Effect of inactivation and reactivation conditions on activity recovery of enzyme catalysts

Enzymes are labile catalysts with reduced half-life time that can be however improved by immobilization and, furthermore, already inactivated catalyst can be recovered totally or partially, therefore allowing the large scale application of enzymes as process catalysts. In recent years a few studies about reactivation of enzyme catalysts have been published as a strategy to prolong the catalyst lifetime. Reported results are very good, making this strategy an interesting tool to be applied to industrial process. These studies have been focused in the evaluation of different variables that may have a positive impact both in the rate and level of activity recovery, being then critical variables for conducting the reactivation process at productive scale. The present work summarizes the studies done about reactivation strategies considering different variables: type of immobilization, enzyme-support interaction, level of catalyst inactivation prior to reactivation, temperature and presence of modulators.

Whey upgrading by enzyme biocatalysis

Whey is a co-product of processes for the production of cheese and casein that retains most of the lactose content in milk. World production of whey is estimated around 200 million tons per year with an increase rate of about 2 percent/per year. Milk production is seasonal, so surplus whey is unavoidable. Traditionally, whey producers have considered it as a nuisance and strategies of whey handling have been mostly oriented to their more convenient disposal. This vision has been steadily evolving because of the upgrading potential of whey major components (lactose and whey proteins), but also because of more stringent regulations of waste disposal. Only the big cheese manufacturing companies are in the position of implementing technologies for their recovery and upgrading, so there is a major challenge in incorporating medium and small size producers to a platform of whey utilization, conciliating industrial interest with environmental protection within the framework of sustainable development. Within this context, among the many technological options for whey upgrading, transformation of whey components by enzyme biocatalysis appears as prominent. In fact, enzymes are green catalysts that can perform a myriad of transformation reactions under mild conditions and with strict specificity, so reducing production costs and environmental burden. This review pretends to highlight the impact of biocatalysis within a platform of whey upgrading. Technological options are shortly reviewed and then an in-depth and critical appraisal of enzyme technologies for whey upgrading is presented, with a special focus on newly developed enzymatic processes of organic synthesis, where the added value is high, being then a powerful driving force for industrial implementation.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Esterificación quimioselectiva de fitosteroles / Chemoselective transesterification of wood steroles by lipases

La esterificacion selectiva de fitosteroles de madera representa una aplicación novedosa de las lipasas, que se inscribe dentro de una plataforma tecnológica de valoración del licor negro en el proceso Kraft. Los fitosteroles de madera son mezclas de esteroles y estanoles (esteroles saturados) donde más del 90 por ciento es beta-sitosterol y beta-sitostanol. Ambos productos tienen mercados potenciales diferentes por lo que el fraccionamiento de los fitosteroles que los contienen implica un valor agregado considerable. Ambas sustancias son muy similares, lo que impide su separación por métodos físicos, siendo la esterificación selectiva de esta mezcla con ésteres de ácidos grasos mediante lipasas quimioselectivas una interesante opción tecnológica para separarlas. Se evaluaron diversas lipasas comerciales en su capacidad de esterificar selectivamente los estanoles, seleccionándose una enzima inmovilizada y una no soportada. Se optimizó el proceso con la enzima inmovilizada obteniéndose grados de esterificación de estanoles por sobre el 90 por ciento y de esteroles en torno al 20 por ciento, lo que satisface el criterio de selectividad establecido. La enzima inmovilizada comercial tuvo baja estabilidad operacional debido a la desorción de la proteína por lo que se desarrollaron estrategias de inmovilización de la lipasa comercial no soportada, obteniéndose los mejores resultados con butil Sepabeads® como soporte. Con dicho biocatalizador se realizó la reacción de transesterificacion en modalidad de lotes repetidos demostrándose la elevada estabilidad de la enzima y comprobándose que es posible realizar cinco lotes productivos sin merma de la conversión ni de la productividad, lo que satisface los criterios de rentabilidad del proceso. La tecnología ha sido transferida al sector productivo y se ha presentado una patente de invención sobre la elaboración del biocatalizador.

Peptide synthesis: chemical or enzymatic

Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective.

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Recombinant CHO TF70R cells are able to grow and produce t-PA on serum-free medium BIOPRO1 (BioWhitaker Europe, Belgium). The purpose of the present study was to determine the effect of medium supplementation with vitamins, lipids, and specific amino acids on cell growth, t-PA production and biological functionality. Among vitamins, only biotin, folic acid, cobalamine and benzoic acid were required for improving growth and t-PA production. Lipid supplement allowed a significant increase cell concentration and t-PA specific activity and concentration, though its specific production rate decreased slightly. Medium supplementation with proline, serine and asparagine had also positive effects on cell growth. Besides, the addition of asparagine (even in the presence of glutamine) was essential for the production and biological quality of the t-PA. This systematic approach for media supplementation produced an increase in cell concentration around 100 percent and in t-PA production around 80 percent, with no detrimental effect on its biological activity. The effect of asparagine on t-PA production was unexpected and needs to be further studied. The above modifications of the production medium did not produce a significant effect on the metabolism of the main carbon and energy sources (glucose and glutamine) and the level of by-product formation (lactate and ammonia).

Evaluation of penicillin acylase production by two strains of Bacillus megaterium

Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics. The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized. The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature. A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media. The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined

Inmovilización de lactasa microbiana / Immobilization of microbial lactase

La lactasa tiene un gran potencial de aplicación en la industria láctea como modificador de propiedades funcionales y nutricionales de leche y derivados y como agente de recuperación del suero de quesería. Este último aspecto se ha abordado a fin de producir y utilizar catalizadores enzimáticos para la hidrólisis continua de permeado de suero. La selección de enzima y soportes y la optimización de la metodología de inmovilización ya han sido reportadas. Los resultados obtenidos con lactasa inmovilizada en quitina han motivado el intento de escalar los procedimientos a nivel productivo, Un primer objetivo ha sido el desarrollo de un sistema de inmovilizacion en el reactor (in situ) adecuado a la operación en planta. Se presentan los resultados de inmovilización in situ en sistemas por lote cerrado y con recirculacióon. Los resultados del protocolo de inmovilización original optimizado son reproductibles, si bien se observan gradientes de actividad enzimática a lo largo del reactor, las que no se logra eliminar por recirculación de reactantes. Se presenta los resultados de operación de reactores continuos de lecho fijo con lactasa inmovilizada in situ, operando a distintos flujos y concentraciones de lactosa. Los grados de conversion y productividad obtenidos se comparan con un modelo simple, desarrollado en base a la expresión cinética correspondiente y un regimen de flujo piston. La desviación es significativa a flujos de operación altos, lo que se atribuye a retromezclamiento y canalización a través del lecho catalítico, obteniéndose un mejor ajuste a flujos bajos y altas concentraciones de sustrato de alimentación. La productividad fue máxima a 120 g/1 de lactosa y 40 ml/h e igual a 58 g de glucosa/1.h. La estabilidad del catalizador fue evaluada en el reactor a pH 4,0 y 40 C con permeado de suero, calculándose un tiempo de vida media operacional de 120 dias