Sepabeads EC-EP Research Articles

Sepabeads EC-EP immobilized α-galactosidase: Immobilization, characterization and application in the degradation of raffinose-type oligosaccharides

Raffinose family oligosaccharides (RFOs) are mainly containing raffinose and stachyose. They are antinutrient compounds and the major factors responsible for the flatulence upon ingestion of soymilk. α-Galactosidases can hydrolyse RFOs to digestible oligosaccharides. α-Galactosidase was partially purified from watermelon and then immobilized on Sepabeads EC-EP with 56% of activity yield by determined best immobilization conditions. The optimum temperature and pH for both enzymes were found as 65 °C and pH 6.0, respectively. They showed an excellent stability between pH 3.0–7.0 and below 70 °C. The effect of several sugars and metal ions showed that both enzymes affected at different rates. Reusability of the immobilized enzyme with pNPG and raffinose as substrates indicated that the enzyme can be used for a long time with over 50% recovered activity. The enzymatic hydrolysis of the RFOs was realized and HPLC analysis demonstrated that 77.5% and 72.7% of the stachyose and raffinose presented in soymilk is hydrolyzed by the immobilized enzyme during 24 h incubation, respectively. This practical and alternative approach may be a potential to improve the nutritional value of soymilk by eliminating RFOs. Because of its high activity and stability properties the immobilized α-galactosidase may also find several applications in food and feed industries.

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

Optimization of covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports for cellulose hydrolysis, in buffer and ionic liquids/buffer media

The covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports were carried out. The optimal immobilization conditions were determined using response surface methodology. The hydrolysis of cellulose using the free and immobilized cellulase preparations in ionic liquids (IL) using cosolvents was investigated. The hydrolytic activities in buffer medium containing 25% (v/v) of 1-butyl-3-methylimidazolium hexafluorophosphate were around 2.6-, 1.6-, and 5.5-fold higher than the activities in buffer medium. The retained initial activities were 32% and 57%, respectively for cellulase preparations immobilized onto Sepabeads EC-EP support and onto modified ReliZyme HA403 support after 5 reuses.

Continuous‐Flow Reactor‐Based Enzymatic Synthesis of Phosphorylated Compounds on a Large Scale

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads-150 or Sepabeads EC-EP). After immobilization 70% of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D-glucose-6-phosphate, N-acetyl-D-glucosamine-6-phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol-1-phosphate, and inosine-5'-monophosphate from the corresponding primary alcohol on gram scale using either a fed-batch reactor or a continuous-flow packed-bed reactor.

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

BackgroundIndustrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.ResultsThe DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.ConclusionsFluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.

Immobilization of β-galactosidase from Bacillus circulans onto epoxy-activated acrylic supports

Abstract The enzyme β-galactosidase from Bacillus circulans was immobilized onto the epoxy-activated acrylic supports Sepabeads EC-EP and Sepabeads EC-HFA. The influence of immobilization conditions on the process yield was studied. Optimal conditions for Sepabeads EC-EP were 1.4 M potassium phosphate buffer pH 8.5, enzyme loads in the range 3–30 mg/g of gel, and an incubation period of 48 h, achieving protein immobilization yields of 98–100% with activity yields of 85–98%. For Sepabeads HFA, low ionic strength (0.02 M potassium phosphate buffer), enzyme loads in the range 1.5–15 mg/g of gel, and shorter incubation periods (24 h) were required; however, when the whole process was performed at pH 8.5, maximum yields achieved were below 60%. This process was improved by performing a fast adsorption at pH 7.5 and then raising the pH to 8.5 to favor covalent coupling, achieving protein immobilization yields of 100% and enzyme activity yields of 83%. The remarkable thermal stability of derivatives obtained at low enzyme loads, and the fact that the thermal stability of medium load enzyme derivatives improved when a post-immobilization alkaline treatment was performed, support the hypothesis that stabilization was achieved through multipoint covalent attachment.

Immobilization of lipase from Candida rugosa on Sepabeads®: the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads(®) support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads(®) EC-EA and Sepabeads(®) EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads(®) EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.

