Abstract A variety of glycosides with interesting biological properties suitable for application ... more Abstract A variety of glycosides with interesting biological properties suitable for application in cosmetic and pharmaceutical industry can be found in nature. However, the demand exceeds the quantity and purity which can be obtained naturally and therefore enzymatic glycosylation has been of great interest in recent decades. In this study, crude Kluyveromyces lactis enzyme preparation Lactozym 3000 L was examined for the production of two phenolic glycosides – tyrosol β-fructoside and tyrosol β-galactoside. Its performance was compared to other commercial fungal and yeast enzyme preparations. It was found that the key enzyme, β-galactosidase is unsuitable for tyrosol β-galactoside production. However, one of the Lactozym 3000 L components, β-fructofuranosidase, performed very well in the tyrosol β-fructoside production from sucrose and tyrosol. Lactozym 3000 L was effectively immobilized onto activated carrier Lifetech ECR8309 via covalent binding of the enzyme, which is a promising method to reduce very high enzyme consumption in its free form. Potential of deep-eutectic solvents to enhance transfructosylation selectivity has been studied proving their interesting stabilization effect on Lactozym 3000 L β-fructofuranosidase.
Effective development of production of any therapeutic protein requires a relatively fast, reliab... more Effective development of production of any therapeutic protein requires a relatively fast, reliable, and economical method of target protein monitoring in the outlet streams of cultivation and separation steps. The aim of this work was to develop a method for the determination of recombinant human erythropoietin (rhEPO) produced by human embryonic kidney (HEK) cells. An indirect ELISA method was designed that meets all requirements to fulfill this goal. It was found that an HEK cell-cultivation medium, containing plant hydrolyzate, is a suitable coating buffer, which solves the problem of competitive binding of byproducts. The method has a linear range of 0.078-4 μg/mL allowing accurate determination of rhEPO in relatively concentrated solutions (about 200 μg/mL) without excessive dilution. For the multicomponent rhEPO concentrate at the 400-fold dilution, the inter-assay coefficient of variation was found to be 3.3%. The cost analysis showed that the designed indirect ELISA assay is by one-two orders of magnitude cheaper than the existing commercial sandwich ELISA kits.
This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of pr... more This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of proteins in a concentrated postcultivation supernatant. Cation-exchange multimodal adsorbent Capto MMC ImpRes was selected as potential chromatographic separation material. Its equilibrium properties were investigated in batch adsorption experiments. The effect of pH in the range of 5.5—7.5 and NaCl concentration in the range of 0—300 mM on the adsorption of rhEPO and contaminant proteins was examined. Optimal conditions found in these equilibrium experiments were applied to rhEPO adsorption in a chromatographic column. Several experiments were carried out at different elution conditions to optimize the rhEPO yield and selectivity.
... Milan Polakovič Corresponding Author Contact Information , Carl-Fredrik Mandenius. ... 50+0.0... more ... Milan Polakovič Corresponding Author Contact Information , Carl-Fredrik Mandenius. ... 50+0.00 ELSEVIER 0032-9592(94)00019.0 Production of Red Pigments by Monascus ruber in Synthetic Media with a Strictly Controlled Nitrogen Source L. Pastrana,* PJ Blanc, AL Santerre ...
The thermal stability of a biocatalyst in the form of whole cells of the fungi Aureobasidium pull... more The thermal stability of a biocatalyst in the form of whole cells of the fungi Aureobasidium pullulans exhibiting fructosyltransferase activity was investigated during a batch catalytic process of fructooligosaccharide production. The stability of fructosyltransferase was monitored during 8 h with respect to temperature and overall saccharide concentration. A fructosyltransferase activity assay was developed for the monitoring of enzyme activity that was based on the rate of formation of 1-kestose measured by an HPLC method. It was found that the biocatalyst inactivated rather quickly at temperatures above 60oC even at high saccharide concentrations. The stabilizing effect of saccharide concentration on the fructosyltransferase activity was investigated at 55oC. A kinetic equation of enzyme inactivation, based on a reversible one-step reaction and simple exponential relationships of the rate constants and on the overall saccharide concentration, was suggested and statistically verif...
