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Roberto Fernandezlafuente - One of the best experts on this subject based on the ideXlab platform.
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modulating the properties of the lipase from thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions
Enzyme and Microbial Technology, 2020Co-Authors: Yuliya Lokha, Nathalia Saraiva Rios, Luciana Rocha Barros Gonçalves, Sara Aranapena, Carmen Mendezsanchez, Fernando Lopezgallego, Roberto FernandezlafuenteAbstract:Abstract The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: Stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final Enzyme properties, altering Enzyme Stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the Enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final Enzyme Stability. The biocatalyst Stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized Enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.
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immobilization stabilization of ficin extract on glutaraldehyde activated agarose beads variables that control the final Stability and activity in protein hydrolyses
Catalysts, 2018Co-Authors: Elhocine Siar, Sara Aranapena, Oveimar Barbosa, Mohammed Nasreddine Zidoune, Roberto FernandezlafuenteAbstract:Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered Enzyme, with no significant effects on Enzyme Stability. The treatment with glutaraldehyde of this ionically exchanged Enzyme produced an almost full Enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized Enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized Enzyme. The maximum Enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the Enzyme Stability or Enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized Enzyme molecules may generate some steric hindrances for the casein hydrolysis.
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stabilization of dimeric β glucosidase from aspergillus niger via glutaraldehyde immobilization under different conditions
Enzyme and Microbial Technology, 2018Co-Authors: Perla Guadalupe Vazquezortega, Maria Teresa Alcarazfructuoso, Juan Antonio Rojascontreras, Javier Lopezmiranda, Roberto FernandezlafuenteAbstract:Abstract The dimeric Enzyme β-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The Stability of the free Enzyme depended on Enzyme concentration. Immobilization via ion exchange improved Enzyme Stability/activity, depending on the immobilization pH. However, the Enzyme was desorbed in 75 mM NaCl at pH 7 and some Stability/Enzyme concentration dependence still existed. Treatment: of these biocatalysts with glutaraldehyde increased Enzyme Stability (e.g. at pH 5, after incubation under conditions where the Enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated Enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in Enzyme activity, but the stabilization was lower. While when measuring the Enzyme activity at pH 4 there were no changes after immobilization, all immobilized Enzymes were more active than the free Enzyme at pH 6 and 7 (2–3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid Enzyme subunit desorption, being the immobilization pH a key point in their design.
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effect of immobilization rate and Enzyme crowding on Enzyme Stability under different conditions the case of lipase from thermomyces lanuginosus immobilized on octyl agarose beads
Process Biochemistry, 2017Co-Authors: Hadjer Zaak, Elhocine Siar, Jakub F Kornecki, Laura Fernandezlopez, Sara G Pedrero, Jose J Virgenortiz, Roberto FernandezlafuenteAbstract:Abstract Lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl agarose (OC). Three different TLL-OC biocatalysts were prepared: one lowly loaded using a low Enzyme concentration, one fully loaded using a low Enzyme concentrations, and a final one using a large excess of Enzyme at a higher concentration. The activities after immobilization increased (180%), although diffusion limitations reduced the hyperactivation of the fully loaded preparations (140%). The stabilities of both preparations using low Enzyme concentrations were similar under all studied conditions discounting the diffusional limitations of the biocatalyst. However, the biocatalyst prepared using a large concentration of Enzyme was less stable that the other preparations at pH 7.0, more stable at pH 5.0 and with a similar Stability at pH 9.0. Adding 3 M NaCl, the Stability of the fully loaded preparations significantly increased; while the lowly loaded preparation slightly improved Enzyme Stability. This produced that the biocatalyst prepared under using high Enzyme concentration become significantly more stable than the other two TLL preparations. Glycerin increased immobilized TLL Stability, in this case all OC-TLL preparations became with similar stabilities. Results show that the TLL concentration during immobilization may greatly affect TLL properties, perhaps due to altering Enzyme packing.