Use of Candida rugosa lipase immobilized on sepabeads for the amyl caprylate synthesis: batch and fluidized bed reactor study

Lipase from Candida rugosa was covalently immobilized on Sepabeads EC-EP for application for amyl caprylate synthesis in an organic solvent system. Several solvents were tested in terms of biocatalyst stability and the best result was obtained with isooctane. The lipase-catalyzed esterification in the selected system was performed in batch and fluidized bed reactor systems. The influence of several important reaction parameters including temperature, initial water content, enzyme loading, acid/alcohol molar ratio, and time of addition of molecular sieves is carefully analyzed by means of an experimental design. Almost complete conversion (> 99%) of the substrate to ester could be performed in a batch reactor system, using lipase loading as low as 37 mg g -1 dry support and in a relatively short time (24 hrs) at 37oC, when high initial substrate molar ratio of 2.2 is used. Kinetics in a fluidized bed reactor system seems to still have a slightly better profile than in the batch system (90.2% yields after 14 hrs). The fluidized bed reactor operated for up 70 hrs almost with no loss in productivity, implying that the proposed process and the immobilized system could provide a promising approach for the amyl caprylate synthesis at the industrial scale.

An efficient enzymatic synthesis of 5-aminovaleric acid

The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free conditions. l-Lysine was oxidized by the action of l-lysine α-oxidase from Trichoderma viride followed by spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the reaction medium. l-Lysine α-oxidase was immobilized on an epoxy-activated solid support (Sepabeads EC-EP) and the activity of both solution-based and immobilized enzyme in this reaction was determined.

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier−1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t1/2=625 h vs. t1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.

A study on the applicability of l-aspartate α-decarboxylase in the biobased production of nitrogen containing chemicals

β-Alanine could serve as an intermediate in the biobased production of nitrogen containing chemicals from L-aspartic acid. Following the biorefinery concept, L-aspartic acid could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. Since α-decarboxylation of L-aspartic acid is difficult to perform chemically, the applicability of Escherichia coliL-aspartate α-decarboxylase (EC 4.1.1.11) (ADC) for the production of β-alanine was studied. With an increasing activity up to 90 °C and maintaining its activity upon storage for 24 hours at 60 °C, ADC showed a remarkably high thermostability. ADC has an optimum at pH 7.5 and starts to lose activity upon storage below pH 6. An inhibiting effect by β-alanine was not observed. Immobilization on Sepabeads EC-EP and EC-HFA epoxy supports did not result in an increased thermostability, but did improve operational stability. Nonetheless, enzyme inactivation occurs during catalysis, probably caused by irreversible transamination of the catalytically essential pyruvoyl group.

Role of the support surface on the loading and the activity of Pseudomonas fluorescens lipase used for biodiesel synthesis

The present work investigates the influence of the support surface on the loading and the enzymatic activity of the immobilized Pseudomonas fluorescens lipase. Different porous materials, polypropylene (Accurel), polymethacrylate (Sepabeads EC-EP), silica (SBA-15 and surface modified SBA-15), and an organosilicate (MSE), were used as supports. The immobilized biocatalysts were compared towards sunflower oil ethanolysis for the sustainable production of biodiesel. Since the supports have very different structural (ordered hexagonal and disordered) and textural features (surface area, pore size, and total pore volume), in order to consider only the effect of the support surface, experiments were performed at low surface coverage. The different functional groups occurring on the support surface allowed either physical (Accurel, MSE, and SBA-15) or chemical adsorption (Sepabeads EC-EP and SBA-15–R-CHO). The surface-modified SBA-15 (SBA-15–R-CHO) allowed the highest loading. The lipase immobilized on the MSE was the most active biocatalyst. However, in terms of catalytic efficiency (activity/loading) the lipase immobilized on the SBA-15, the support that allowed the lowest loading, was the most efficient.

Practical application of different enzymes immobilized on sepabeads

The immobilization of an endoglucanase, benzoylformate decarboxylase (BFD) from Pseudomonas putida, as well as of lipase B from Candida antarctica (CALB) onto the carrier supports Sepabeads EC-EP, Sepabeads EC-EA, and Sepabeads EC-BU was accomplished. It is shown that via these immobilized biocatalysts the synthesis of both fine and bulk chemicals is possible. This is illustrated by the syntheses of polyglycerol esters and (S)-hydroxy phenyl propanone. The benefit of immobilization is illustrated by repetitive use in a bubble column reactor as well as in a stirred tank reactor. High stability of two biocatalysts was achieved and reusability up to eight times was demonstrated. The comparison of CALB immobilized on Sepabeads EC-EP to Novozym 435 shows similar activity.