Fructo-oligosaccharides (FOS), namely kestose, nystose and fructo-furanosylnystose, are prebiotic... more Fructo-oligosaccharides (FOS), namely kestose, nystose and fructo-furanosylnystose, are prebiotic sugars that can be obtained from sucrose by fermentation. The resulting fermentative broth is a complex mixture containing also salts and other smaller sugars that must be removed. In this study, a Dowex Monosphere calcium resin was evaluated for the separation of sugars. The adsorption behavior of FOS contained in a fermentative broth was compared with FOS from purified mixtures. Experiments were performed in static batch mode at 60ºC. Experimental isotherms data were analyzed using linear, Langmuir/anti-Langmuir, Freundlich, Redlich & Peterson, and Toth type models. Isotherm parameters were estimated using linear and non-linear correlations for the minimization of several error functions. The non-linear correlations were found to provide the best isotherm parameters for the models. FOS adsorption showed different behaviors depending on the complexity of the liquid phase of the FOS mixtures studied. The experimental adsorption data of FOS contained in purified mixtures were well fitted with the anti-Langmuir isotherm, while FOS in a fermentative broth were better fitted with Toth and Langmuir isotherms
The e ect of the initial sucrose concentration (50 g dm−3, 200 g dm−3, and 350 g dm−3) and time o... more The e ect of the initial sucrose concentration (50 g dm−3, 200 g dm−3, and 350 g dm−3) and time of cultivation (1 d, 2 d, and 4 d) on the fructosyltransferase production by Aureobasidium pullulans CCY 27-1-1194 in shaken flask cultivations was investigated. It was found that the overall fructosyltransferase production depended strongly on the initial sucrose concentration when the highest value of enzyme activity (130 000 U dm−3) was achieved at 350 g dm−3 in the fourth cultivation day. The maximum of speci c activity per dry cell mass was achieved at the same sucrose concentration, but already in the second day. Since the second cultivation day, the ratio of the enzyme activity contained in cells and released into medium was 1.3—1.6, independently of sucrose concentration. In order to discuss the fructosyltransferase production in relation to cell growth and state of the culture, the sucrose conversion, produced amount of fructooligosaccharides and organic acids, and the yield fact...
This article reviews the information about fructosyltransferases, the enzymes producing important... more This article reviews the information about fructosyltransferases, the enzymes producing important prebiotic compounds, fructooligosaccharides. The chemical structure, safety and physiological effects, preparation and application of fructooligosaccharides are discussed. Plant and microbial sources of fructosyltransferases are presented. A detailed description is devoted to the mechanisms of action, substrate specificities, and regioselectivities of fructosyltransferases. Their physicochemical and catalytic properties are also discussed. Finally, the methods of purification of fructosyltransferases reported in literature are summarized in respect to the yield and factor of purification.
This study focused on the problem of the systematic errors of the adsorbed/desorbed amount in col... more This study focused on the problem of the systematic errors of the adsorbed/desorbed amount in column experiments when the parameters of the Langmuir isotherm were obtained from independent experiments. The effect of this discrepancy on the estimation of the mass transfer coefficient in the linear driving force model was investigated. The systematic errors were introduced by using the negatively or positively biased values of the partition coefficient from 1 % to 10 %. Simulated experiments were carried out with the biased values using a dimensionless form of the adsorption column model having three parameters: the dimensionless mass transfer coefficient (d), the partition coefficient (Λ), and the separation factor (r) that characterizes the degree of saturation of adsorbent. It was found interesting to vary a single model parameter, the product of d and Λ. Three values of dΛ, 50, 500, and 5000, were used. They covered the situations from an almost immediate breakthrough of the solute in the outlet to a sharp bend at negligible mass transfer resistance. The deviations of the estimated and true values of d were presented as functions of the systematic errors both for adsorption and desorption. The accuracies of the estimates of d were characterized by the t-values calculated from the residual sum of squares function.
