose) . The immobilization of the thiolated enzyme through reversible disulfide bonds on both adsorbenta showed similar results. A close analysis reveals that immobilization of proteins on thiolsulfonate-agarose is a very promising technique.

Research paper thumbnail of beta-Galactosidase from Kluyveromyces lactis immobilized on to thiolsulfinate/thiolsulfonate supports for lactose hydrolysis in milk and dairy by-products

Biotechnology Techniques, 1998

After reversible immobilization of neutral beta-galactosidase from Kluyveromyces lactis on to thi... more After reversible immobilization of neutral beta-galactosidase from Kluyveromyces lactis on to thiolsulfinate/thiolsulfonate supports, more than 80 % of the activity was retained. Blocking the remaining reactive groups with glutathione increased the thermal stability of the derivatives almost two-fold. These derivatives achieved a high degree of conversion (85-90 %) of lactose (50g / l) in saline solution, whey, whey permeates, and

Research paper thumbnail of A novel extracellular β-glucosidase from Issatchenkia terricola: Isolation, immobilization and application for aroma enhancement of white Muscat wine

Process Biochemistry, 2011

Since most Saccharomyces strains show no ␤-glucosidase activity, the importance of non-Saccharomy... more Since most Saccharomyces strains show no ␤-glucosidase activity, the importance of non-Saccharomyces ␤-glucosidases in the development of wine aroma has been highlighted. However, only a few enzymes from yeasts isolated from grape-must and enological ecosystems are active in the stringent conditions of wine and the wine-making process (low pH, high concentrations of ethanol or glucose). A purified extract of extracellular ␤-glucosidase from Issatchenkia terricola, proved to be very active in the presence of glucose (100 g/L), ethanol (18%) and metabisulfite (60 mg/L). It is also active and relatively stable at acidic pH over 3.0. Immobilization onto Eupergit C greatly improved its stability, allowing the aromatization of white Muscat wine over a 16-day experiment. The released aroma compounds of control and treated wine were analyzed by GC-MS. The enzymatic treatment significantly increased the amount of monoterpenes and norisoprenoids, showing the potential of the immobilized enzyme for aroma development in wines.

Research paper thumbnail of Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

Journal of Molecular Catalysis B: Enzymatic, 1998

. The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two differ... more . The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose Ž . via a cyanylating agent CDAP was optimized. Gel-bound protein and gel-bound activity were both measured directly, Ž . allowing the determination of apparent specific activities S.A. . Higher amounts of b-gal were immobilized on the activated Ž y1 . CPC-silica maximum capacity, 23 mg ml of packed support than on the CDAP-activated agarose. For the lower enzyme Ž y1 . loading assayed 12.6 mg ml packed support , 100% of the enzyme was immobilized but only 34% of its activity was Ž y1 expressed. This inactivation during immobilization was confirmed by the S.A. values 22-29 EU mg for the CPC-deriva-y1 . Ž . tives and 80 EU mg for soluble b-gal . The K 3.4 mM for the CDAP-derivative with ONPG as substrate was higher app Ž . than the K value for soluble b-gal 2 mM . When the enzyme loading was increased five-fold, the K increased M app four-fold, to 13 mM. The V values for the CPC-derivatives were remarkably lower than the V for soluble app max b-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 48C, the activity of both derivatives remained stable for at least 2 months. Ž . Both derivatives displayed high percentages of lactose conversion 90% in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved. q 1998 Elsevier Science B.V. All rights reserved.

Research paper thumbnail of Development of a continuous solid phase process for reduction and thiol-dependent immobilization of yeast β-galactosidase

Journal of Molecular Catalysis B: Enzymatic, 2009

... Karen Ovsejevi Corresponding Author Contact Information , a , E-mail The Corresponding Author... more ... Karen Ovsejevi Corresponding Author Contact Information , a , E-mail The Corresponding Author , Karina Cuadra a and FranciscoBatista-Viera a. ... In addition, it could be reused four times without losing its lactose hydrolysis capacity. ...