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Assessing the enzymatic synthesis of amoxicillin using different ionic liquids
New Biotechnology · Volume 29S · September 2012
stearin fat in the ratio of 70:30 (w/w), respectively. The increase in solid fat content (SFC) as a function of the crystallization time was monitored by nuclear magnetic resonance spectrometer. The measurement was made every minute for 60 min. The induction period and maximum SFC values were determined for the original and interesterified blends. The induction period for the original blend was 3 min and maximum SFC was 21%. In interesterified blend with commercial lipase, free lipase and with the mixture, the induction period and maximum SFC were respectively: 4 min and 11%; 8 min and 4%; 3 min and 10%. The change in the profile of crystallization of the samples indicates the possible change in the structure of lipids, thus demonstrating the potential of this new enzyme, acting alone or in combination with the commercial enzyme. http://dx.doi.org/10.1016/j.nbt.2012.08.266 Poster 1.5.44 A new active and easy recovered biocatalyst: penicillin G acylase superparamagnetic aggregates Willian Kopp, Rodrigo F.C. Marques, Sandra C. Pereira, Roberto C. Giordano, Fernando M. Araújo-Moreira, Raquel L.C. Giordano ∗ Federal University of São Carlos, São Carlos-SP, Brazil This work describes the preparation of superparamagnetic enzymes aggregates (SEAs) of penicillin G acylase (PGA) using amino-superparamagnetic iron oxide nanoparticles (SPION). After adding a thin layer of silica, amine groups were added to SPION through reaction with 3-aminopropyl-triethoxysilane. For PGA-SEAS preparation, a precipitant agent (dimethoxyethane, tert-butanol or polyethylene glycol) was added to a solution containing PGA and amino-SPION. After forming the supramolecular complex, the amino-SPION and enzymes were cross-linked with glutaraldehyde (GLU). Traditional cross-linked enzyme aggregates (CLEAs) were also prepared with PGA under the same reaction conditions for SEAs, to be used as reference. While all PGA-SEAs derivatives could be easily and quickly recovered by the application of an external magnetic field, the recovery of PGA-CLEAS was very difficult. The highest recovery activity was 50%, for SEAsTBA. Similar enzyme recovery activities, for each precipitant agent, were obtained for SEAs and CLEAs when the substrate was penicillin G. However, when 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) was used, enzyme recoveries for PGA-SEAs were four to seven times higher than the ones measured for PGA-CLEAs. The presence of severe diffusion effects in the NIPAB hydrolysis, when the derivative was PGA-CLEA, was confirmed by measuring again the derivative activity after milling the catalyst. Both, SEAs and CLEAs kept 2 and 14 fold more activity than the soluble enzyme after incubation at 55◦ C and in 50% methanol, respectively. The increase of GLU concentration led to still higher thermal stabilities. Therefore, besides an easy recovery, PGA-SEAs showed better catalytic properties than PGA-CLEAs. http://dx.doi.org/10.1016/j.nbt.2012.08.267
Poster 1.5.45 Assessing the enzymatic synthesis of amoxicillin using different ionic liquids Sandra Cerqueira Pereira 1 , Roberta Bussamara 2 , Graciane Marin 2 , Raquel de Lima Camargo Giordano 1 , Jairton Dupont 2 , Roberto de Campos Giordano 1,∗ 1
Department of Chemical Engineering, Federal University of São Carlos, Via Washington Luiz, km 235, 13565-905, São Carlos, SP, Brazil 2 Laboratory of Molecular Catalysis, Institute of Chemistry, Federal University of Rio Grande do Sul, Avenue Bento Gonc¸alves, 9500, 91501970, Porto Alegre, RS, Brazil The industrial implementation of the enzymatic synthesis of -lactam antibiotics has been hindered mostly by economical reasons, in view of the high yields of the classical chemical route. During the kinetically controlled synthesis of amoxicillin a major problem is to improve the selectivity of the process, minimizing undesirable hydrolytic reactions. This problem can be minimized by reducing water activity in the medium. Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability and negligible vapor pressure. This work assesses the catalytic activity of penicillin G acylase (E.C.3.5.1.11) for the synthesis of amoxicillin using media with different Ils. The 1-butyl-3-methylimidazolium cation is common to all tested ILs. An increase of 400% in selectivity (synthesis/hydrolysis, S/H ratio) was observed for the reactions carried out with BMI.PF6 at 75% (vIL /vwater ) when compared to the totally aqueous medium (in phosphate buffer). For BMI.NTf2 a 350% increase in S/H was observed, while for BMI.BF4 there was only a slight increase in selectivity. The highest conversion of the -lactam nucleus (6-APA) was achieved using BMI.NTf2 at 71% (v/v), more than 36% above the one in water. No deactivation of the enzyme after the reactions was observed in any of the ILs, and the physical integrity of the biocatalyst particles was preserved. http://dx.doi.org/10.1016/j.nbt.2012.08.268 Poster 1.5.46 Potential application of protease from Aspergillus oryzae lba 01 in whey protein hydrolysis to maximize the antioxidant activity of bioactive peptides: a comparative study with commercial proteases R.J.S. de Castro ∗ , H.H. Sato Laboratory of Food Biochemistry, Department of Food Science, Faculty of Food Engineering, State University of Campinas - UNICAMP, Rua Monteiro Lobato 80, 13083-862, Campinas, São Paulo, Brazil Enzymatic hydrolysis for the generation of bioactive peptides with antioxidant properties has attracted much attention by researchers. The aim of this study was to evaluate the effects of the enzymatic hydrolysis of bovine whey proteins (WHEY) on their antioxidant properties. The hydrolysates were prepared with protease from Aspergillus oryzae LBA 01 (AO) produced in solid-state fermentation and two commercial proteases: protease from Aspergillus oryzae (CAO), and Bacillus licheniformis (CBL). The hydrolysis was carried out under the optimum conditions www.elsevier.com/locate/nbt S95
stearin fat in the ratio of 70:30 (w/w), respectively. The increase in solid fat content (SFC) as a function of the crystallization time was monitored by nuclear magnetic resonance spectrometer. The measurement was made every minute for 60 min. The induction period and maximum SFC values were determined for the original and interesterified blends. The induction period for the original blend was 3 min and maximum SFC was 21%. In interesterified blend with commercial lipase, free lipase and with the mixture, the induction period and maximum SFC were respectively: 4 min and 11%; 8 min and 4%; 3 min and 10%. The change in the profile of crystallization of the samples indicates the possible change in the structure of lipids, thus demonstrating the potential of this new enzyme, acting alone or in combination with the commercial enzyme. http://dx.doi.org/10.1016/j.nbt.2012.08.266 Poster 1.5.44 A new active and easy recovered biocatalyst: penicillin G acylase superparamagnetic aggregates Willian Kopp, Rodrigo F.C. Marques, Sandra C. Pereira, Roberto C. Giordano, Fernando M. Araújo-Moreira, Raquel L.C. Giordano ∗ Federal University of São Carlos, São Carlos-SP, Brazil This work describes the preparation of superparamagnetic enzymes aggregates (SEAs) of penicillin G acylase (PGA) using amino-superparamagnetic iron oxide nanoparticles (SPION). After adding a thin layer of silica, amine groups were added to SPION through reaction with 3-aminopropyl-triethoxysilane. For PGA-SEAS preparation, a precipitant agent (dimethoxyethane, tert-butanol or polyethylene glycol) was added to a solution containing PGA and amino-SPION. After forming the supramolecular complex, the amino-SPION and enzymes were cross-linked with glutaraldehyde (GLU). Traditional cross-linked enzyme aggregates (CLEAs) were also prepared with PGA under the same reaction conditions for SEAs, to be used as reference. While all PGA-SEAs