Fructo-oligosaccharides (FOS) namely, kestose, nystose and fructofuranosylnystose, are prebiotics... more Fructo-oligosaccharides (FOS) namely, kestose, nystose and fructofuranosylnystose, are prebiotics that can be obtained by fermentation. The resulting fermentative broth is a complex mixture consisting also of salts and other sugars that must be removed. Adsorption equilibrium studies were conducted using the static method in batch mode onto Dowex Monosphere calcium resin, at 60ºC, for FOS contained in a fermentative broth and pure mixtures. Experimental isotherms data were analyzed using linear, Langmuir/anti-Langmuir, Freundlich, Redlich & Peterson, and Toth models. Isotherm parameters were determined using linear and nonlinear correlations for the minimization of several error functions. The nonlinear correlations were found to provide the best isotherm parameters for the models. FOS contained in a fermentative broth were well fitted with anti-Langmuir isotherms, while FOS in pure mixtures were better fitted with Toth, Langmuir and Redlich & Peterson isotherms. A detailed error an...
Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular ... more Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.
A set of chromatographic materials for bioseparation were characterised by various methods. Both ... more A set of chromatographic materials for bioseparation were characterised by various methods. Both commercial materials and new supports presenting various levels of rigidity were analysed. The methods included size-exclusion and capillary phenomena based techniques. Both batch exclusion and inverse size-exclusion chromatography were used. Gas adsorption, mercury porosimetry and thermoporometry were applied as well as a new method based on water desorption starting from the saturated state. When the rigidity of adsorbents is high enough, the agreement is reasonable between the values of the structural parameters that were determined (surface area, porosity, and pore size) by various methods. Nevertheless, a part of macroporosity may not be evidenced by inverse size-exclusion chromatography whereas it is visible by batch exclusion and the other methods. When the rigidity decreases, for example with soft swelling gels, where standard nitrogen adsorption or mercury porosimetry are no more reliable, two main situations are encountered: either the methods based on capillary phenomena (thermoporometry or water desorption) overestimate the pore size with an amplitude that depends on the method, or in some cases it is possible to distinguish water involved in the swelling of pore walls from that involved in pore filling by capillary condensation.
... Basics and Applications 1996 Elsevier Science BV Pitfalls of initial reaction rate measuremen... more ... Basics and Applications 1996 Elsevier Science BV Pitfalls of initial reaction rate measurements M. Polakovic, G. Handrikova, P. Acai, V. Stefuca ... This is the only extra parameter needed for a generalized solution of and unsteady state process compared to the analogous steady ...
ABSTRACT This publication reviews the results of the authors' studies dealing with the id... more ABSTRACT This publication reviews the results of the authors' studies dealing with the identification of mechanisms of enzyme inactivation. It presents both a critical assessment of traditional approaches of evaluation of enzyme inactivation kinetics from uniresponse experiments as well as the procedures of better design and evaluation of experiments which improve the analysis of inactivation mechanism and its corresponding kinetics. These methods are based on the simultaneous evaluation of inactivation experiments carried out at several temperatures or pHs. The inactivation mechanism is thus evaluated from the data set containing larger amount of information compared to a single inactivation curve when the influence of the factor of variation is unequivocally defined, for example, by the Arrhenius equation in the case of temperature or protonation and deprotonation equilibrium constants in the case of pH. It is demonstrated furthermore that the complexity of the mechanism of the thermal inactivation of free enzyme can be enhanced by utilising the combination of selective immobilization and multitemperature evaluation when the immobilization prevents some reactions in the ordinary inactivation pathway to occur. All methods are illustrated on case studies using yeast invertase.
