Rational design of biocatalysts based on covalent immobilization of acylase enzymes.

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst's total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA-Sepharose CL 4B supports, for the use in bioconversion of residual colorants.

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

Rational Design Strategy as a Novel Immobilization Methodology Applied to Lipases and Phospholipases.

Immobilization of lipases and phospholipases, mainly on water-insoluble carriers, helps in their economic reusing and in the development of continuous bioprocesses. Design of efficient lipase and phospholipase-immobilized systems is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop efficient immobilized systems. We conceived a new strategy for the rational design of immobilized derivatives (RDID) in favor of the successful synthesis of optimal lipase and phospholipase-immobilized derivatives, aiming the prediction of the immobilized derivative's functionality and the optimization of load studies. The RDID strategy begins with the knowledge of structural and functional features of synthesis components (protein and carrier) and the practical goal of the immobilized product. The RDID strategy was implemented in a software named RDID1.0. The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.

Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes.

Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2 µmol aldehyde/cm2). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH2, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV-GCM showed the highest maximal immobilized protein load (2.1 µg/cm2), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.

Diseño asistido por computadora de la inmovilización covalente de bromelina y papaína / Computer-aided design of bromelain and papain covalent immobilization

Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The aim of this work was to optimize the covalent immobilization of bromelain and papain via rational design of immobilized derivatives strategy (RDID) and RDID1.0 program. Were determined the maximum protein quantity to immobilize, the optimum immobilization pH (in terms of functional activity retention), was predicted the most probable configuration of the immobilized derivative and the probabilities of multipoint covalent attachment. As support material was used Glyoxyl-Sepharose CL 4B. The accuracy of RDID1.0 program´s prediction was demonstrated comparing with experimental results. Bromelain and papain immobilized derivatives showed desired characteristics for industrial biocatalysis, such as: elevate pH stability retaining 95% and 100% residual activity at pH 7.0 and 8.0, for bromelain and papain, respectively; high thermal stability at 30 °C retaining 90% residual activity for both immobilized enzymes; a catalytic configuration bonded by immobilization at optimal pH; and the ligand load achieve ensure the minimization of diffusional restrictions.

Las enzimas inmovilizadas han sido ampliamente utilizadas en las industrias Alimentaria, Agroquímica, Farmacéutica y Biotecnológica. La inmovilización de proteínas es, probablemente, la tecnología más empleada para elevar la estabilidad operacional de estas moléculas. La bromelina (Ananas comosus) y la papaína (Carica papaya) son cisteín proteasas extensamente usadas como biocatalizadores inmovilizados con disímiles aplicaciones en la terapéutica, resolución de mezclas racémicas, cromatografía de afinidad, entre otros escenarios industriales. El objetivo del presente trabajo fue optimizar la inmovilización covalente de las enzimas bromelina y papaína a través de la estrategia de diseño racional de derivados inmovilizados (RDID) y el programa RDID1.0. Se predijo la cantidad máxima de proteína a inmovilizar, el pH óptimo de inmovilización (en términos de retención de la actividad funcional), la configuración más probable del derivado inmovilizado y la probabilidad de enlazamiento covalente multipuntual. Como soporte de inmovilización de empleó Glioxil-Sepharose CL 4B. La precisión de las predicciones llevadas a cabo con el programa RDID1.0 fue validada comparando con los resultados experimentales obtenidos. Los derivados inmovilizados de bromelina y papaína mostraron características deseadas para la biocatálisis a nivel industrial, tales como: elevada estabilidad al pH reteniendo el 95% y 100% de actividad residual a pH 7.0 y 8.0, para la bromelina y la papaína, respectivamente; una elevada estabilidad térmica con la retención del 90% de actividad residual a 30 °C para ambas enzimas; al pH de inmovilización óptimo la configuración obtenida es catalíticamente competente; y la carga de ligando alcanzada asegura la disminución de las restricciones difusionales.

A thermostable exo-ß-fructosidase immobilised through rational design.

Thermotoga maritima exo-ß-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl-Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 °C was achieved with a productivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5 days at 60 °C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

Rational design of immobilized lipases and phospholipases.

Immobilization of lipases and phospholipases on, mainly, water insoluble carriers, helps in their economic reuse and in the development of continuous bioprocesses. Design of efficient lipases and phospholipases-immobilized system is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop an efficient immobilized system. A new rational design of immobilized derivatives strategy (RDID) has been conceived in favor of the successful synthesis of optimal lipases and phospholipases-immobilized derivatives, aiming prediction of the immobilized derivative's functionality and the optimization of load studies. RDID begins with the knowledge of structural and functional features of synthesis components (protein and carrier), and the practical goal of immobilized product. RDID was implemented in software named RDID ( 1.0 ). The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.