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stabilization of the hexameric glutamate dehydrogenase from escherichia coli by cations and polyethyleneimine
Enzyme and Microbial Technology, 2013Co-Authors: Cristina Garciagalan, Oveimar Barbosa, Roberto FernandezlafuenteAbstract:Abstract The Enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The Stability of this Enzyme was increased in the presence of Li + in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate. A very significant dependence of the Enzyme Stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of Enzyme concentration on its Stability was not significantly decreased by the presence of 10 mM Li + . Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the Enzyme subunits. This incubation does not have a significant effect on Enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2 h under conditions where the untreated Enzyme retained only 20% of the initial activity, and the effect of the Enzyme concentration on Enzyme Stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified Enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li + on Enzyme Stability was maintained after Enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the Enzyme.
Fernández-lafuente Roberto - One of the best experts on this subject based on the ideXlab platform.
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Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads
'Japanese Society of Applied Entomology & Zoology', 2021Co-Authors: Ait Braham Sabrina, Morellon-sterling Roberto, Siar El-hocine, Diandra De ,andrades, Pedroche Justo, Millán-linares, María Del Carmen, Rodrigues, Rafael C., Aksas Ali, Fernández-lafuente RobertoAbstract:5 Figuras.-- 1 TablaTris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal Stability of the obtained immobilized biocatalysts. The results indicate a reduction of the Enzyme Stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the Enzymes assayed. The reduction of Enzyme Stability increased with the Tris concentration. Another interesting result is that the Stability reduction was more noticeable for immobilized PGA than in the other immobilized Enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.The research has been supported by Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R).Peer reviewe
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Effect of amine length in the interference of the multipoint covalent immobilization of Enzymes on glyoxyl agarose beads
'Japanese Society of Applied Entomology & Zoology', 2021Co-Authors: Morellon-sterling Roberto, Siar El-hocine, Ait Braham Sabrina, Diandra De ,andrades, Pedroche Justo, Millán-linares, María Del Carmen, Fernández-lafuente RobertoAbstract:1 Tabla.-- 17 FigurasTrypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized Enzyme Stability. As expected, the presence of amines reduced the intensity of the Enzyme-support multipoint covalent attachment, and therefore the Enzyme Stability. However, it is clear that this effect is higher using octyl amine for all Enzymes (in some cases the Enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the Enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease Enzyme Stability. The results suggested that, if the composition of the Enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the Enzyme-support reaction.We gratefully recognize the financial support from Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R). RMS thank to Ministerio de Educacion -Spanish Government for a FPU fellowship, SAB and EHS thank Algerian Ministry of higher education and scientific research for their fellowships. The help and suggestions from Dr. Ángel Berenguer (Departamento de Química Inorgánica, Universidad de Alicante) are gratefully recognized.Peer reviewe
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Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions
'Elsevier BV', 2020Co-Authors: Lokha Yuliya, Arana-peña Sara, Rios, Nathalia S., Mendez-sanchez Carmen, Gonçalves, Luciana R. B., López-gallego Fernando, Fernández-lafuente RobertoAbstract:The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: Stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final Enzyme properties, altering Enzyme Stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the Enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final Enzyme Stability. The biocatalyst Stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized Enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.We gratefully recognize the support from the MICIU from Spanish Government, (project number CTQ2017-86170-R). FLG thanks ARAID for granting his permanent position at the University of Zaragoza. NSR thanks to CNPq for a predoctoral fellowship (CNPq scholarship – Brazil).Peer reviewe
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Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol
'Elsevier BV', 2019Co-Authors: García-galán Cristina, Gonçalves, Luciana R. B., Dos Santos, José C. S., Barbosa Oveimar, Torres Sáez Rodrigo, Pereira, Ernandes B., Cortés Corberán Vicente, Fernández-lafuente RobertoAbstract:Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized Enzymes (including Stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the Enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled Enzyme is even negative. Most of the modifications presented a positive effect on some Enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated Enzyme permitted to improve Enzyme Stability of both immobilized Enzymes at pH 7 and 9 (around a 10-fold), but only the aminated Enzyme improved the Enzyme Stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the Stability of octyl-Lecitase, while it reduced the Stability of the covalent preparation. Modification with TNBS only improved Enzyme Stability of the covalent preparation at pH 9 (by a 10-fold factor).We gratefully recognize the support from the Spanish Government, grant CTQ2009-07568, grant VIE cod 5706 (UIS-Colombia) and CNPq (Brazil). The predoctoral fellowships for Ms. García-Galán (Spanish Government) and Mr dos Santos (CNPq, Brazil) are also recognized. Dr Pereira thanks Fundación Carolina and UNIFAL-MG.Peer Reviewe
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Stabilizing effects of cations on lipases depend on the immobilization protocol
'Royal Society of Chemistry (RSC)', 2019Co-Authors: Fernández-lópez Laura, Bartolomé-cabrero Rocío, Rodriguez, María Daniela, Sousa Dos Santos, José Cleiton, Rueda Nazzoly, Fernández-lafuente RobertoAbstract:The effect of an additive on Enzyme Stability used to be considered an intrinsic feature of a lipase. However, in this paper we have found that the effect of additive on Enzyme Stability depends on the immobilization protocol. After assaying the effects of diverse chloride salts with different cations on different lipases activity, no relevant effect was detected. Free Enzymes or the covalently immobilized Enzymes are not stabilized by these cations for any of the studied lipases. However, Mn2+ and Ca2+ (at a concentration of 5 mM) are able to greatly stabilize the lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present during the inactivation, but only if the Enzymes are immobilized on octyl-agarose (stabilization factor ranging from 20 to 50). The effect was only detected when using more than 2.5 mM of the cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action. The stabilization seemed to be based on a specific mechanism, and required the recognition sites to be saturated by the cations. Mg2+ has no effect on Enzyme Stability for both Enzymes, but it is able to suppress the stabilization promoted by the other two cations using CRL; while it has no effect on the cation stabilization when using RML. This is the first report of a cation induced Enzyme stabilization effect that depends on the lipase immobilization protocol.We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). The predoctoral fellowships for Ms Rueda (Colciencias, Colombian Government and Becas Iberoamérica “Jóvenes Investigadores”, Banco Santander) and for Ms Rodríguez (CONICET and SPU, Argentine Government) and Dr dos Santos (CNPq, Brazil) are also recognized.Peer Reviewe
Oveimar Barbosa - One of the best experts on this subject based on the ideXlab platform.
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immobilization stabilization of ficin extract on glutaraldehyde activated agarose beads variables that control the final Stability and activity in protein hydrolyses
Catalysts, 2018Co-Authors: Elhocine Siar, Sara Aranapena, Oveimar Barbosa, Mohammed Nasreddine Zidoune, Roberto FernandezlafuenteAbstract:Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered Enzyme, with no significant effects on Enzyme Stability. The treatment with glutaraldehyde of this ionically exchanged Enzyme produced an almost full Enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized Enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized Enzyme. The maximum Enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the Enzyme Stability or Enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized Enzyme molecules may generate some steric hindrances for the casein hydrolysis.
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reversible immobilization of lipases on octyl glutamic agarose beads a mixed adsorption that reinforces Enzyme immobilization
Journal of Molecular Catalysis B-enzymatic, 2016Co-Authors: Nazzoly Rueda, Cleiton Dos S Santos, Maria Daniela Rodriguez, Tiago Lima De Albuquerque, Oveimar Barbosa, Rodrigo TorresAbstract:Abstract A new octyl-glutamic(OCGLU) heterofunctional agarose bead has been prepared. It has been compared to octyl-agarose (OC) in their performance to immobilize 5 different lipases, those from Candida antarctica (A (CALA) and B (CALB)), from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and a phospholipase (Lecitase ultra, LU). The immobilization rate was very similar using both supports, and the increase of activity versus p-nitrophenyl butyrate were also very similar. The effects on Enzyme Stability of the immobilization on OCGLU compared to the conventional OC was quite diverse, in some cases reducing the Enzyme Stability while in other examples the Enzyme Stability improved more than hundredfold. Curiously, the highest stabilizations were found under pH conditions where the free Enzyme could not be adsorbed on a support just bearing glutamic groups on its surface, suggesting that the mechanism of stabilization may be a quite complex one that should consider the hydrophilization of the support surface, the cationic and anionic groups of glutamic, the likely partition of organic solvents from the support surface, positive and negative Enzyme-support interactions, etc. Even though the lipases adsorption was very strong, the support could be regenerated and reused by incubation in ionic detergents.