Abstract A variety of glycosides with interesting biological properties suitable for application ... more Abstract A variety of glycosides with interesting biological properties suitable for application in cosmetic and pharmaceutical industry can be found in nature. However, the demand exceeds the quantity and purity which can be obtained naturally and therefore enzymatic glycosylation has been of great interest in recent decades. In this study, crude Kluyveromyces lactis enzyme preparation Lactozym 3000 L was examined for the production of two phenolic glycosides – tyrosol β-fructoside and tyrosol β-galactoside. Its performance was compared to other commercial fungal and yeast enzyme preparations. It was found that the key enzyme, β-galactosidase is unsuitable for tyrosol β-galactoside production. However, one of the Lactozym 3000 L components, β-fructofuranosidase, performed very well in the tyrosol β-fructoside production from sucrose and tyrosol. Lactozym 3000 L was effectively immobilized onto activated carrier Lifetech ECR8309 via covalent binding of the enzyme, which is a promising method to reduce very high enzyme consumption in its free form. Potential of deep-eutectic solvents to enhance transfructosylation selectivity has been studied proving their interesting stabilization effect on Lactozym 3000 L β-fructofuranosidase.
Effective development of production of any therapeutic protein requires a relatively fast, reliab... more Effective development of production of any therapeutic protein requires a relatively fast, reliable, and economical method of target protein monitoring in the outlet streams of cultivation and separation steps. The aim of this work was to develop a method for the determination of recombinant human erythropoietin (rhEPO) produced by human embryonic kidney (HEK) cells. An indirect ELISA method was designed that meets all requirements to fulfill this goal. It was found that an HEK cell-cultivation medium, containing plant hydrolyzate, is a suitable coating buffer, which solves the problem of competitive binding of byproducts. The method has a linear range of 0.078-4 μg/mL allowing accurate determination of rhEPO in relatively concentrated solutions (about 200 μg/mL) without excessive dilution. For the multicomponent rhEPO concentrate at the 400-fold dilution, the inter-assay coefficient of variation was found to be 3.3%. The cost analysis showed that the designed indirect ELISA assay is by one-two orders of magnitude cheaper than the existing commercial sandwich ELISA kits.
This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of pr... more This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of proteins in a concentrated postcultivation supernatant. Cation-exchange multimodal adsorbent Capto MMC ImpRes was selected as potential chromatographic separation material. Its equilibrium properties were investigated in batch adsorption experiments. The effect of pH in the range of 5.5—7.5 and NaCl concentration in the range of 0—300 mM on the adsorption of rhEPO and contaminant proteins was examined. Optimal conditions found in these equilibrium experiments were applied to rhEPO adsorption in a chromatographic column. Several experiments were carried out at different elution conditions to optimize the rhEPO yield and selectivity.
... Milan Polakovič Corresponding Author Contact Information , Carl-Fredrik Mandenius. ... 50+0.0... more ... Milan Polakovič Corresponding Author Contact Information , Carl-Fredrik Mandenius. ... 50+0.00 ELSEVIER 0032-9592(94)00019.0 Production of Red Pigments by Monascus ruber in Synthetic Media with a Strictly Controlled Nitrogen Source L. Pastrana,* PJ Blanc, AL Santerre ...
The thermal stability of a biocatalyst in the form of whole cells of the fungi Aureobasidium pull... more The thermal stability of a biocatalyst in the form of whole cells of the fungi Aureobasidium pullulans exhibiting fructosyltransferase activity was investigated during a batch catalytic process of fructooligosaccharide production. The stability of fructosyltransferase was monitored during 8 h with respect to temperature and overall saccharide concentration. A fructosyltransferase activity assay was developed for the monitoring of enzyme activity that was based on the rate of formation of 1-kestose measured by an HPLC method. It was found that the biocatalyst inactivated rather quickly at temperatures above 60oC even at high saccharide concentrations. The stabilizing effect of saccharide concentration on the fructosyltransferase activity was investigated at 55oC. A kinetic equation of enzyme inactivation, based on a reversible one-step reaction and simple exponential relationships of the rate constants and on the overall saccharide concentration, was suggested and statistically verif...