derivatives could be easily and quickly recovered by the application of an external magnetic field, the recovery of PGA-CLEAS was very difficult. The highest recovery activity was 50%, for SEAsTBA. Similar enzyme recovery activities, for each precipitant agent, were obtained for SEAs and CLEAs when the substrate was penicillin G. However, when 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) was used, enzyme recoveries for PGA-SEAs were four to seven times higher than the ones measured for PGA-CLEAs. The presence of severe diffusion effects in the NIPAB hydrolysis, when the derivative was PGA-CLEA, was confirmed by measuring again the derivative activity after milling the catalyst. Both, SEAs and CLEAs kept 2 and 14 fold more activity than the soluble enzyme after incubation at 55◦ C and in 50% methanol, respectively. The increase of GLU concentration led to still higher thermal stabilities. Therefore, besides an easy recovery, PGA-SEAs showed better catalytic properties than PGA-CLEAs. http://dx.doi.org/10.1016/j.nbt.2012.08.267
Poster 1.5.45 Assessing the enzymatic synthesis of amoxicillin using different ionic liquids Sandra Cerqueira Pereira 1 , Roberta Bussamara 2 , Graciane Marin 2 , Raquel de Lima Camargo Giordano 1 , Jairton Dupont 2 , Roberto de Campos Giordano 1,∗ 1
Department of Chemical Engineering, Federal University of São Carlos, Via Washington Luiz, km 235, 13565-905, São Carlos, SP, Brazil 2 Laboratory of Molecular Catalysis, Institute of Chemistry, Federal University of Rio Grande do Sul, Avenue Bento Gonc¸alves, 9500, 91501970, Porto Alegre, RS, Brazil The industrial implementation of the enzymatic synthesis of -lactam antibiotics has been hindered mostly by economical reasons, in view of the high yields of the classical chemical route. During the kinetically controlled synthesis of amoxicillin a major problem is to improve the selectivity of the process, minimizing undesirable hydrolytic reactions. This problem can be minimized by reducing water activity in the medium. Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability and negligible vapor pressure. This work assesses the catalytic activity of penicillin G acylase (E.C.3.5.1.11) for the synthesis of amoxicillin using media with different Ils. The 1-butyl-3-methylimidazolium cation is common to all tested ILs. An increase of 400% in selectivity (synthesis/hydrolysis, S/H ratio) was observed for the reactions carried out with BMI.PF6 at 75% (vIL /vwater ) when compared to the totally aqueous medium (in phosphate buffer). For BMI.NTf2 a 350% increase in S/H was observed, while for BMI.BF4 there was only a slight increase in selectivity. The highest conversion of the -lactam nucleus (6-APA) was achieved using BMI.NTf2 at 71% (v/v), more than 36% above the one in water. No deactivation of the enzyme after the reactions was observed in any of the ILs, and the physical integrity of the biocatalyst particles was preserved. http://dx.doi.org/10.1016/j.nbt.2012.08.268 Poster 1.5.46 Potential application of protease from Aspergillus oryzae lba 01 in whey protein hydrolysis to maximize the antioxidant activity of bioactive peptides: a comparative study with commercial proteases R.J.S. de Castro ∗ , H.H. Sato Laboratory of Food Biochemistry, Department of Food Science, Faculty of Food Engineering, State University of Campinas - UNICAMP, Rua Monteiro Lobato 80, 13083-862, Campinas, São Paulo, Brazil Enzymatic hydrolysis for the generation of bioactive peptides with antioxidant properties has attracted much attention by researchers. The aim of this study was to evaluate the effects of the enzymatic hydrolysis of bovine whey proteins (WHEY) on their antioxidant properties. The hydrolysates were prepared with protease from Aspergillus oryzae LBA 01 (AO) produced in solid-state fermentation and two commercial proteases: protease from Aspergillus oryzae (CAO), and Bacillus licheniformis (CBL). The hydrolysis was carried out under the optimum conditions www.elsevier.com/locate/nbt S95