The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis

BackgroundImmobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.ResultsTo provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50°C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.ConclusionMulti-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.

The immobilization of lipase on Sepabeads: Coupling, characterization and application in geranyl butyrate synthesis in a low aqueous system

Lipase from Candida rugosa immobilized on Sepabeads EC-EP was shown to catalyze the esterification of geraniol with butyric acid in a predominantly organic system. The immobilization procedure was adjusted to optimize the enzyme activity and the immobilized enzyme was then used for a geranyl butyrate synthesis as a study model. The immobilized enzyme showed favorable performances in an aqueous system and increased the stability in the presence of organic solvents. The response surface methodology and a 5-level-5-factor central composite rotatable design were performed to identify the factors that influence the ester production and to verify whether any changes should be made in their settings to improve this reaction. The initial water content, the reaction temperature, the enzyme concentration, acid/alcohol molar ratio and time of addition of molecular sieves were the variables investigated. The production of the ester was optimized and an ester production response equation was obtained, making it possible to predict ester yields from the known values of the five main factors. The temperature during the esterification reaction was identified as the factor having the greatest impact on the ester yield.

Immobilization of lipase on sepabeads and its application in pentyl octanoate synthesis in a low aqueous system

The object of the study was to investigate the process conditions relevant for the pentyl octanoate production with the lipase from Candida rugosa immobilized on Sepabeads EC-EP carrier. This is an epoxide-containing commercial polymethacrylic carrier with suitable characteristics for enzyme immobilization. The immobilized lipase suitable for pentyl octanoate synthesis has been prepared by a direct lipase binding to polymers via their epoxide groups. The enzymatic activity was determined by both hydrolysis of olive oil in an aqueous system and esterification of n-pentanol with octanoic acid in a low aqueous system. The influence of several important reaction parameters such as temperature, initial water content, initial substrate molar ratio, enzyme loading and time of adding of molecular sieves in the system is carefully analyzed by means of an experimental design. Production of the ester was optimized and an ester production response equation was obtained, making it possible to predict ester yields from known values of the five main factors. Almost complete conversion (&gt;99%) of the substrate to ester could be realized, using lipase loading as low as 37 mg/g dry support and in a relatively short time (24 h) at 45?C, when high initial substrate molar ratio of 2.2 is used.

Immobilization of penicillin acylase from Escherichia coli on commercial sepabeads EC-EP carrier

This paper describes the covalent immobilization of penicillin G acylase from Escherichia coli on sepabeads EC-EP, an epoxy-activated polymethacrylic carrier and kinetic properties of the immobilized enzyme. The selected enzyme belongs to a class of biocatalysts whose industrial interest is due to their versatility to mediate hydrolysis of penicillins and semi-synthetic ?-lactam antibiotics synthesis reactions. About 2.7 mg of the pure enzyme was immobilized onto each gram of sepabeads with an enzyme coupling yield of 96.9%. However, it seems that the activity coupling yield is not correlated with the amount of enzyme bound and the maximum yield of 89.4% can be achieved working at low enzyme loading (0.14 mg g-1). Immobilization of the penicillin acylase resulted in slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted structural and conformational stability of this enzyme. It appears that both free and immobilized penicillin acylase followed simple Michaelis-Menten kinetics, implying the same reaction mechanism in both systems.

Preparation and characterization of penicillin acylase immobilized on sepabeads EC-EP carrier

This paper reports the covalent immobilization of penicillin G acylase from E. coli on Sepabeads EC-EP, an epoxy-activated polymethacrylic carrier, and describes the properties of the immobilized enzyme. Due to its versatility to mediate hydrolysis of penicillins and semi-synthetic B-lactam antibiotics synthesis reactions, the selected enzyme belongs to a class of biocatalysts of great industrial interest. The immobilized enzyme was characterized in its pH and thermal stability and reaction kinetics. The immobilization of penicillin acylase resulted in a slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted the structural and conformational stability to this enzyme. The immobilized enzyme also retained a high catalytic activity and showed the increased thermal stability compared with a free enzyme. By comparison of decimal reduction time values obtained at 50?C, it can be concluded that the immobilized enzyme was approximately 5-fold more stable than a free enzyme. The immobilization procedure developed is quite simple and easily reproduced, and provides a promising solution for the application of penicillin acylase for the purpose of 6-aminopenicillanic acid production.