Browsing by Author "Fernandes, Pedro"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- From Inulin to Fructose Syrups Using Sol–Gel Immobilized InulinasePublication . Santa, Gonçalo L. M.; Bernardino, Susana M. S. A.; Magalhães, Salomé; Mendes, Vanessa; Marques, Marco P. C.; Fonseca, Luís P.; Fernandes, PedroThe present work aims to provide the basic characterization of sol-gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature- and pH- activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis-Menten kinetics suggest that immobilization in sol-gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol-gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24- hour batch runs without noticeable decay in product yield. The performance of sol-gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol-gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.
- Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro‐particlesPublication . Bernardino, Susana M. S. A.; Fernandes, Pedro; Fonseca, Luís P.There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h-1gbiocatalyst-1 at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol-1 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin.
- A new biocatalyst: Penicillin G acylase immobilized in sol‐gel micro‐particles with magnetic propertiesPublication . Bernardino, Susana M. S. A.; Fernandes, Pedro; P. Fonseca, Luís; Bernardino, SusanaThe present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/gdry weight) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.
- Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic propertiesPublication . Bernardino, Susana; Estrela, Nídia; Ochoa-Mendes, Vanessa; Fernandes, Pedro; Fonseca, Luís P.Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from E. coli is currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol-gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol-gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65-67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein g dry sol-gel -1 and 930 Ug dry sol-gel -1 for specific activity were obtained.