Fructo-oligosaccharides (FOS), namely kestose, nystose and fructo-furanosylnystose, are prebiotic... more Fructo-oligosaccharides (FOS), namely kestose, nystose and fructo-furanosylnystose, are prebiotic sugars that can be obtained from sucrose by fermentation. The resulting fermentative broth is a complex mixture containing also salts and other smaller sugars that must be removed. In this study, a Dowex Monosphere calcium resin was evaluated for the separation of sugars. The adsorption behavior of FOS contained in a fermentative broth was compared with FOS from purified mixtures. Experiments were performed in static batch mode at 60ºC. Experimental isotherms data were analyzed using linear, Langmuir/anti-Langmuir, Freundlich, Redlich & Peterson, and Toth type models. Isotherm parameters were estimated using linear and non-linear correlations for the minimization of several error functions. The non-linear correlations were found to provide the best isotherm parameters for the models. FOS adsorption showed different behaviors depending on the complexity of the liquid phase of the FOS mixtures studied. The experimental adsorption data of FOS contained in purified mixtures were well fitted with the anti-Langmuir isotherm, while FOS in a fermentative broth were better fitted with Toth and Langmuir isotherms
The e ect of the initial sucrose concentration (50 g dm−3, 200 g dm−3, and 350 g dm−3) and time o... more The e ect of the initial sucrose concentration (50 g dm−3, 200 g dm−3, and 350 g dm−3) and time of cultivation (1 d, 2 d, and 4 d) on the fructosyltransferase production by Aureobasidium pullulans CCY 27-1-1194 in shaken flask cultivations was investigated. It was found that the overall fructosyltransferase production depended strongly on the initial sucrose concentration when the highest value of enzyme activity (130 000 U dm−3) was achieved at 350 g dm−3 in the fourth cultivation day. The maximum of speci c activity per dry cell mass was achieved at the same sucrose concentration, but already in the second day. Since the second cultivation day, the ratio of the enzyme activity contained in cells and released into medium was 1.3—1.6, independently of sucrose concentration. In order to discuss the fructosyltransferase production in relation to cell growth and state of the culture, the sucrose conversion, produced amount of fructooligosaccharides and organic acids, and the yield fact...
This article reviews the information about fructosyltransferases, the enzymes producing important... more This article reviews the information about fructosyltransferases, the enzymes producing important prebiotic compounds, fructooligosaccharides. The chemical structure, safety and physiological effects, preparation and application of fructooligosaccharides are discussed. Plant and microbial sources of fructosyltransferases are presented. A detailed description is devoted to the mechanisms of action, substrate specificities, and regioselectivities of fructosyltransferases. Their physicochemical and catalytic properties are also discussed. Finally, the methods of purification of fructosyltransferases reported in literature are summarized in respect to the yield and factor of purification.
This study focused on the problem of the systematic errors of the adsorbed/desorbed amount in col... more This study focused on the problem of the systematic errors of the adsorbed/desorbed amount in column experiments when the parameters of the Langmuir isotherm were obtained from independent experiments. The effect of this discrepancy on the estimation of the mass transfer coefficient in the linear driving force model was investigated. The systematic errors were introduced by using the negatively or positively biased values of the partition coefficient from 1 % to 10 %. Simulated experiments were carried out with the biased values using a dimensionless form of the adsorption column model having three parameters: the dimensionless mass transfer coefficient (d), the partition coefficient (Λ), and the separation factor (r) that characterizes the degree of saturation of adsorbent. It was found interesting to vary a single model parameter, the product of d and Λ. Three values of dΛ, 50, 500, and 5000, were used. They covered the situations from an almost immediate breakthrough of the solute in the outlet to a sharp bend at negligible mass transfer resistance. The deviations of the estimated and true values of d were presented as functions of the systematic errors both for adsorption and desorption. The accuracies of the estimates of d were characterized by the t-values calculated from the residual sum of squares function.