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stabilization of the hexameric glutamate dehydrogenase from escherichia coli by cations and polyethyleneimine
Enzyme and Microbial Technology, 2013Co-Authors: Cristina Garciagalan, Oveimar Barbosa, Roberto FernandezlafuenteAbstract:Abstract The Enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The Stability of this Enzyme was increased in the presence of Li + in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate. A very significant dependence of the Enzyme Stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of Enzyme concentration on its Stability was not significantly decreased by the presence of 10 mM Li + . Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the Enzyme subunits. This incubation does not have a significant effect on Enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2 h under conditions where the untreated Enzyme retained only 20% of the initial activity, and the effect of the Enzyme concentration on Enzyme Stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified Enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li + on Enzyme Stability was maintained after Enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the Enzyme.
Nazzoly Rueda - One of the best experts on this subject based on the ideXlab platform.
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reversible immobilization of lipases on octyl glutamic agarose beads a mixed adsorption that reinforces Enzyme immobilization
Journal of Molecular Catalysis B-enzymatic, 2016Co-Authors: Nazzoly Rueda, Cleiton Dos S Santos, Maria Daniela Rodriguez, Tiago Lima De Albuquerque, Oveimar Barbosa, Rodrigo TorresAbstract:Abstract A new octyl-glutamic(OCGLU) heterofunctional agarose bead has been prepared. It has been compared to octyl-agarose (OC) in their performance to immobilize 5 different lipases, those from Candida antarctica (A (CALA) and B (CALB)), from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and a phospholipase (Lecitase ultra, LU). The immobilization rate was very similar using both supports, and the increase of activity versus p-nitrophenyl butyrate were also very similar. The effects on Enzyme Stability of the immobilization on OCGLU compared to the conventional OC was quite diverse, in some cases reducing the Enzyme Stability while in other examples the Enzyme Stability improved more than hundredfold. Curiously, the highest stabilizations were found under pH conditions where the free Enzyme could not be adsorbed on a support just bearing glutamic groups on its surface, suggesting that the mechanism of stabilization may be a quite complex one that should consider the hydrophilization of the support surface, the cationic and anionic groups of glutamic, the likely partition of organic solvents from the support surface, positive and negative Enzyme-support interactions, etc. Even though the lipases adsorption was very strong, the support could be regenerated and reused by incubation in ionic detergents.
Nathalia Saraiva Rios - One of the best experts on this subject based on the ideXlab platform.
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modulating the properties of the lipase from thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions
Enzyme and Microbial Technology, 2020Co-Authors: Yuliya Lokha, Nathalia Saraiva Rios, Luciana Rocha Barros Gonçalves, Sara Aranapena, Carmen Mendezsanchez, Fernando Lopezgallego, Roberto FernandezlafuenteAbstract:Abstract The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: Stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final Enzyme properties, altering Enzyme Stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the Enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final Enzyme Stability. The biocatalyst Stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized Enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.
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Chitosan activated with divinyl sulfone: a new heterofunctional support for Enzyme immobilization. Application in the immobilization of lipase B from Candida antarctica.
International journal of biological macromolecules, 2019Co-Authors: Bruna B. Pinheiro, Nathalia Saraiva Rios, Elena Rodríguez Aguado, Roberto Fernandez-lafuente, Tiago M. Freire, Pierre Basílio Almeida Fechine, José C. S. Dos Santos, Luciana Rocha Barros GonçalvesAbstract:Abstract A novel heterofunctional support for Enzyme immobilization, chitosan-divinyl sulfone, was assessed in this study. The activation of chitosan with DVS was carried out at three different pHs (10.0, 12.5 and 14.0) and a Candida antarctica Lipase B (CALB) was selected as the model Enzyme. After immobilization, the biocatalysts were incubated under alkaline conditions in a buffer to facilitate the multipoint covalent attachment, followed by incubation in ethylenediamine (EDA) aiming at blocking the remaining reactive groups. The highest thermal Stability was obtained when pH 10.0 was used during support activation. These results were shown to be better than those obtained when using glutaraldehyde as the support-activating reagent. Subsequently, the immobilization pH was investigated (5.0, 7.0 and 10.0) prior to alkaline incubation, with the highest Enzyme Stability levels found at pH 10.0. Finally, the selected biocatalyst was used in the hydrolysis of ethyl hexanoate and presented an activity of 14,520.37 U/g of immobilized lipase at pH 5.0. These results show that chitosan activated with divinyl sulfone is a very promising support for Enzyme immobilization and the proposed protocol is able to successfully improve Enzyme Stability.