Fructo-oligosaccharides (FOS) namely, kestose, nystose and fructofuranosylnystose, are prebiotics... more Fructo-oligosaccharides (FOS) namely, kestose, nystose and fructofuranosylnystose, are prebiotics that can be obtained by fermentation. The resulting fermentative broth is a complex mixture consisting also of salts and other sugars that must be removed. Adsorption equilibrium studies were conducted using the static method in batch mode onto Dowex Monosphere calcium resin, at 60ºC, for FOS contained in a fermentative broth and pure mixtures. Experimental isotherms data were analyzed using linear, Langmuir/anti-Langmuir, Freundlich, Redlich & Peterson, and Toth models. Isotherm parameters were determined using linear and nonlinear correlations for the minimization of several error functions. The nonlinear correlations were found to provide the best isotherm parameters for the models. FOS contained in a fermentative broth were well fitted with anti-Langmuir isotherms, while FOS in pure mixtures were better fitted with Toth, Langmuir and Redlich & Peterson isotherms. A detailed error an...
Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular ... more Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.
A set of chromatographic materials for bioseparation were characterised by various methods. Both ... more A set of chromatographic materials for bioseparation were characterised by various methods. Both commercial materials and new supports presenting various levels of rigidity were analysed. The methods included size-exclusion and capillary phenomena based techniques. Both batch exclusion and inverse size-exclusion chromatography were used. Gas adsorption, mercury porosimetry and thermoporometry were applied as well as a new method based on water desorption starting from the saturated state. When the rigidity of adsorbents is high enough, the agreement is reasonable between the values of the structural parameters that were determined (surface area, porosity, and pore size) by various methods. Nevertheless, a part of macroporosity may not be evidenced by inverse size-exclusion chromatography whereas it is visible by batch exclusion and the other methods. When the rigidity decreases, for example with soft swelling gels, where standard nitrogen adsorption or mercury porosimetry are no more reliable, two main situations are encountered: either the methods based on capillary phenomena (thermoporometry or water desorption) overestimate the pore size with an amplitude that depends on the method, or in some cases it is possible to distinguish water involved in the swelling of pore walls from that involved in pore filling by capillary condensation.
... Basics and Applications 1996 Elsevier Science BV Pitfalls of initial reaction rate measuremen... more ... Basics and Applications 1996 Elsevier Science BV Pitfalls of initial reaction rate measurements M. Polakovic, G. Handrikova, P. Acai, V. Stefuca ... This is the only extra parameter needed for a generalized solution of and unsteady state process compared to the analogous steady ...
ABSTRACT This publication reviews the results of the authors' studies dealing with the id... more ABSTRACT This publication reviews the results of the authors' studies dealing with the identification of mechanisms of enzyme inactivation. It presents both a critical assessment of traditional approaches of evaluation of enzyme inactivation kinetics from uniresponse experiments as well as the procedures of better design and evaluation of experiments which improve the analysis of inactivation mechanism and its corresponding kinetics. These methods are based on the simultaneous evaluation of inactivation experiments carried out at several temperatures or pHs. The inactivation mechanism is thus evaluated from the data set containing larger amount of information compared to a single inactivation curve when the influence of the factor of variation is unequivocally defined, for example, by the Arrhenius equation in the case of temperature or protonation and deprotonation equilibrium constants in the case of pH. It is demonstrated furthermore that the complexity of the mechanism of the thermal inactivation of free enzyme can be enhanced by utilising the combination of selective immobilization and multitemperature evaluation when the immobilization prevents some reactions in the ordinary inactivation pathway to occur. All methods are illustrated on case studies using yeast invertase.
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